MXPA00010811A - Indolicidin analogs and methods of using same - Google Patents

Abstract

The present invention relates to 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-NH2 ("Indol 1-13;"SEQ ID NO:1). The indolicidin analogs of the invention include, for example, analogs such as Indol 2-13 (SEQ ID NO:2) and Indol 3-13 (SEQ ID NO:3), which are truncated at the amino terminus by one and two amino acids, respectively, as compared to Indol 1-13 (SEQ ID NO:1);analogs in which at least one Trp residue in an amino terminal truncated indolicidin analog is replaced by a Phe residue ("Indol/F"analogs);indolicidin analogs comprising, at the carboxy terminus, a homoserine residue;and fusion polypeptides comprising an indolicidin analog. In addition, the invention provides nucleic acid molecules encoding the indolicidin analogs of the invention or precursors of such analogs. The invention also relates to methods of using the indolicidin analogs to reduce or inhibit microbial growth or survival by contacting an environment capable of sustaining microbial growth with the indolicidin analog.

Description

INDOLICIDINE ANALOGS AND METHODS FOR USING THEMSELVES DESCRIPTION OF THE INVENTION "The present invention relates generally to antimicrobial agents and, more specifically, to indolicidin analogs and methods for using the analogs to reduce or inhibit microbial growth or subsistence. 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 infection, the patient and the elderly are particularly susceptible, for example, patients who are hospitalized frequently. they acquire secondary infections due to a combination of their weakened condition and the prevalence of microorganisms in a hospital, such opportunistic infections result in the patient's increased suffering, the increased length of hospitalization and, consequently, increased costs to the patient and the care system of the patient. health. 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 deteriorating 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 and some 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 analogues which, when administered to an individual, have increased selectivity against infectious microorganisms as 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 subsistence of microorganisms. The present invention satisfies this need and provides additional advantages. The present invention relates to indolicidin analogues, 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 indolicidin analogues of the invention have the structure: X1-X2-X3-X4-X5-X6-P6-X6-X6-P- X6-X7-X7-X8, 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 or absent, X5 is Arg, Lys or absent, X6 is Trp, or Phe, X7 is Arg, Lys or absent, and X8 is homoserine, Met, Met-X9-Met or absent, where X9 is at least one amino acid; the condition that if X1 is present, X8 is either homoserine, Met or Met-X9-Met where X9 is at least one amino acid or both events of X7 are absent, and with the additional proviso that if 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 For example, the invention provides analogs of truncated amino terminal indolicidin, including Indole 2-13 (SEQ ID NO: 2), Indo l 3-13 (SEQ ID. NO: 3), Indole 4-13 (SEQ ID NO: 4) and Indole 5-13 (SEQ ID NO: 5), each of which has broad spectrum antimicrobial activity. The invention also provides indolicidin analogs, wherein at least one of the Trp residues in a truncated amino terminal indolicidin analog is replaced by a Phe residue ("Indol / F" analogs). For example, the invention provides Indole 2-13 (SEQ ID NO: 2) in which each of the residues Trp in Indole 2-13 (SEQ ID NO: 2) is replaced with a residue of Phe. Truncated amino terminal indolicidin analogs lacking a carboxy terminal amide are also provided. In addition, the invention provides analogs of indolicidin comprising, at the carboxy terminus, a homoserin residue ("Hse") ("Indol-Hse"), such analogs having antimicrobial activity. For example, the invention provides analogues of Indol-Hse such as H2N-Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-Hse (SEQ ID NO: 24; "Indole (1-13) -Hse"), Indole (2-13) -Hse and the like, and similarly provides analogs of Indole / F, including Indole 1-13 / F-Hse. In addition, the invention provides nucleic acid molecules that encode the indolicidin analogs of the invention or precursors of such analogues. The ability of an Indol-Hse or Indol / F-Hse analog to exhibit antimicrobial activity provides a means to express poly-indolicidin analog polypeptides in vitro or in vivo, wherein each indolicidin analog in the polypeptide is separated by a residue Met or by the Met-X9-Met sequence, such that treatment of the polypeptide with cyanogen bromide results in the production of Indol-Hse or Indol / F-Hse analogs. For example, Met-X9-Met can be Met-Gly-Ser-Glu-Met (SEQ ID NO: 20) or Met-Ala-Arg-Ile-Ala-Met (SEQ ID NO: 21) ) or similar. The expression, for example, of one mole of Indol l-13 / F- (MGSEM) -Indol 1-13 / FM (SEQ ID NO: 22), which consists of two separate un-amidated Indole 1-13 peptides by the sequence Met-Gly-Ser-Glu-Met (SEQ ID NO: 20) and including a carboxy terminal Met, followed by treatment with cyanogen bromide results in the production of two moles of Indole 1-13 / F-Hse (SEQ ID NO: 23). Expression of such poly-indolicidin analog polypeptides provides a convenient means to produce substantial amounts of indolicidin analog peptides from a host organism, which includes a microorganism, because the polypeptide form of the analog has no substantial antimicrobial activity. In addition, the invention provides fusion polypeptides comprising an indolicidin analog and a peptide of interest, which may be useful, for example to facilitate the purification of an expressed indolicidin analog. The invention also relates to methods for using an indolicidin analog to reduce or inhibit microbial growth or subsistence in an environment capable of sustaining microbial growth or subsistence by contacting the environment with the indolicidin analog. As such, the invention provides methods for reducing or inhibiting microbial growth or subsistence 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 subsistence 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 indolicidal analogue to the individual, thereby reducing the severity of the pathological condition. BRIEF DESCRIPTION OF THE DRAWINGS Figures IA to ID compare the antimicrobial activity of Indol 1-13 (SEQ ID NO: 1) synthetic and various truncated amino terminal indolicidin analogs, as indicated, using an agar diffusion test. . The antimicrobial activity was examined against Escherichia coli (Figure 1), Staphylococcus aureus (Figure IB), Cryptococcus neofor ans (Figure 1C) or Candida albicans (Figure ID). The peptides were added at concentrations ranging from 5.25 μM to 157.4 μM and the zones of inhibition were measured (millimeters "mm"). Figures 2A to 2D compare the antimicrobial activity of Indole 1-13 (SEQ ID NO: 1) and various truncated amino terminal indolicidin analogs, as indicated, using a suspension test. The antimicrobial activity was examined against Escherichia coli (Figure 2A), Staphylococcus aureus (Figure 2B), Cryptococcus neoformans (Figure 2C) or Candida albicans (Figure 2D). Peptides were added at concentrations ranging from 0.52 μM to 15.74 μM and the mortality rate was determined. Figures 3A to 3C compare the antimicrobial activity of Indole 1-13 (SEQ ID NO: 1); open circles) with the homoserine acid lactone (closed circles) and the Indol-H lactone analogues produced after cleavage with cyanogen bromide from Indole (1-13) -Met-Gly-Ser-Glu (SEQ ID. NO: 35) using an agar diffusion test (Figures 3A and 3B) or a bactericidal test (Figure 3C). The peptides were examined at the indicated concentrations against S. to ureus (Figure 3A) or E. coli (Figures 3B and 3C). The zones of inhibition (mm) were determined for the agar diffusion tests and the colony forming units (CFU) / ml were determined for the bactericidal tests. Figures 4A to 4F compare the antimicrobial activities of Indol 1-13 (SEQ ID NO: 1) and the various truncated amino terminal Indol-Hse analogues, as indicated, using an agar diffusion test. The antibacterial activity was determined against E. coli (Figures 4A to 4C) or S. to ureus (Figures 4D to 4F). The zones of inhibition were determined for peptide concentrations ranging from 5.25 μM to 157.4 μM (nmol / ml). Figures 5A and 5B compare the antimicrobial activities of the Indole (1-13) -Hse / Phe analogs, in which all the Trp residues of Indole 1-13 (SEQ ID NO: 1) are replaced by the Phe residues and each peptide contains a carboxy terminal homoserin (Hse) residue, using an agar diffusion test. Peptides were tested at concentrations ranging from 1.57 μM to 52.5 μM against E. coli (Figure 5A) or S. aureus (Figure 5B). Figure 6 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: 21). The coding strand (sense) is shown in uppercase letters, the antisense strand is shown in lower case and the encoded amino acid sequence is shown using the three letter code ("Stp" indicates STOP codon). Eco Rl and Sal I are indicated sites of endonuclease restriction. The enterokinase recognition site is simply underlined, with the double arrow indicating the cleavage site. Simple arrows denote cleavage sites by cyanogen bromide. The dotted underlined tetranucleotide sequences correspond to overlays in the oligonucleotides used for binding. The double underlined sequences denote sebators used for PCR amplification (see Example I.B). Figure 7 compares the antimicrobial activity of In 1-13 (SEQ ID NO: 1, open circles), In (1-13) -H synthetic (diamonds) and In (1-13) -H is derived from poly (In (1-13) - Met-Ala-Arg-Ile-Ala-Met) 3 (closed circles, see Figure 6) using an agar diffusion test. The peptides were examined at various concentrations, as indicated against S. aureus and the zones of inhibition were determined (mm). The present invention provides isolated incidin analogues having the general structure: X1-X2-X3-X4-X5-X6-P-X6-X6-P-X6-X7-X7-X8, wherein Xl 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 or absent; X5 is Arg, Lys or absent; X6 is Trp, or Phe; X7 is Arg, Lys or absent; and X8 is homoserine or absent with the proviso that if XI is present, X8 is either homoserine (Hse), Met or Met-X9-Met where X9 is at least one amino acid or both X7 events are absent; and with the additional proviso that if X2 is absent, XI is absent, if X3 is absent X1 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. In particular, the invention provides the truncated amino terminal incidin analogs, which include analogs wherein 1 or more Trp residues in the analogue is replaced by Phe (see Tables 1 and 2); for incidin analogues, including full-length incidin analogs, containing in the carboxy terminal a homoserin residue (Hse), a Met residue or the amino acid sequence Met-X9-Met, for example, Met-Gly-Ser -Glu-Met (SEQ ID NO: 20), Met-Ala-Arg-Ile-Ala-Met (SEQ ID NO: 21) or the like and for fusion polypeptides comprising such incidin analogue. Incidin is a naturally occurring polypeptide, having the amino acid sequence Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-CONH2 ("In1-13;" ID SECTION NO: 1). Incidin (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 incidin analogs having the general structure H2N-ILPWKWPWWPWX (SEQ ID NO: 23), wherein X designates one or two independently selected amino acids, have been described (see US Pat. No. 5,534,939, issued August 20). of 1996). Such incidin analogues, such as incidin (SEQ ID NO: 1), are peptides rich in tryptophan and are characterized, in part, by having improved selectivity when compared to incidin (SEQ ID NO: 1) . Additional analogs of incidin have also been described (International Publication No. WO 97/08199, published March 6, 1997). These previously described incidin analogs are distinguishable from those of the present invention in that the previously described analogs contain either an amino acid residue, as represented by XI at X1-X2-X3-X4-X5-X6-P-X6- X6-X7-X7-X8 or lack of Hse, Met or Met-X9-Met in the carboxy terminus. As described herein, an incidin analog of the invention can be a truncated amino terminal incidin analog such as In 2-13 (SEQ ID NO: 2), In 3-13 (SEQ ID NO. NO: 3), In 4-13 (SEQ ID NO: 4) or In 5-13 (SEQ ID NO: 5, see Table 1), each of which exhibits antimicrobial activity (see, for example, example, Figures 1 and 2). In addition, an incidin analog may be an analog of truncated amino incidin wherein, in addition, one or more residues of Trp is replaced by a Phe residue (see Table 2). For example, substitution of all Trp residues in In 2-13 (SEQ ID NO: 2) with Phe results in the incidin analogue In 2-13 / F (SEQ ID NO: 11; see Table 2), which presents antimicrobial activity (see Figure 5). An incidin analog of the invention is based on the general structure of incidin (SEQ ID NO: 1), except that various deletions, substitutions or additions of amino acids are made with respect to indolicidin. 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, which include amino acid analogs. Thus, the reference herein to an amino acid includes, for example, (L) -naturally occurring proteinaceous amino acids, as well as (D) -amino acids, chemically modified amino acids such as amino acid analogues, naturally occurring non-proteogenic amino acids, such such as norleucine, 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 on an indolicide analog having the structure shown as X1-X2-X3-X4-X5-X6-P-X6-X6-P-X6-X7-X7-X8-X8 can be independently selected. 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 influence the selection of the amino acid residue at any other position on the analogue. Thus, the selection of a Trp residue for X6 shown at the position of X1-X2-X3-X4-X5-X6-P-X6-X6-P-X6-X7-X7-X8 does not influence if, for example, the amino acid present in X6 shown in position 8 is a Trp residue or a Phe residue. Reference is made herein to an amino acid position in an indolicidin analogue to the position of the amino acid in the naturally occurring indolicidin (SEQ ID NO: 1). As such, the positions are referred to as positions 1 to 13, starting with the residue lie in SEQ ID NO: 1 (position 1) and terminated with the carboxy terminal arginine (position 13). As a result, although Leu is the first amino acid in Indole 2-13 (SEQ ID NO: 2), this residue of Leu is referred to as being located in position 2 because it is the location of the corresponding Leu in SEC. FROM IDENT. NO: 1. It is observed that SEC. FROM IDENT. NO: 2 is referred to as Indole 2-13 because it starts with an amino acid corresponding to the second amino acid (Leu) of indolicidin (Indole 1-13); SEC. FROM IDENT. NO: 1; see Table 1). An indolicidin analogue containing at least one substitution of a Phe residue for a Trp residue is generally referred to herein as an "Indole (Trp / Phe)" or an "Indole / F" analogue. Since the exemplified indolicidin analogs contain a global substitution of Phe for each Trp in the peptide, no specific designation for the position of the substitution is made to name the exemplified analogs. Thus, the designation "Indol 2-13 / F" indicates an indolicidin analog that lacks an amino terminal amino acid when compared to the naturally occurring indolicidin (SEQ ID NO: 1) and contains a Phe substitution for each Trp in another form present in indolicidin. However, the present invention encompasses indolicidin analogs containing as few substitutions as a Phe-for-Trp, for example, in the 4 or 6 or 8 or 9 or 11 position. Where a few of all the Trp residues in an analogue are replaced with a Phe, the nomenclature for the analogue includes a number indicating the position of the substitution. For example, "Indol 2-13 / 6F" indicates an indolicidin analog that lacks an amino terminal amino acid when compared to naturally occurring indolicidin (SEQ ID NO: 1) and contains a substitution of Phe for Trp only in position 6; similarly, "Indol 2-13 / 6, 11F" contains Phe by substitutions of Trp at positions 6 and 11. As described herein, the amino terminal truncation of indolicidin that occurs naturally results in the production of analogs of indolicidin having antimicrobial activity (see Example II). In view of this description, the skilled artisan will recognize that various amino acid substitutions can be made in certain positions without destroying the antimicrobial activity of the. analogue derivative. Thus, considering that indole 1-13 (SEQ ID NO: 1) contains a residue lie in position 1, the skilled artisan, knowing that this is a lie can be suppressed without destroying the antimicrobial activity, would recognize that the lie also it can be substituted conservatively with an amino acid such as Leu, Val, Ala or Gly without destroying the antimicrobial activity of the indolicidin analog. Similarly, conservative amino acid substitutions are permissible for the Leu at position 2. In addition, the substitution of an Arg residue for Lys at position 5 is allowed, as indicated by the description that the substitution of a Lys by an Arg-12 or by Arg-13 results in indolicidin analogs having antimicrobial activity (see U.S. Patent No. 5,534,939, supra, 1996). In addition, since indolicidin analogs lacking a carboxy terminal Arg residue or containing a Lys substitution for one or both of the carboxy terminal Arg residues have antimicrobial activity (US Patent No. 5,547,939, supra, 1996), such Deletions or conservative amino acid substitutions are permissible in the indolicidin analogs of the present invention, provided that, when an Arg residue is deleted or when one or both of the Arg residues are replaced with a Lys, at least XI is absent (see X1-X2-X3-X4-X5-X6-P-X6-X6-P-X6-X7-X7-X8) or a carboxy terminus Met, Met-X9-Met or Hse is present (see Table 3). Such indolicidin analogs may additionally be analogues of Indole / F, as desired, and refer, for example, as "Indole 2-12 / F" or "Indole 2-12 / F / 12K" or "Indole 1-13". / F-Hse "or similar, within the restrictions of the nomenclature indicated above. As described herein, such truncated amino and carboxy terminal indolicidin analogues have antimicrobial activity. For example, Indole 3-12 (SEQ ID NO: 27), Indole 4-12 (SEQ ID NO: 28), Indole 5-12 (SEQ ID NO: 29) and Indole 6 -12 (SEQ ID NO: 30) have substantial antimicrobial activity against fungi such as Cryptococcus. An indolicidin analogue of the invention can be expressed from a nucleic acid molecule encoding in vitro or in vivo in a host cell or can be chemically synthesized. With respect to the expression of analogs, the nucleic acid sequences encoding the various indolicidin analogues 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 indolicidin analogs can be cloned into an appropriate vector, particularly an expression vector, and the encoded analog 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 poly-indolicidin analog polypeptide, which can be cloned into an expression vector and expressed in vivo in a bacterium, insect or mammalian host cells (see Example IB). An advantage of expressing a poly-indolicidin analog polypeptide 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 polypeptide can be split to produce active analogs of indolicidin. An 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). for reference; see, also, Example I. A.). An advantage of using chemical synthesis to prepare an indolicidin analogue 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 indolicidin analog can confer desirable characteristics on the peptide, for example, increased stability in vitro 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 indolicidin analogs were synthesized using an automated peptide synthesizer such as an Eppendorf Synostat (Madison Wl) or a Milligen 9050 (Milford MA, Example IA), although manual methods of peptide synthesis can also be used in Solution: Indolicidin analogues were synthesized on an Fmoc resin (5- (4-Fmoc-aminomethyl-3,5-dimethoxyphenoxy) valeric acid-polyethylene glycol-polystyrene (Fmoc-PAL-PEG-PS) (Fmoc is 9-fluorenylmethyloxycarbonyl) Milligen, see Example I. A) However, the skilled artisan will recognize that other resins, amino acid derivatives and methods for modifying reactive amino acid groups or the amino terminus, for example, by acetylation, or the term carboxy can be used to obtain a desired indolicidin analog (see, for example, Protein Engineering: A practical approach (IRL Press 1992); Bodanszky, Principies of Peptide Synthesis (Springer-Verlag 1984), each of which is incorporated herein by reference). The synthesized indolicidin analogs were purified by reverse phase HPLC and characterized by fast atom bombardment mass spectroscopy, acid-urea gel electrophoresis and analytical HPLC (see Example IA) or can be purified and characterized using other routine methods of purification and analysis of peptides. Selective modification of a reactive group 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 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 analog against a microorganism when compared to its activity against the environment to which it is administered, particularly its activity in against normal cells in a treated individual. For example, an indolicidin analog which is characterized by having an antimicrobial activity that is equivalent to indolicidin, but which has decreased emollitic activity when compared to indolicidin, s-e considers that it has higher antimicrobial selectivity than indolicidin. Indolicidin analogues that have higher antimicrobial selectivity than 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, the indolicidin analogs in which all Trp residues were substituted with Phe but not the analogs having Ala by Pro substitutions, had higher antimicrobial selectivity than indolicidin (Subbalakshmi et al., FEBS Lett, 395: 48-52 (1996), which is incorporated herein by reference). Indolicidin analogues 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, are truncated amino terminal analogs of indolicidin. The antimicrobial selectivity of an indolicidin analog can be determined using the methods described herein (see Example II) or using routine methods such as those described in the references cited above (see, also, Selsted, ME Investigational approaches for studying the structures and Biological functions of myeloid antimicrobial peptides In: Genetic Engineering, Principies and Methods Vol 15 (Setlow, ed., Plenum Press, New York 1993), see pages 131-147, which is incorporated herein by reference). The indolicidin analogs of the invention have broad spectrum antimicrobial activity (see Figures 1 to 5, Example II). As used herein, the term "broad spectrum" when used with reference to the antimicrobial activity of an indolicidin analog, refers to the ability of the analog to reduce or inhibit the subsistence or proliferative capacity of various prokaryotic and eukaryotic microorganisms. . For example, the indolicidin analogs of the invention may exhibit antimicrobial activity against protozoa such as Giardia lambia, Chlamydia sp. and Acan thamoeba sp.; viruses, particularly enveloped viruses, such as HIV-I; fungal yeasts 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 indolicidin analog to reduce or inhibit the subsistence of a microorganism by irreversibly killing or damaging it, or through microbiostatic inhibition, which is referred to to the ability of an indolicidin analog to reduce or inhibit the growth or proliferative capacity of an objective microorganism without necessarily killing it. As described herein, various truncated amino terminal indolicidin analogues (Tables 1 and 2); truncated amino terminal and carboxy terminal indolicidin analogues (Table 3); and indolicidin analogs having a carboxy terminal Hse residue, Indole (1-13) -Hse (see Figure 4) were prepared and examined for antimicrobial activity. Indole 2-13 (SEQ ID NO: 2), Indole 5-13 (SEQ ID NO: 5) and Indole (1-13) -Hse, for example, demonstrated similar selectivity, specificity and antimicrobial potency as the Indole 1-13, as did various analogs of Indole / F, which include analogs of Indole / F-Hse, and analogs truncated from the amino terminus and the carboxy terminus (see Example ID - The determination that the Indol-Hse analogues are Indole / F-H a significant antimicrobial activity is maintained because a Hse group remains in the carboxy terminus of a peptide after cleavage with cyanogen bromide of the peptide in a Met residue. Since the indolicidin analogs of the invention do not contain internal Met residues, the analogues do not unfold upon exposure to cyanogen bromide. The Hse in the carboxy terminus of a peptide typically exists as a state of equilibrium between the lactone and carboxylated forms and, as described herein, both forms of the Hse analogs exhibit substantial antimicrobial activity (see Figure 3). If desired, an Indol-Hse analog may be amidated at the carboxy terminus. The described ability of an Indol-Hse analog to maintain antimicrobial activity provides a means to produce substantial quantities of the analog by expressing a poly- (Indol-Met) N polypeptide where "N" is the number of times the Indol-Met sequence repeat, and unfold the polypeptide with cyanogen bromide to produce N-Indol-Hse analog peptides (see Example IB, see, also, Figure 6). In particular, such a method can be performed in vivo in a host cell because the polypeptide poly- (Indol-Met) N does not exhibit substantial antimicrobial activity. Such a method is performed, for example, by synthesizing a nucleic acid sequence encoding the indole portion of the analog and a carboxy terminal Met; ligating 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; transforming 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 analogues. Thus, the invention provides Indol-Hse and Indol / F-Hse analogs, including Indol analogs (1-13) -Hse and Indole 1-13 / F-Hse, which have antimicrobial activity (see Figure 7). 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 bound 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 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 analogs. Thus, the invention provides fusion polypeptides comprising an indolicidin analog linked to a peptide of interest. The invention also provides nucleic acid molecules that encode the indolicidin analogs of the invention. Thus, the invention provides nucleic acid molecules encoding truncated amino terminal indolicidin analogs, including truncated amino terminal Indole / F analogs, as well as nucleic acid molecules encoding precursors of indolicidin analogues containing at least one methionine residue. carboxy terminal, such precursors result in the formation of Indol Hse or Indol / F-Hse analogs of the invention. A nucleic acid molecule of the invention can also encode a poly-indolicidin analog polypeptide or a fusion polypeptide comprising an indolicidin analog, as described herein. 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 indolicidin analog 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 indolicidin analog encoding the sequence. The nucleic acid molecules of the invention are useful, for example, to produce indolicidin analogues in vitro using an appropriate transcription / translation system or in vivo using an appropriate expression system. 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 proper structure. An indolicidin analog having antimicrobial activity can be applied to an environment capable of sustaining the subsistence or growth of a microorganism or an environment at risk of supporting such subsistence or growth, thus providing a means to reduce or inhibit microbial growth or subsistence. . Accordingly, the invention relates to methods for using an indolicidin analog to reduce - or inhibit microbial growth by contacting the environment capable of sustaining microbial growth or subsistence with the indolicidin analogue. As used herein, reference to "an environment capable of sustaining the subsistence or growth of a microorganism" suggests 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 subsistence or growth of microorganisms in so diverse, for example, as viruses, bacteria and fungi, and also in view of the described effectiveness of the claimed indolicidin analogues 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 indolicidin analogue such that the analogue can contact a microorganism with the environment, thereby reducing or inhibiting the body's ability to grow or survive. The indolicidin analogs can be used in a variety of methods to reduce or inhibit the subsistence or growth of microorganisms, which includes the microbicidal inhibition of the subsistence of a microorganism as well as the microbiostatic inhibition of growth. As such, an indolicidin analog may be used, for example, as a therapeutic agent, a food preservative, a disinfectant or a medicament. An 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 analogue. Thus, the invention provides methods for treating an individual suffering from a pathology caused, at least in part, by microbial infection, by administering an indolicidin analogue to the individual under conditions that allow the analog to contact the infectious microorganisms, reducing or inhibiting with that the subsistence or growth of the microorganisms and alleviating the severity of the infection. For use as a therapeutic agent, the indolicidin analog may be formulated with a pharmaceutically acceptable carrier to produce a pharmaceutical composition, which may be administered to the individual, which may 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 indolicidin analog. Such physiologically acceptable compounds include, for example, carbohydrates such as glucose, sucrose or dextrans; antioxidants such as ascorbic acid, glutathione; chelating agents such as EDTA, which disrupt the microbial membranes; divalent metal ions such as calcium magnesium; low molecular weight proteins; or other stabilizers and excipients. One skilled in the art will know that the section 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 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 indolicidin analog may be incorporated into a liposome, a non-liposome lipid complex, or another polymer matrix, which may additionally be incorporated therein, for example a second drug useful for 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 indolicidin analog that is administered. Food and food products can also be treated with 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 subsistence of such microorganisms can be reduced or inhibited by contacting the product with an indolicidin analogue. Food crops such as fruits, vegetables and grains can be treated with an 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 indolicidin analogue of the invention into the embryonic cell line of such organisms. Methods for producing transgenic plants and animals are well known and routine in the art. An indolicidin analog can also be used as a disinfectant to reduce or inhibit the subsistence or growth of microorganisms on an object, in a solution or in a gas. An indolicidin analog can be used to treat essentially any object or solution that can support microbial growth, wherein the subsistence 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 indolicidin analogue. In such methods, the indolicidin analog can be applied locally to the object or it can be added to the solution or it can be in an aerosol form in a gas. To exhibit antimicrobial activity in an environment, an effective amount of an indolicidin analog is administered to the environment. As used herein, the term "effective amount" refers to the amount of an indolicidin analog that reduces or inhibits the subsistence 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 indolicidin analog can be administered to a subject such as a human systemically at a dose ranging from 1 to 100 mg / kg body weight, for example, at a dose of about 10 to 80 mg / kg, particularly about 10 mg / kg. at 50 mg / kg, and the indolicidin analog can be incorporated into liposomes, if desired. In addition, an 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 about 0.5. at 5 mg / ml. Although indolicidin analogs are generally effective in such 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 indolicidin analogue, in addition to having antimicrobial activity, may have hemolytic activity as a side effect. The skilled artisan will recognize that the level of such secondary defects can be considered when prescribing a treatment and should be retained during the treatment period, and will adjust in 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 indolicidin analog administered. In addition, an effective amount depends on the manner in which the indolicidin analog is administered. For example, the incorporation of indolicidin into liposomes allows 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, nineteen ninety five) .

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 chemically synthesizing indolicidin analogues and for expressing indolicidin analogues in a host cell. A. Chemical Synthesis of Indol and Indol / F Analogs: The indolicidin peptide chain and various indolicidin analogs were assembled on a Fmoc-PAL-PEG-PS resin at 0.2 mmoles on a Millipore 9050 Plus continuous-flow peptide synthesizer. The resin was swelled for 30 minutes in DMF before starting the synthesis. Completely chemical Fmoc was 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 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 cleaved 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 removed by filtration, and the resin was cooled to 0 ° C. The protection groups were removed 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. A C-18 Vydac inverse phase column (25 x 100 mm) was used for purification and a Vydac C-18 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. Appropriate HPLC fractions were combined, concentrated by centrifugation, and lyophilized. The purity of the final synthetic product was established by HPLC analysis and electrophoresis in acid-urea polyacrylamide gel. The molecular mass was determined by MALDI-TOF mass spectrometry, and the composition and amount of the peptide was determined by amino acid analysis. The indolicidin analogs synthesized and characterized in the above manner are shown in Tables 1 to 3.

TABLE 1 ANALOGS TRUNCATED FROM INDOLICIDINE NAME AMINO ACID SEQUENCE SEC ID NO: Indole 1-13 * H2N-ILPWKWPWWPWRR-CONH2 1 Indole 2-13 H2N-LPWKWPWWPWRR-CONH2 2 Indole 3-13 H2N-PWKWPWW ~ PWRR-CONH2 3 Indole 4-13 H2N-WKWPWWPWRR- CONH2 4 Indole 5-13 H2N-KWPWWPW ~ RR-CONH2 5 l_) Indole 6-13 H2N-WPWWPWRR-CONH2 6 Indole 7-13 H2N-PWWPW ~ RR-CONH2 7 Indole 8-13 H2N-WWPWRR-CONH2 8 Indole 9 -13 H2N-WPWRR-CONH2 9 indolicidin (which occurs naturally) Table 2 ANALOGUES OF INDOLICIDIN INDOL (TRP / PHE) ("INDOL F") NAME ACID AMINO SEQUENCE SEC ID NO: Indole 1-13 'H2N-ILPWKWPWWPWRR-CONH2 1 Indole 1-13 / F H2N-ILPFKFPFFPFRR-CONH2 10 Indole 2-13 / F H2N-LPFKFPFFPFRR-CONH2 11 Indole 3-13 / F H2N-PFKFPFFPFRR-CONH2 12 Indole 4- 13 / F H2N-FK -FPFFPFRR-CONH2 13 or Indole 5-13 / F H2N-KFPFFPFRR-CONH2 14 Indole 6-13 / F H2N-FPFFPFRR-CONH2 15 Indole 7-13 / F H2N-PFFPFRR-CONH2 16 Indole 8 -13 / F H2N-FFPFRR-CONH2 17 Indole 9-13 / F H2N-FPFR ~ R-CONH2 18 indolicidin (which occurs naturally) Table 3 ANALOGUES OF INDOL1CIDIN AMINO AND CARBOXI TERMINAL TRUNCADOS NAME SEQUENCE OF AMINO ACIDO SEC ID NO Indole 1-13 * H2N-ILPWKWPWWPWRR-CONH2 1 Indole 1-12 H2N-ILPWKWPWWPWR-CONH2 25 Indole 2-12 H2N-LPWKWPWWP- -R-CONH2 26 Indole 3-12 H2N-PWKWPWWPWR- CONH2 27 Indole 4-12 H2N- -KWPWWP- -R-CONH2 28 -1 Indole 5-12 H2N-KWPWWPWR-CONH2 29 Indole 6-12 H2N-WPWWPWR-CONH2 30 Indole 1-11 H2N-ILPWKWPWWPW-CONH2 31 Indole 2 -11 H2N-LPWLWPWWPW-CONH2 32 Indol 3-11 H2N-PWKWPWWPW-CONH2 33 Indol 1-10 H2N-ILPWKWPWWP-CONH2 34 indolicidin (which occurs naturally) B. Expression of Indol-Hse analogues: Indol-Hse was expressed from a recombinant construct encoding three replicates of the mature peptide, each separated by a hexapeptide spacer sequence; poly- (Indole (1-13) -Met-Ala-Arg-Ile-Ala-Met) 3 (see Figure 6). Recombinant indolicidin was produced as a fusion polypeptide with a maltose binding protein (MBP) and recovered by cleavage with cyanogen bromide. 'The multicopy indolicidin encoding the DNA sequence was assembled from six synthetic oligonucleotides (see Figure 6). The oligonucleotides were phosphorylated and assembled on annealing and ligation of each fragment (Ikehara et al., Proc. Nati, Acad. Sci., USA 81: 2956-5960 (1984), which is incorporated herein by reference). The 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 Englans BioLabs, Beverly MA). The transformation of INVctF 'E. coli was carried out with the TA cloning team according to the instructions of the manufacturers (Invitrogen, La Jolla CA). The DNA sequence shown in Figure 6 was formed by dideoxy sequencing. INV F 'cells containing the poly-indolicidin analog pMAL-c2 fusion polypeptide was grown overnight in 15 ml of LB medium containing 100 μg / ml ampicillin at 37 ° C with constant agitation. Ten ml of the culture overnight was transferred into one liter of freshly prepared 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-cooled lysis buffer (0.01 M Tris-HCl, pH 8.0; 1 mM each of PMSF, DTT and EDTA; 2 mg / ml lysozyme), then slowly mixed for 30 minutes on ice. 1.6 ml of 10% sodium deoxycholate was added and 63 μl of a 2 mg / ml solution of DNAse I was 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 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) was approximately 50% of the theoretical yield. Alternatively, a MBP-indolicidin fusion polypeptide as in Figure 6 can be prepared, except that it has the sequence Met-Ala-Arg-Ile-Ala-Met (SEQ ID NO: 21) instead of the Met residue shown between the cleavage site of enterokinase and the first cleavage site CNBr (see Figure 6). Such a MBP-indolicidin fusion polypeptide can be first cleaved with enterokinase, to release the MBP portion of the fusion polypeptide. The poly-indolicidin portion can then be treated with CNBr, to release the Indol-Hse analogs, which can be purified as above. The results discussed above indicate that the poly-indolicidin analog polypeptides can be produced in vivo in a bacterial expression system, without killing the host microorganism and, therefore, provides a means to produce substantial amounts of Indol-Hse analogues.

EXAMPLE II ANTIMICROBIAL ACTIVITY OF INDOLICIDIN ANALOGS This example demonstrates that various indolicidin analogs of the invention exhibit broad spectrum antimicrobial activity. The antimicrobial activity of the indolicidin analogues was determined using microbicidal or microbial inhibition tests. The indolicidin analogs were initially 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 a selected target microorganism and peptide samples (5-10 μl) were placed into small wells formed in the solid medium. After an initial incubation interval of 1 to 4 hours, the layer containing p'ozos 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). The microbicidal activity was measured by first incubating the target organism with an indolicidin analogue in an aqueous suspension, then sowing the suspension to quantify the surviving microorganisms. The 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. Peptide stock solutions were usually made in 0.01% acetic acid, diluted in the incubation buffer to a final concentration of 1 μg / ml to 100 μg / ml, and incubation started by the addition of an appropriate volume of the fungal or bacterial stock suspension to the preheated mixture (37 ° C) of regulator-peptide. At time intervals, 50 μl or 100 μl samples were removed and serially diluted, then seeded onto plates containing nutrient agar. The destructive activity was quantified determined the reduction in CFU relative to the appropriate incubation control. Indolicidin analogs lacking up to 5 amino terminal amino acids are as effective as synthetic Indole 1-13 (SEQ ID NO: 1) to inhibit the growth of E. coli and S. aureus (Figures 1A-1B) . Additional amino terminal deletion reduced the antimicrobial activity of the analogues, although activity was maintained at the highest doses examined. In comparison, substantial activity was maintained against fungi, including C. neoformans and C. albicans by indolicidin analogs lacking as many as seven amino terminal amino acids (Figures 1C and ID). Similar results were obtained when truncated amino terminal indolicidin analogues were examined in a suspension test to determine the destruction activity of the peptides. The activity of destruction of the peptides against E. coli and S. a ureus was similar to the results obtained for the agar diffusion test (compare Figures 2A and 2B) with Figures IA and IB). In addition, the indolicidin analogues are particularly effective in killing C. neoformans (Figure 2C), although there was less destructive activity by the shorter peptides against C. albicans (Figure 2D). These results indicate that truncated amino terminal indolicidin analogues have antimicrobial activity which is equivalent to that of naturally occurring indolicidin. The antimicrobial activity of Indol-Hse analogs was also examined. The Indol-Hse analogues were produced by cleavage with cyanogen bromide of Indole (1-13) -Met-Gly-Ser-Glu (SEQ ID NO: 35), followed by the isolation of the carboxylic acid form from the lactone form, which otherwise exists in equilibrium. As shown in Figures 3A and 3B, both forms of the Indol-Hse analog have antimicrobial activity equivalent to Indole 1-13 (SEQ ID NO: 1) against S. aureus and E. coli, as well as the ability to destroy E. coli (Figure 3C). Indole (1-13) -He was prepared from a recombinantly expressed poly-indolicidin polypeptide also had antimicrobial activity equivalent to that of synthesized Indole 1-3 (SEQ ID NO: 1) and Indole (1-13) -Hse synthesized (Figure 7). Truncated amino terminal Indole-Indol analogs were also examined for antimicrobial activity. The Indol-Hse analogs lacking as many as 5 amino acid terminal amino acids had antimicrobial activity against S. aureus and E. coli (Figure 4), although the activity began to decrease in the analogue of Indole (6-13) -H compared to the analogue of Indole (5-13) -Hse (see Figures 4C and 4F). These results demonstrate that the Indol-Hse analogs, which include truncated amino terminal analogs, exhibit antimicrobial activity equivalent to that of Indole 1-13 (SEQ ID NO: 1) that occurs naturally. The antimicrobial activity of Indol / F-Hse analogues against E. coli and S. aureus was also examined. As shown in Figure 5, the truncated amino terminal Indole / F-Hse analogs lacking as many as 4 amino terminal amino acid residues had antimicrobial activity equivalent to that of Indole (1-13) / F. These results indicate that the Trp in the indolicidin that occurs naturally can be replaced with Phe without destroying the antimicrobial activity of the peptide. 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 (46)

1. CLAIMS 1. An indolicidin analog characterized by having the amino acid sequence: Xaal-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Pro-Xaa6-Xaa6-Pro-Xaa6-Xaa7-Xaa7-Xaa8, wherein: 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 or absent; Xaa5 is Arg, Lys or absent; Xaa6 is Trp or Phe; Xaa7 is Arg or Lys; and Xaa8 is homoserine (Hse), Met, or Met-Xaa9-Met; wherein Xaa9 is at least one amino acid; with the proviso 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 indolicidin analogue according to claim 1, 45 or 46, characterized in that it also comprises a C-terminal amide.
3. 3. The indolicidin analogue according to claim 1, characterized in that it is selected from the group consisting of: H2N-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-CONH2 (SEQ ID NO: 2); H2N-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-CONH2 (SEQ ID NO: 4); and • H2N-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-CONH2 (SEQ ID NO: 5).
4. 4. The indolicidin analogue according to claim 1, characterized in that it is selected from the group consisting of: H2N-Leu-Pro-Phe-Lys-Phe-Pro-Phe-Phe-Pro-Phe-Arg-Arg-CONH2 (SEQ ID NO: 11); H2N-Pro-Phe-Lys-Phe-Pro-Phe-Phe-Pro-Phe-Arg-Arg-CONH2 (SEQ ID NO: 12); H2N-Phe-Lys-Phe-Pro-Phe-Phe-Pro-Phe-Arg-Arg-CONH2 SEC. of IDENT. NO: 13); H2N-Lys-Phe-Pro-Phe-Phe-Pro-Phe-Arg-Arg-CONH2 (SEQ ID. NO: 14)
5. 5. The indolicidin analogue according to claim 1, 45 or 46, characterized in that X8 is Hse.
6. 6. The indolicidin analog according to claim 1, characterized in that it comprises the sequence: H2N-Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-Hse. (SEQ ID NO: 24).
7. 7. The indolicidin analogue according to claim 1, characterized in that it is selected from the group consisting of: H2N-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-CONH2 (SEQ. IDENT. NO: 27); H2N-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-CONH2 (SEQ ID NO: 28); H2N-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-CONH2 (SEQ ID NO: 29); and H2N-Trp-Pro-Trp-Trp-Pro-Trp-Arg-CONH2 (SEQ ID NO: 30).
8. 8. The fusion polypeptide, characterized in that it comprises the indolicidin analogue according to claim 1, 45 or 46 linked to a peptide.
9. 9. The fusion polypeptide according to claim 8, characterized in that the peptide is capable of specifically binding to 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, 45 or 46, 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, 45 or 46, characterized in that it has antimicrobial activity against a protozoan.
15. 15. The indolicidin analogue according to claim 14, characterized in that the protszoan is selected from the group consisting of Giardia sp. and Acanthamoeba sp.
16. 16. The indolicidin analogue according to claim 1, 45 or 46, 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 a human immunodeficiency virus-1.
18. 18. The pharmaceutical composition, characterized in that it comprises the indolicidin analogue according to claim 1, 45 or 46 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 lipid complex without liposome.
21. 21. The method for reducing or inhibiting the growth or subsistence of a microorganism capable of sustaining the growth or subsistence of the microorganism, characterized in that it comprises administering an effective amount of the indolicidin analogue according to claim 1, 45 or 46 to the environment, reducing or inhibiting the growth or subsistence 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 nesformans.
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 human immunodeficiency virus-1.
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 of compliance with the claim 21, characterized in that the environment is a solution.
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 of compliance with the claim 21, characterized in that the environment is a mammal.
32. 32. The method according to claim 31, characterized in that the administration is topical.
33. 33. The method according to claim 31, characterized in that the administration is by injection.
34. 34. The method according to claim 31, characterized in that the administration is oral.
35. 35. A nucleic acid molecule encoding an indolicidin analog having the amino acid sequence: Xaal-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Pro-Xaa6-Xaa6-Pro-Xaa6-Xaa7-Xaa7-Xaa8, wherein : 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 or absent; Xaa5 is Arg, Lys or absent; Xaa6 is Trp or Phe; 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 if Xaal is present, Xaa8 is Met or Met-Xaa9-Met, or both Xaa7 events are absent; 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 indolicidin analog is selected from the group consisting of: H2N-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp- Arg-Arg-CONH2 (SEQ ID NO: 2); H2N-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-CONH2 (SEQ ID NO: 3); H2N-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-CONH2 (SEQ ID NO: 4); and H2N-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-CONH2 (SEQ ID NO: 5)
37. 37. The nucleic acid molecule according to claim 35, characterized in that the analogue of indolicidin is selected from the group consisting of: H2N-Leu-Pro-Phe-Lys-Phe-Pro-Phe-Phe-Pro-Phe-Arg-Arg-CONH2 (SEQ ID NO: 11); H2N-Pro-Phe-Lys-Phe-Pro-Phe-Phe-Pro-Phe-Arg-Arg-CONH2 (SEQ ID NO: 12); H2N-Phe-Lys-Phe-Pro-Phe-Phe-Pro-Phe-Arg-Arg-CONH2 (SEQ ID NO: 13); - H2N-Lys-Phe-Pro-Phe-Phe-Pro-Phe-Arg-Arg-CONH2 (SEQ. of IDENT. NO: 14)
38. 38. The nucleic acid molecule according to claim 35, characterized in that the indolicidin analog is selected from the group consisting of: N2N-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-CONH2 (SEQ ID NO: 27); H2N-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-CONH2 (SEQ ID NO: 28); H2N-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-CONH2 SEC. of IDENT. NO: 29); and H2N-Trp-Pro-Trp-Trp-Pro-Trp-Arg-CONH2 (SEQ ID NO: 30).
39. 39. The nucleic acid molecule according to claim 35, characterized in that X8 in the indolicidin analog is selected from the group consisting of Met and Met-Xaa9-Met.
40. 40. The nucleic acid molecule according to claim 39, characterized in that the indolicidin analog is selected from the group consisting of: 'H2N-Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro -Trp- Arg-Arg-Met (SEQ ID NO: 36); and H2N-Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-Met-Xaa9-Met (SEQ ID NO: 37).
41. 41. The nucleic acid molecule according to claim 35, characterized in that it further comprises a nucleotide sequence encoding a peptide of interest.
42. 42. The nucleic acid molecule according to claim 41, characterized in that the peptide of interest is capable of specifically binding by a molecule.
43. 43. The nucleic acid molecule according to claim 42, characterized in that the molecule is an antibody that binds specifically to the peptide.
44. 44. The nucleic acid molecule according to claim 42, characterized in that the peptide and the molecule, respectively, are selected from the group consisting of: glutathione-S-transferase and glutathione; protein that binds maltose and maltose; and His-6 and a metallic ion.
45. 45. An indolicidin analog characterized by having the amino acid sequence: Xaal-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Pro-Xaa6-Xaa6-Pro-Xaa6-Xaa7-Xaa7-Xaa8, wherein: Xaal is Ala, Gly or absent; Xaa2 is Ala or Gly; Xaa3 is Pro; Xaa4 is Trp or Phe; Xaa5 is Arg or Lys; Xaa6 is Trp or Phe; Xaa7 is Arg, Lys or absent; and Xaa8 is homoserine (Hse), Met, Met-Xaa9-Met or absent; wherein Xaa9 is at least one amino acid; with the proviso that if Xaal is present, Xaa8 is Hse, Met or Met-Xaa9-Met.
46. 46. An indolicidin analog characterized in having the amino acid sequence: Xaa4-Xaa5-Xaa6-Pro-Xaa6-Xaa6-Pro-Xaa6-Xaa7-Xaa7-Xaa8, wherein: Xaa4 is Trp, Phe or absent; 'Xaa5 is Arg, Lys or absent; Xaa6 is Trp or Phe; Xaa7 is Arg, Lys or absent; and Xaa8 is homoserine (Hse), Met, Met-Xaa9-Met or absent; wherein Xaa9 is at least one amino acid; with the proviso that if Xaal is present, Xaa8 is Hse, Met or Met-Xaa9-Met; and with the additional condition that if Xaa5 is absent, Xaa4 is absent.