WO2007044998A1 - Retrocyclins: antiviral and antimicrobial peptides - Google Patents

Abstract

Retrocyclin peptides are small antimicrobial agents with potent activity against bacteria and viruses. The peptides are nonhemolytic, and exhibit minimal in vitro cytotoxicity. A pharmaceutical composition comprising retrocyclin as an active agent is administered therapeutically to a patient suffering from an infection by a vancomycin resistant enterococcus.

Description

RETROCYCLINS: ANTIVIRAL AND ANTIMICROBIAL PEPTIDES

[01] This invention was made with Government support under contract R01 AI056921 :01 A2 awarded by the NIH. The Government has certain rights in this invention. Background

[02] Retrocyclins are circular peptides, whose structure was initially based on nucleotide sequences found in the human genome and/or in mRNA expressed by human bone marrow. Similar peptides have been found in certain Old World monkeys. Retrocyclins have been shown to be active in inhibiting viral pathogens including human immunodeficiency virus (HIV-1), herpes simplex virus-1 and herpes simplex virus-2 (HSV-2).

[03] Enterococci are enteric gram-positive cocci in the genus, Enterococcus. E. faecalis has been the predominant pathogenic enterococcal species, followed by E. faecium. Other Enterococcus species include E. gallinarum, E. casseliflavus, E. durans, E. avium, and E. raffinosis. Enterococci are primarily nosocomial pathogens, having become the second most common organisms recovered from nosocomial urinary tract and wound infections and the third most common cause of nosocomial bacteremia in the United States. These organisms have an intrinsic resistance to several commonly used antibiotics and can readily acquire resistance to all currently available antibiotics, either by mutation or through the transfer of plasmids and transposons. Because most enterococci are tolerant to the bactericidal activity of β-lactam and glycopeptide antibiotics, bactericidal synergy between one of these antibiotics and an aminoglycoside is needed to treat most serious enterococcal infections such as endocarditis and meningitis. However, synergistic bactericidal effect between aminoglycosides and β-lactam and glycopeptide antibiotics is lost if there is high-level resistance to either class of drug. Resistance to high concentrations of aminoglycoside antibiotics is usually mediated by aminoglycoside- modifying enzymes, and it is widespread among enterococci. Also, many isolates of E. faecium are highly resistant to penicillins, because their penicillin binding proteins (PBP) have low affinity for penicillins. Until recently, vancomycin was virtually the only drug that could be consistently relied on for the treatment of infections caused by multidrug-resistant enterococci. [04] In 1988 the first vancomycin-resistant E. faecalis and E. faecium (VRE) were isolated. Subsequently, VRE have spread with unanticipated rapidity and are now encountered by hospitals in most states. There are five recognized phenotypes of vancomycin resistance, VanA, VanB, VanC, VanD, and VanE. Two of these (VanA and VanB) are mediated by newly acquired gene clusters not previously found in enterococci.

[05] The vanA gene and other genes involved in the regulation and expression of vancomycin resistance (vanR, vanS, vanH, vanX, and vanZ) are located on a 10,581-bp transposon (Tn 1546) of E. faecium, which often resides on a plasmid. Expression of these genes results in the synthesis of abnormal peptidoglycan precursors terminating in D-AIa-D- lactate instead of D-AIa-D-AIa. Vancomycin binds to D-Ala-D-Lac with markedly lower affinity than it does to the normal dipeptide product. A variety of core protein functions favor synthesis of pentadepsipeptide terminating in D-Ala-D-Lac. VanA protein is a ligase of altered substrate specificity which produces D-Ala-D-Lac in preference to D-AIa-D-AIa. VanH protein is a D- hydroxy acid dehydrogenase which creates a pool of D-lactate for use in the above reaction. VanX protein is a D,D-dipeptidase lacking activity against D-Ala-D-Lac. This enzyme reduces pools of D-AIa-D-AIa produced by the native enterococcal ligase, thereby minimizing the competing synthesis of normal pentapeptide. VanR and VanS proteins constitute a two- component regulatory system that regulates the transcription of the vanHAX gene cluster. [06] VanB glycopeptide resistance in enterococci is mediated by an abnormal ligase (VanB) that is structurally related to VanA ligase. VanB protein also favors the production of the pentadepsipeptide terminating in D-Ala-D-Lac. Genes analogous to their class A resistance counterparts are designated vanH_B, vanXβ, vanY_B, vanR_B, and vanS_B. Levels of D,D-dipeptidase activity (VanX_B) correlate with levels of vancomycin resistance. There is a high degree of sequence identity between VanHAX and VanH_BBX_B but considerably less homology between the RS and Y proteins of VanA and VanB VRE. There is no gene counterpart of vanZ in these organisms. >___ ^?

[07] Early studies dealing with the emergence of VRE in the United States revealed that most patients with VRE were in ICUs. However, VRE are now being seen with increasing frequency among patients with chronic renal failure or cancer, organ transplant recipients, and patients who experience prolonged hospitalization. Risk factors specifically associated with VRE infections such as bacteremia include malignancy, increased Acute Physiology and Chronic Health Evaluation (APACHE) Il score, neutropenia, prolonged hospital stay, antibiotic therapy and preceding therapy with agents active against anaerobes, mean number of days on antibiotic therapy, renal insufficiency, and hospitalization on a hematologic malignancy/bone marrow transplantation service.

[08] Oral vancomycin use may also be a risk factor for VRE colonization, and this has led to recommendations discouraging the use of this agent for the primary treatment of antibiotic- associated diarrhea. Vancomycin most probably predisposes patients to colonization and infection with VRE by inhibiting the growth of the normal gram-positive bowel flora and by providing a selective advantage for VRE that may be present in small numbers in the individual's bowel.

[09] The emergence of vancomycin resistance in enterococci in addition to the increasing incidence of high-level enterococcal resistance to penicillin and aminoglycosides presents a serious challenge for physicians treating patients with infections due to these microorganisms. Treatment options are often limited to combinations of antimicrobials or experimental compounds with unproven efficacy. Once VRE are established in the hospital environment, their frequent resistance to multiple antibiotics makes it difficult to avoid further selective pressure in their favor. In addition to the existing problem with VRE, the potential emergence of vancomycin resistance in other gram-positive microorganisms is a concern.

[10] Treatment of infections due to VRE is extremely problematic because these organisms are resistantto multiple antibiotics. Treatment options for multiple-drug-resistant enterococci are extremely limited, and agents to which they may appear susceptible are at best bacteristatic, of unproven efficacy, or associated with toxicity. Thus, optimal therapy for these patients remains unknown.

[11] Bacillus anthracis is a large, Gram-positive, spore-forming, rod-shaped bacterium that infects mainly herbivores. The blood of an animal dying of anthrax can contain >10⁹ vegetative bacteria per ml. Post mortem, these bacteria will form highly infectious endospores that can remain viable for years and infect additional animals to perpetuate the infectious cycle. [12] Under normal circumstances, human anthrax is rare - occurring only after close contact with infected farm animals or their contaminated wool or hides. Cutaneous anthrax ("wool sorters disease") is initiated when spores infect abraded skin. It is usually contained locally, and resolves without major sequelae. Gastrointestinal and oropharyngeal anthrax follow ingestion of spore-contaminated meat, and are rare, but often fatal, lnhalational anthrax, the most dangerous form, results when aerosolized spores reach pulmonary air spaces, germinate, and are carried to regional lymph nodes and the blood.

[13] Bacillus anthracis owes its lethality to its antiphagocytic poly-D-glutamic acid capsule and to a tri-component toxin composed of protective antigen (PA; 83 kDa), lethal factor (LF; 90 kDa), and edema factor (EF; 89 kDa). Individually, none of its three proteins is toxic. However, lethal toxin (LeTx), a mixture of PA and LF, causes lethal shock in experimental animals, and edema toxin; (EdTx), a mixture of PA and EF, induces local edema. LeTx and EdTx are binary toxins, because each requires an additional factor (PA) to elicit toxicity. Genes encoding the three anthrax toxin proteins reside on a large plasmid called pXO1 , and those for capsule synthesis are on another plasmid called pXO2. Strains of B. anthracis that carry only one of these plasmids are essentially avirulent, implying in vivo synergy between the capsular and protein-toxin virulence factors.

[14] Anthrax toxin is an unusual binary toxin because its components interact only after their secretion from the bacteria. EF and LF require assistance from PA to enter a host cell. EF, an adenylate cyclase, induces high levels of cAMP after it enters mammalian target cells. Assembly of the toxin is initiated when PA binds a cellular receptor and is activated by a furin-like (or other) cellular protease that cleaves it into two parts: PA63 (63 kDa), and PA20 (20 kDa). The PA20 fragment, which derives from the N terminus of the holoprotein, dissociates from PA63, and diffuses into the surrounding medium. Residual, receptor-bound PA63 molecules then self- associate into ring-shaped, heptameric oligomers. These bind EF or LF to high-affinity sites that span the interface of the PA63 subunits, forming complexes that contain 1 to 3 molecules of EF and/or LF per PA63 heptamer.

[15] Oligomerization of PA63 induces endocytosis that transports the complexes to an acidic compartment. Here, the heptamer changes from a "pre-pore" to an ion-conductive, integral- membrane pore that translocates EF and LF into the cytosol. Within the cytosol, EF catalyzes the conversion of ATP to cyclic AMP (cAMP), and LF acts proteolytically to cleave certain MAP- kinase-kinases. EF and LF enter many types of cells, including "professional phagocytes". Elevated cAMP levels can inhibit phagocytic performance, and LeTx can lyse murine macrophages. Immunization against PA is protective, confirming the importance of PA in pathogenesis.

[16] There is a clinical need for novel antimicrobial agents that have low toxicity against mammalian cells and are active against anthrax and/or VRE. The present invention addresses this need. Relevant literature

[17] Defensins are reviewed by Lehrer et a/. (1992) Ann. Rev. Immunol. 11 :105-128. Other endogenous antimicrobials are reviewed in Schonwetter et a/. (1995) Science 267:1645-1648; Schroder (1999)Cθ// MoI Life Sci. 56:32-46 (1999); and Harwig etal. (1994) FEBS Lett 342:281- 285.

[18] Specific defensins are described in Tang et a/. (1999) Science 286:498-502; Zimmermann et al. (1995) Biochemistry 34:13663-13671 ; Liu et al. (1997) Genomics 43:316- 320; and Palfree & Shen (1994) GenBank U10267; Polley et al. GenBank AF238378 disclose the sequence of Homo sapiens chromosome 8p23 clone SCb-561b17.

SUMMARY OF THE INVENTION

[19] Methods and compositions are provided for the use of retrocyclin peptides in the treatment of infection by vancomycin resistant enterococci (VRE) and/or B. anthracis. Retrocyclin peptides are small antimicrobial agents that kill VRE. Retrocyclin peptides also kill B. anthracis cells, and also are shown herein to have potent activity in the neutralization of anthrax toxins. These circular peptides are nonhemolytic and generally exhibit little or no in vitro cytotoxicity. Retrocyclins are equally effective against growing and stationary phase bacteria. [20] A pharmaceutical composition comprising retrocyclin or other circular mini-defensins as an active agent is administered to a patient suffering from VRE or anthrax infection. Alternatively, a pharmaceutical composition comprising retrocyclin or other circular mini- defensins or is administered as a protective agent to a normal individual facing potential exposure to VRE and/or anthrax. Retrocyclin is also effective at killing VRE and/or anthrax in vitro. Retrocyclin may be administered alone, or in combination with other bacteriocidal agents, e.g. antibiotics and/or other antiviral agents, and antiviral agents as a cocktail of effective peptides, etc. Retrocyclin-mediated killing is also useful for modeling and screening novel antibiotics.

BRIEF DESCRIPTION OF THE DRAWINGS

[21] Figure 1. Structure:Activity Relationships: The inhibitory activity of 5 μg/ml (~ 2.5 μM) of retrocyclin-1 is compared to that of its retro, enantio and retmenantio analogs in Figure 1a. Despite their identical amino acid compositions, their potency varied considerably (RC110>RC100>RC112>RC110). Fig. 1 b compares the inhibitory activity of retrocyclins 1-3 and rhesus theta-defensins (RTDs) 1 -3. Their similar activities suggest that activity resides primarily in residues that are common to all of them (Fig 1b).

[22] Figure 2A-B. Figure 2A depicts the activity of retrocyclins against anthrax toxins. Figure 2B depicts the binding of retrocyclins to anthrax toxins.

[23] Figures 3A-3C depict the activity of retrocyclins in protection of cells against anthrax toxins.

[24] Figure 4. Activity of retrocyclins against vancomycin-resistant enterococci. Three different VREF strains were tested in radial diffusion assays.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[25] Methods and compositions are provided for the use of retrocyclins and retrocyclin analogs as therapeutic and/or prophylactic agents against vancomycin resistant enterococci and/or β. anthracis. The peptides are effective at killing VRE and/or anthrax by direct microbicidal activity.

[26] The peptide form of retrocyclins provides a basis for further therapeutic development, by modification of the polypeptide structure to yield modified forms having altered biological and chemical properties. The native or modified forms are formulated in a physiologically acceptable carrier for therapeutic uses, or are otherwise used as an antimicrobial agent.

VANCOMYCIN RESISTANT ENTEROCOCCI

[27] Enterococci are enteric gram-positive cocci. E. faecalis has been the predominant pathogenic enterococcal species, followed by E. faecium. Species of enterococci include, without limitation, Enterococcus aquimarinus; Enterococcus asini; Enterococcus avium; Enterococcus azikeevi; Enterococcus caccae; Enterococcus canintestini; Enterococcus canis; Enterococcus casseliflavus; Enterococcus cecorum; Enterococcus columbae; Enterococcus devriesei; Enterococcus dispar; Enterococcus durans; Enterococcus faecalis; Enterococcus faecalis subsp. liquefaciens; Enterococcus faecalis V583; Enterococcus faecium; Enterococcus faecium DO; Enterococcus flavescens; Enterococcus gallinarum; Enterococcus gilvus; Enterococcus haemoperoxidus; Enterococcus hermanniensis; Enterococcus hirae; Enterococcus inusitatus; Enterococcus italicus; Enterococcus lactis; Enterococcus malodoratus; Enterococcus moraviensis; Enterococcus mundtii; Enterococcus pallens; Enterococcus phoeniculicola; Enterococcus pseudoavium; Enterococcus raffinosus; Enterococcus ratti; Enterococcus rattus; Enterococcus rottae; Enterococcus saccharolyticus; Enterococcus saccharominimus; Enterococcus sulfureus; and Enterococcus villorum. [28] There are five currently recognized phenotypes of vancomycin resistance, VanA, VanB, VanC, VanD, and VanE. VanA-resistant strains possess inducible, high-level resistance to vancomycin, where the MICs are from about >64 μg/ml. Resistance can be induced by glycopeptides and by nonglycopeptide agents. VanB-resistant strains have varying levels of resistance, where the MIC is from about 4 μg/ml to about 1 ,000 μg/ml. VanC resistant strains have an MIC of from about 4 to about 32 μg/ml.

[29] In some embodiments of the invention, a VRE is an enterococcal microorganism, wherein the MIC of vancomycin for the microorganism is at least about 4 mg/ml; at least 10 mg/ml; at least 25 mg/ml; at least 50 mg/ml; at least 250 mg/ml; or greater.

RETROCYCLIN COMPOSITIONS

[30] For use in the subject methods, a naturally occurring or synthetic retrocyclin may be used. As used herein, retrocyclins are cyclic polypeptides comprising the amino acid sequence:

X-I X₂ X3 X₄ X5 XQ X₇ Xe X9 Xio X11 X12 X13 Xi₄ X15 X16 X17 Xiβ wherein X1 and X18 are linked through a peptide bond, disulfide crosslinks are formed between at least one of: X₃ and Xi₆; X₅ and X₁₄, and X₇ and X₁₂, usually between at least two of such pairs, and preferably between the three pairs of amino acids, with the proviso that when such a crosslink is present, the crosslinked amino acids are both cysteines; at least about three of amino acids X₁ to Xi₈ are arginine or lysine, and the number of arginine or lysine residues may be four or more, five or more, or six or more. Preferred residues for arginine or lysine are X₄, Xg, X₈, X₁₃, and Xi₈;

X_2» X_δ> X-₁₁. X-₁₅ are preferably aliphatic amino acids, e.g. isoleucine, leucine, valine, phenylalanine, and alanine;

X₁₁ Xβi X₁₀ and X₁₇ are preferably glycine or alanine, usually glycine.

[31] Retrocyclins are octadecapeptides that contain two linked nonapeptides that may be identical or different. A consensus nonapeptide has the sequence shown below, where the bolded and underlined residues are invariant among the primate sequences identified herein. Substitutions found in the nonapeptide regions of other circular minidefensin precursors are shown below the consensus nonapeptide.

Residue No 1 3 5 7 9

Consensus nonapeptide RCICGRGIC

Variant L RLRV Variant T F Variant V Variant R

[32] From the consensus peptide and these variants, one can generate unique nonapeptide sequences (herein termed n1 , n2 . . . etc. ). Thus, n1 could be linked to itself or any of the other nonapeptides (n1 :n1 , n1 :n2, n1 :n3 . . . etc.), to generate unique octadecapeptides. To continue the process, n2 could be linked to itself or to any other nonapeptide except n1, to generate additional unique octadecapeptides, and so forth.

[33] Two naturally occurring human nonapeptide sequences are RCICGRGIC; and RCICGRRIC. The set of nonapeptides derived from these sequences and variants (which are also provided in the sequence listing as SEQ ID NO:19-64; 74-119 and 136) is as follows:

78 R C I C V R R I C V5 101 R C L C R R R F C L3, R5, F8

79 R C I C G L R I C L6 102 R C L C T L R I C L3, T5, L6

80 R C I C G R R V C V8 103 R C L C G R R V C L3, T5, V8

81 R C I C G R R F C F8 104 R C L C T R R F C L3, T5, F8

82 R C L C R R R V C L3, R5 105 R C L C V L R I C L3, V5, L6

83 R C L C T R R I C L3, T5 106 R C L C V R R V C L3, V5, V8

84 R C L C V R R I C L3, V5 107 R C I C G R R I C L3, V5, F8

85 R C L C G L R V C L3, L6 108 R C I C R L R V C R5, L6, V8

86 R C L C G R R V C L3, V8 109 R C I C R L R F C R5, L6, F8

87 R C L C G R R F C L3, F8 110 R C I C T L R V C T5, L6, V8

88 R C I C R R R V C R5, V8 111 R C I C T L R F C T5, L6, F8

89 R C I C R R R F C R5.F8 112 R C I C V L R V C V5, L6.V8

90 R C I C T R R V C T5, V8 113 R C I C V L R F C V5, L6, F8

91 R C I C T R R F C T5,F8 114 R C L C G L R V C L3, R5, L6, V8

92 R C I C T L R I C T5, L6 115 R C L C G L R I C L3, R5, L6, F8

93 R C I C V L R F C V5, L6 116 R C L C T L R V C L3.T5, L6, V8

94 R C I C R L R I C R5, L6 117 R C L C T L R I C L3, T5, L6, F8

95 R C I C V R R V C V5, V8 118 R C L C V L R V C L3, V5, L6, V8

96 R C I C G R R F C V5, F8 119 R C L C V L R I C L3, V5, L6, F8

136 R C I C G K G I C

^* residue modifications are shown in this column. NP# is a reference number for the nonamers.

[34] Retrocyclins of interest include cyclic peptides derived from the peptide sequence set forth in SEQ ID NO. 12, in particular a circular homodimer comprising a dimer of the amino acid sequence SEQ ID NO: 12, aa 48-56. This retrocyclin has the structure (SEQ ID NO:1 ; RC100): G l C R C I C G R G I C R C I C G R

X-| X2 X3 X₄ X5 Xδ X7 Xδ X9 XiO Xi1 Xi2 Xi3 Xi4 Xi5 Xi6 Xi7 Xi8

Wherein X₁ and Xi₈ are joined by a peptide bond, X₂ and Xn; X₄ and Xg, and Xi₃ and Xi₈ are disulfide bonded.

Another retrocyclin of interest is the synthetic analog (SEQ ID NO:2, RC101 ) G l C R C I C G K G I C R C I C G R

X-I X₂ X3 X₄ X5 Xθ X₇ X₈ X9 XiO Xi1 Xi2 Xi3 Xi4 Xi5 Xi6 Xi7 Xi8

wherein Xi and Xi₈ are joined by a peptide bond, X₂ and Xn; X₄ and Xg, and X₁3 and X₁₈ are disulfide bonded. Other synthetic analogs, or congeners, of retrocyclin are set forth in SEQ ID NO:3-SEQ ID NO:10.

[35] Some retrocyclins comprising lysine residues or analogs thereof are of particular interest. Lysine analogs include lysine, diaminohexynoic acid, N-epsilon-methyllysine, N-alpha- methyilysine, diaminopimelic acid, 5-aminopentanoic acid, and 7-aminoheptanoic acid and their D-amino acid counterparts. Such sequences are modified from those set forth in SEQ ID NO:19-64; and SEQ ID NO:94-119 by replacing at least one arginine residue with a lysine residue, and in a nonamer, may replace 1 or 2 arginines with lysines. Of particular interest are retrocyclins where at least one of the nonamers set forth in SEQ ID NO: 19-64; and SEQ ID NO:94-119 comprises lysine or lysine analog at residue 6.

[36] Six positions found to be important in modulating the specificity and activity of retrocyclins are residues 1 , 4, 9, 10, 13, & 18 corresponding to the sequence GiCRCiCGRGiCRCiCGR. Analogs of particular interest include those having one or more R→K substitutions, e.g. the RC101 peptide. Such peptides include those having a lysine at position 4; at position 9, at position 13, at position 18, or combinations thereof. Such combinations include analogs comprising two lysine residues at positions selected from residue 4, 9, 13 and 18; analogs comprising 3 lysine residues at positions selected from 4, 9, 13 and 18; and analogs comprising 4 or more lysine residues, including positions 4, 9, 13 and 18. [37] In some instances, such nonamers have the consensus sequence:

where the retrocyclin molecule itself comprises two independently selected nonamers. Such sequences are desirably reduced in hemagglutination relative to the arginine containing sequence. Among the specific lysine containing retrocyclins of interests are the following:

Identifier Alternative Name Sequence ( linear )

SEQ ID NO:126 RC114 RC101 /103 hybrid GICRCICGK GICRCYCGR

SEQ ID NO:127 RC119 R->K Retrocyclin-1 GICKCICGK GICKCICGR

SEQ ID NO:128 RC123A Retrocyclin 2A GICRCICGK RICRCICGR

SEQ ID NO:129 RC123B Retrocyclin 2B GICRCICGK KICRCICGR

SEQ ID NO:130 RC123C Retrocyclin 2C GICRCICGR KICRCICGR

SEQ ID NO:131 RC123D Retrocyclin 2D GICRCICGR RICKCICGR

SEQ ID NO:132 RC123E Retrocyclin 2E GICKCICGR RICRCICGR

SEQ ID NO:133 RC123F Retrocyclin 2F GICRCICGR RICRCICGK

[38] Also of interest are the sequences SEQ ID NO:134: GVCRC ICGRG VCRCI CRR; and SEQ ID NO:135: GVCRC ICGRG VCRCI CGR . Retrocyclins of this type are comprised, or consist, of two linked nonamers independently selected from SEQ ID NO:25 and SEQ ID NO:33. [39] The sequence of the retrocyclin polypeptides may be altered in various ways known in the art to generate targeted changes in sequence. The polypeptide will usually be substantially similar to the sequences provided herein, i.e. will differ by one amino acid, and may differ by two amino acids. The sequence changes may be substitutions, insertions or deletions. [40] The protein may be joined to a wide variety of other oligopeptides or proteins for a variety of purposes. By providing for expression of the subject peptides, various post- translational modifications may be achieved. For example, by employing the appropriate coding sequences, one may provide famesylation or prenylation. In this situation, the peptide will be bound to a lipid group at a terminus, so as to be able to be bound to a lipid membrane, such as a liposome.

[41] Modifications of interest that do not alter primary sequence include chemical derivatization of polypeptides, e.g., acetylation, or carboxylation. Also included are modifications of glycosylation, e.g. those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g. by exposing the polypeptide to enzymes which affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences that have phosphorylated amino acid residues, e.g. phosphotyrosine, phosphoserine, or phosphothreonine.

[42] Also included in the subject invention are polypeptides that have been modified using ordinary molecular biological techniques and synthetic chemistry so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g. D-amino acids or non-naturally occurring synthetic amino acids.

[43] The subject peptides may be prepared by in vitro synthesis, using conventional methods as known in the art. Various commercial synthetic apparatuses are available, for example, automated synthesizers by Applied Biosystems, Inc., Foster City, CA, Beckman, etc. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like.

[44] If desired, various groups may be introduced into the peptide during synthesis or during expression, which allow for linking to other molecules or to a surface. Thus cysteines can be used to make thioethers, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, amino groups for forming amides, and the like. [45] The polypeptides may also be isolated and purified in accordance with conventional methods of recombinant synthesis. A lysate may be prepared of the expression host and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. For the most part, the compositions which are used will comprise at least 20% by weight of the desired product, more usually at least about 75% by weight, preferably at least about 95% by weight, and for therapeutic purposes, usually at least about 99.5% by weight, in relation to contaminants related to the method of preparation of the product and its purification. Usually, the percentages will be based upon total protein. Genetic sequences encoding demi-defensins are provided herein, e.g. SEQ ID NO:4, 7 and 9. [46] In one embodiment of the invention, the antimicrobial peptide consists essentially of a polypeptide sequence as set forth herein. By "consisting essentially of in the context of a polypeptide described herein, it is meant that the polypeptide is composed of the sequence set forth in the seqlist, which sequence may be flanked by one or more amino acid or other residues that do not materially affect the basic characteristic(s) of the polypeptide. [47] Retrocyclin coding sequences can be generated by methods known in the art, e.g. by in vitro synthesis, recombinant methods, etc. to provide a coding sequence to corresponds to a linear retrocyclin polypeptide that could serve as an intermediate in the production of the cyclic retrocyclin molecule. Using the known genetic code, one can produce a suitable coding sequence. For example, the circular polypeptide of retrocyclin (SEQ ID NO: 1 ) is encoded by the sequence (SEQ ID NO: 18) AGG TGC ATT TGC GGA AGA GGA ATT TGC AGG TGC ATT TGC GGAAGA GGAATT TGC, but since the peptide is circular, it is somewhat arbitrary which codon is selected to be first, allowing this to be based on other criteria, e.g. relative efficiency in purification or cyclization of the predicted product. The polypeptide set forth in SEQ ID NO:2 is encoded by a similar sequence, wherein one of the arginine codons (AGA) is substituted with a lysine codon (AAA or AAG).

[48] The nucleic acids can be cDNAs or genomic DNAs, as well as fragments thereof, particularly fragments that encode a biologically active polypeptide and/or are useful in the methods disclosed herein. The term "cDNA" as used herein is intended to include all nucleic acids that share the arrangement of sequence elements found in native mature mRNA species, where sequence elements are exons and 3' and 5' non-coding regions. Normally mRNA species have contiguous exons, with the intervening introns, when present, being removed by nuclear RNA splicing, to create a continuous open reading frame encoding a polypeptide of the invention.

[49] A genomic sequence of interest comprises the nucleic acid present between the initiation codon and the stop codon, as defined in the listed sequences, including all of the introns that are normally present in a native chromosome. It can further include the 3' and 5' untranslated regions found in the mature mRNA. It can further include specific transcriptional and translational regulatory sequences, such as promoters, enhancers, etc., including about 1 kb, but possibly more, of flanking genomic DNA at either the 5' and 3' end of the transcribed region. The genomic DNA can be isolated as a fragment of 100 kbp or smaller; and substantially free of flanking chromosomal sequence. The genomic DNA flanking the coding region, either 3' and 5', or internal regulatory sequences as sometimes found in introns, contains sequences required for proper tissue, stage-specific, or disease-state specific expression. [50] Probes specific to the nucleic acid of the invention can be generated. The probes are preferably at least about 18 nt, 25nt or more of the corresponding contiguous sequence. The probes can be synthesized chemically or can be generated from longer nucleic acids using restriction enzymes. The probes can be labeled, for example, with a radioactive, biotinylated, or fluorescent tag. Preferably, probes are designed based upon an identifying sequence of one of the provided sequences. More preferably, probes are designed based on a contiguous sequence of one of the subject nucleic acids that remain unmasked following application of a masking program for masking low complexity (e.g., BLASTX) to the sequence, i.e., one would select an unmasked region, as indicated by the nucleic acids outside the poly-n stretches of the masked sequence produced by the masking program.

[51] The nucleic acids of the invention are isolated and obtained in substantial purity, generally as other than an intact chromosome. Usually, the nucleic acids, either as DNA or RNA, will be obtained substantially free of other naturally-occurring nucleic acid sequences, generally being at least about 50%, usually at least about 90% pure and are typically "recombinant," e.g., flanked by one or more nucleotides with which it is not normally associated on a naturally occurring chromosome.

[52] Retrocyclin encoding nucleic acids can be provided as a linear molecule or within a circular molecule, and can be provided within autonomously replicating molecules (vectors) or within molecules without replication sequences. Expression of the nucleic acids can be regulated by their own or by other regulatory sequences known in the art. The nucleic acids of the invention can be introduced into suitable host cells using a variety of techniques available in the art, such as transferrin polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated DNA transfer, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, gene gun, calcium phosphate-mediated transfection, and the like.

[53] Expression vectors may be used to introduce a retrocyclin coding sequence into a cell. Such vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences. Transcription cassettes may be prepared comprising a transcription initiation region, the target gene or fragment thereof, and a transcriptional termination region. The transcription cassettes may be introduced into a variety of vectors, e.g. plasmid; retrovirus, e.g. lentivirus; adenovirus; and the like, where the vectors are able to transiently or stably be maintained in the cells, usually for a period of at least about one day, more usually for a period of at least about several days to several weeks. [54] The gene or retrocyclin peptide may be introduced into tissues or host cells by any number of routes, including viral infection, microinjection, orfusion of vesicles. Jet injection may also be used for intramuscular administration, as described by Furth et al. (1992) Anal Biochem 205:365-368. The DNA may be coated onto gold microparticles, and delivered intradermally by a particle bombardment device, or "gene gun" as described in the literature (see, for example, Tang et al. (1992) Nature 356:152-154), where gold microprojectiles are coated with the stresscopin or DNA, then bombarded into skin cells.

SCREENING METHODS

[55] Identification of biologically active retrocyclins analogs may utilize various screening assays. Considerable information is gained by analyzing how bacteria respond to antibiotic exposure. A bacterial genomic gene expression array (Invitrogen) may be used. Conditions (CFU/ml, incubation medium, retrocyclin concentration) are identified that result in gradual but substantial killing (e.g. , 50% over 15 minutes), and conditions that are very slightly subinhibitory. To do so, an aliquot of overnight culture of bacteria 1 :125 is diluted into fresh medium and dispense 100 μl aliquots into a 96 well microplate plate that is incubated at 37⁰C with continuous shaking in a Spectra Max 250 kinetic microplate spectrophotometer (Molecular Devices). Some wells also contain retrocyclin (various concentrations). The growth curves and retrocyclin effects are analyzed. Once the subinhibitory and slightly inhibitory conditions have been determined, the cultures are scaled to 20 ml and verified that the retrocyclin concentrations have the desired (sub)inhibitory effects. Bacteria are quickly fixed at predetermined intervals using Qiagen RNAprotect reagent™ to avoid non-specific gene induction. RNA extraction are done using a Qiagen RNA Mini Kit, and the RNA is retro-transcribed into biotinylated cDNA using Invitrogen Superscriptase. This labeled cDNA is used to probe a bacterial gene expression array (Affymetrix Inc. Santa Clara, CA). The Affymetrix gene array is scanned and data analyzed. Significant gene expression changes may be confirmed by real-time PCR. [56] Screening assays may include detection of binding to S. anthracis lethal factor (LF), edema factor (EF), protective antigen (PA), and the thermolysin-like protease of B. anthracis. [57] For example, recombinant proteins may be synthesized or purchase, and binding measured by surface plasmon resonance (SPR) on a BiaCore3000 instrument. To identify retrocyclin analogs that inactivate the protease activity of LF, a labeled substrate of LF (19-mer peptide containing the MAPKK motif) is commercially available. LF and retrocyclin analogs are preincubated at room temperature for 0-30 minutes. Assays will usually be run in 10 mM HEPES, 15OmM NaCI, 1 mM Ca²⁺, pH 7.4, with substrate added last. Fluorescence is monitored with excitation at 355 nm and emission at 460 nm. The V_max (maximal velocity) will be determined, and used to calculate % inhibition from the formula: (V_max experimental/ V_max control) x 100. \

[58] To identify retrocyclin analogs that inactivate the adenylate cyclase activity of EF, tthe following cell-based and cell-free systems may be used. Qualitative assessment (Cell Round-up Assay) is simple and economical screening assay based on the ability of increased intracellular cAMP levels to induce cytoskeletal rearrangements that make cells round up. The assays are done when the cells reach 50-80% confluence, retrocyclins are tested at 1-25 μM. EF and PA will be added at final concentrations of 3 and 25 ng/ml, respectively. The morphology of the cells is examined by inverted phase microscopy after incubation for 1 h, 4 h, and overnight. If rounding is seen, cAMP levels in cell lysates can be measured with the Applied Biosystems cAMP-Screen Direct™ Chemiluminescent Immunoassay System.

[59] To identify retrocyclin analogs that prevent processing, assembly and delivery of LF & EF each toxin component may be independently titrated in a cell-based intoxication assay to determine which component is limiting. We expect that if anti-PA activity alone is most important, then increasing PA concentration will overcome defensin-mediated blocks, while increasing LF concentration will have little effect. Conversely, if anti-LF activity is more important, then increasing LF concentration will increase cell death, while increasing PA concentration will have no effect. Finally, if targeting both toxin components is necessary for antitoxin activity, then we expect that increasing the concentration of either will result in some ability to overcome the defensin-mediated block.

[60] Before LF enters the cytosol, it traverses a compartment whose acidic pH might cause θ- defensin/LF complexes to dissociate. The ability of retrocyclins to bind LF and inhibit its enzymatic activity may be tested within the pH range of 4.5-6.5. An established assay for LFN internalization may be used to determine if defensins are found in host-cell cytosol in the presence of LeTx. Cells are incubated on ice to block endocytosis, then PA and LF added to allow for binding to host cells in the presence or absence of radiolabeled retrocyclins. In this assay, PA is first "activated" by incubating with trypsin to generate nicked PA (nPA). Trypsin nicking mimics the furin cleavage and thus bypasses the requirement for furin acitivity, which is inefficient at 4°C. Following a binding step, cells are exposed to a low pH buffer to induce pore formation and translocation at the plasma membrane. Cells are then treated with or without a nonspecific protease such as pronase to remove all surface bound (non-translocated) toxin and radiolabeled retrocyclin. Cells are washed with DTT to reduce retrocyclin-encoded disulfide bonds required for plasma membrane binding and incubated with an excess of unlabeled retrocyclin, which will largely remove the remaining reversibly-bound retrocyclins present on the plasma membrane. Following a final wash, cells will be lysed and the total radioactivity associated with the cells will be determined as a measure of the amount of retrocyclin internalized. As a control for specific translocation, the assay will be performed without PA. [61] At least part of the antitoxin effect of θ-retrocyclins may be at the level of blocking PA- pore formation, thereby preventing LF translocation. However, additional blocks to LF translocation may also exist. To determine if there are direct blocks to translocation, a cell- based assay may be employed. Briefly, nPA is bound to cells at 4^°C followed by incubation with radiolabeled LF ± retrocyclins. The cells are exposed to acidic pH to induce pore formation and translocation through the plasma membrane. Next, cells are washed, treated ± pronase to digest non-translocated toxin, washed again, and then lysed. Protein is precipitated with trichloroacetic acid and analyzed by SDS-PAGE followed by autoradiography. Translocation efficiency is determined as the fraction of total radioactivity (with no pronase) present in the pronase-treated samples.

[62] Surface plasmon resonance studies may determine if retrocyclins block any of the protein-protein interactions needed for toxin entry. These include studies on the interactions of PA with its two known receptors to determine if on- or off-rates are affected, and determining if binding in the presence of retrocyclins occurs normally by performing experiments in the absence of divalent cations (required for PA-ANTXR binding) and by using mutant forms of receptors and PA that have reduced affinity for each other. It may be determined if retrocyclins affect the interaction between PA monomers, which would influence heptamer formation. Complementary mutant forms of PA are used that cannot oligomerize with themselves, but can form dimers with each other.

[63] To identify retrocyclin analogs that inactivate the thermolysin-like protease of B. anthracis, ifhermolysin activity is measured spectrophotometrically, using furylacroyl-Ala-Phe- NH2 as the substrate. The thermolysin-like protease of S. anthracis (Sterne or delta Ames) may be isolated from culture supernatants. Briefly, B. anthracis is grown overnight in LB medium. After removing the bacteria by centrifugation at 8000 g, the supernatant is passed through a 0.22 μm cellulose acetate filter, and further concentrated with a 10 kD cut-off Amicon Ultra15 centrifugal filter (Millipore, MA). When additional purification is required (e.g., for binding studies), this is done 4⁰C by FPLC. The enzyme is stored at 4°C and used within a few days. Protein content is determined using the Bradford reagent (Bio-Rad) with BSA as the standard. [64] Anthrax progresses through its early stages with subtle and vague flu-like symptoms. While it is important to develop prophylactics that reduce the infectivity or germination of spores for persons exposed to B. anthracis, it is also necessary to develop therapeutics that target the vegetative form. By the time a patient with inhalation anthrax is clinically ill, vegetative bacilli have already disseminated to initiate a systemic infection. To test if retrocyclins kill vegetative bacilli, they are screened against bacilli grown in bacteriological media such as LB, Brain heart infusion (BHI), or nutrient broth yeast supplemented with 0.8% sodium bicarbonate (NBYCO3) to induce toxin or capsule production. Additionally, antibacterial activity of retrocyclins in co-culture of vegetative bacilli with mammalian cells such as human macrophages (monocyte-derived and alveolar), monocytes, or PMNs is tested.

[65] Because B. anthracis bacilli are relatively large, the opsonic activity of retrocyclins may be assessed by direct phase microscopy. If phagocytosis is seen, it may be verified that the bacteria are internalized by performing transmission electron microscopy, which will also allow assessment of the structural integrity of the ingested bacteria. [66] A modified spore-challenge assay uses macrophages that allows for examination of intracellular germination while reducing complications associated with extracellular germination of β. anthracis spores and extracellular sources of toxin. In this assay, spores are allowed to bind to macrophages then non-bound spores are removed by centrifugation through a Ficoll- Paque gradient. Macrophages are recovered in the "buffy" layer and plated to allow for intracellular germination. As added assurance that extracellular germination and/or growth of vegetative bacilli are minimal, purified PIyG, a phage encoded lysin that efficiently kills germinated spores and vegetative cells of B. anthracis may be included. To block any extracellular toxin, a soluble version of the ANTXR2 ectodomain, which is a highly effective antitoxin may be included. Efficiency of intracellular spore germination in the presence or absence of retrocyclin peptides is measured using, for example: 1 ) differential staining of germinated and non-germinated spores with Gram's stain or malachite green respectively, 2) hypotonic lysis of macrophages followed by plating of spores/bacilli with or without heat treatment (only non-germinated spores will survive and grow into colonies with heat treatment, while all spores/bacilli will grow into colonies without heat), or 3) fluorescence video-microscopy of B. anthracis strains overexpressing GFP.

[67] Considerable information is gained by analyzing how bacteria respond to antibiotic exposure. A Bacillus subtilis bacterial genomic gene expression array (Invitrogen) may be used, given that the responses of B. subtilis and B. anthracis to retrocyclins are likely to be similar. Conditions (CFU/ml, incubation medium, retrocyclin concentration) are identified that result in gradual but substantial killing (e.g., 50% over 15 minutes), and conditions that are very slightly subinhibitory. To do so, an aliquot of overnight culture of B. subtilis 1 :125 is diluted into fresh medium and dispense 100 μl aliquots into a 96 well microplate plate that is incubated at 37 ⁰C with continuous shaking in a Spectra Max 250 kinetic microplate spectrophotometer (Molecular Devices). Some wells also contain retrocyclin (various concentrations). The growth curves and retrocyclin effects are analyzed. Once the subinhibitory and slightly inhibitory conditions have been determined, the cultures are scaled to 20 ml and verified that the retrocyclin concentrations have the desired (sub)inhibitory effects. Bacteria are quickly fixed at predetermined intervals using Qiagen RNAprotect reagent™ to avoid non-specific gene induction. RNA extraction are done using a Qiagen RNA Mini Kit, and the RNA is retro-transcribed into biotinylated cDNA using Invitrogen Superscriptase. This labeled cDNA is used to probe B. subtilis gene expression array (Affymetrix Inc. Santa Clara, CA). The Affymetrix gene array is scanned and data analyzed. Significant gene expression changes may be confirmed by real-time PCR. [68] The poly-D-glutamic acid capsule of β. anthracis allows it to escape ingestion by phagocytic host defense cells: neutrophils, monocytes and macrophages, that possess oxidative and nonoxidative antimicrobial mechanisms. In addition to their abundant α-retrocyclins, the non-oxidative bactericides of human neutrophils (PMNs) include lysozyme, an α-helical cathelicidin called LL-37, a chymotrypsin-like neutral protease called cathepsin G, azurocidin, and phospholipase A₂ - all of which can kill certain Gram-positive bacteria. PMNs also possess NADPH oxidase, an enzyme complex that produces copious amounts of superoxide and hydrogen peroxide, and myeloperoxidase - an enzyme that converts these reactive oxygen intermediates into even stronger oxidants, including hypochlorite and chloramines. Human monocytes lack α-retrocyclins, but possess other oxidative and nonoxidative antimicrobial mechanisms. Such cells may be used to study the effects of retrocyclins on their phagocytic uptake of B. anthracis spores, and encapsulated and nonencapsulated bacilli; and their uptake of fluorescent retrocyclins from the medium.

[69] There are many ways to study phagocytosis, for example based on direct microscopic and ultramicroscopic examination using phase, fluorescent and electron microscopy. Distinguishing surface adherent from truly internalized organisms can be resolved at the light- microscopy level by using fluorescent bacteria and examining them in the presence of a non- permeant dye that can quench the fluorescence of extracellular bacteria, but not the fluorescence of intracellular bacteria. For example, B. anthracis can be labeled with fluorescein isothiocyanate, and the fluorescence of surface-adherent but extracellular organisms can be quenched with trypan blue. Another version of this assay uses ethidium bromide as a quenching agent that added to cells after they have phagocytosed labeled bacteria. Ethidium bromide causes extracellular FITC-labeled bacteria to fluoresce red-orange, whereas intracellular bacteria are not exposed to the dye and remain green. This process allows distinction between intracellular and extracellular bacteria by simultaneous visualization of both populations. With minor modifications, this approach can be used to monitor phagocytosis by flow cytometry and to follow the ingestion of S. anthracis spores. These experiments are done both with the nonencapsulated but toxigenic Sterne strain and with the encapsulated, but nontoxigenic Pasteur strain of B. anthracis.

METHODS OF USE

[70] Formulations of retrocyclins are administered to a host suffering from an ongoing VRE and/or anthrax bacterial infection, or who faces exposure to a VRE and/or anthrax. Administration may be topical, localized or systemic, depending on the specific microorganism. Generally the dosage will be sufficient to decrease the microbial or viral population by at least about 50%, usually by at least 1 log, and may be by 2 or more logs. The compounds of the present invention are administered at a dosage that reduces the pathogen population while minimizing any side-effects. It is contemplated that the composition will be obtained and used under the guidance of a physician for in vivo use. [71] Retrocylins are also useful for in vitro formulations to kill VRE and/or anthrax, particularly where one does not wish to introduce quantities of conventional antibiotics. For example, retrocyclins may be added to animal and/or human food preparations. [72] The susceptibility of a VRE and/or anthrax to killing or inhibition by retrocyclins may be determined by in vitro testing, as detailed in the experimental section and described above. Typically a culture of the microbe is combined with retrocyclins at varying concentrations for a period of time sufficient to allow the protein to act, usually ranging from about one hour to one day. The viable microbes are then counted, and the level of killing determined. Two stage radial diffusion assay is a convenient alternative to determining the MIC or minimum inhibitory concentration of an antimicrobial agent.

[73] Various methods for administration may be employed. The polypeptide formulation may be given orally, or may be injected intravascularly, subcutaneously, peritoneally, by aerosol, opthalmically, intra-bladder, topically, etc. For example, methods of administration by inhalation are well-known in the art. The dosage of the therapeutic formulation will vary widely, depending on the specific retrocyclin or demi-defensin to be administered, the nature of the disease, the frequency of administration, the manner of administration, the clearance of the agent from the host, and the like. The initial dose may be larger, followed by smaller maintenance doses. The dose may be administered as infrequently as weekly or biweekly, or fractionated into smaller doses and administered once or several times daily, semi-weekly, etc. to maintain an effective dosage level. In many cases, oral administration will require a higher dose than if administered intravenously. The amide bonds, as well as the amino and carboxy termini, may be modified for greater stability on oral administration.

Formulations

[74] The compounds of this invention can be incorporated into a variety of formulations for therapeutic administration. More particularly, the compounds of the present invention can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, lotions, and aerosols. As such, administration of the compounds can be achieved in various ways, including oral, vaginal, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intratracheal, etc., administration. The retrocyclins may be systemic after administration or may be localized by the use of an implant or other formulation that acts to retain the active dose at the site of implantation.

[75] The compounds of the present invention can be administered alone, in combination with each other, or they can be used in combination with other known compounds (e.g., perforin, anti- inflammatory agents, antibiotics, etc.) In pharmaceutical dosage forms, the compounds may be administered in the form of their pharmaceutically acceptable salts. The following methods and excipients are merely exemplary and are in no way limiting.

[76] For oral preparations, the compounds can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

[77] The compounds can be formulated into preparations for injections by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives. [78] The compounds can be utilized in aerosol formulation to be administered via inhalation. The compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like. [79] The compounds can be used as lotions, for example to prevent infection of burns, by formulation with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

[80] Furthermore, the compounds can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compounds of the present invention can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.

[81] Unit dosage forms for oral, vaginal or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more compounds of the present invention. Similarly, unit dosage forms for injection or intravenous administration may comprise the compound of the present invention in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.

[82] Implants for sustained release formulations are well-known in the art. Implants are formulated as microspheres, slabs, etc. with biodegradable or non-biodegradable polymers. For example, polymers of lactic acid and/or glycolic acid form an erodible polymer that is well- tolerated by the host. The implant containing retrocyclins is placed in proximity to the site of infection, so that the local concentration of active agent is increased relative to the rest of the body.

[83] The term "unit dosage form", as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with the compound in the host.

[84] The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.

[85] Typical dosages for systemic administration range from 0.1 μg to 100 milligrams per kg weight of subject per administration. A typical dosage may be one tablet taken from two to six times daily, or one time-release capsule or tablet taken once a day and containing a proportionally higher content of active ingredient. The time-release effect may be obtained by capsule materials that dissolve at different pH values, by capsules that release slowly by osmotic pressure, or by any other known means of controlled release.

[86] Those of skill will readily appreciate that dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects. Some of the specific compounds are more potent than others. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means. A preferred means is to measure the physiological potency of a given compound. [87] The use of liposomes as a delivery vehicle is one method of interest. The liposomes fuse with the cells of the target site and deliver the contents of the lumen intracellular^. The liposomes are maintained in contact with the cells for sufficient time for fusion, using various means to maintain contact, such as isolation, binding agents, and the like. In one aspect of the invention, liposomes are designed to be aerosolized for pulmonary administration. Liposomes may be prepared with purified proteins or peptides that mediate fusion of membranes, such as Sendai virus or influenza virus, etc. The lipids may be any useful combination of known liposome forming lipids, including cationic or zwitterionic lipids, such as phosphatidylcholine. The remaining lipid will be normally be neutral or acidic lipids, such as cholesterol, phosphatidyl serine, phosphatidyl glycerol, and the like.

[88] For preparing the liposomes, the procedure described by Kato et a/. (1991) J. Biol. Chem. 266:3361 may be used. Briefly, the lipids and lumen composition containing peptides are combined in an appropriate aqueous medium, conveniently a saline medium where the total solids will be in the range of about 1-10 weight percent. After intense agitation for short periods of time, from about 5-60 sec, the tube is placed in a warm water bath, from about 25-40° C and this cycle repeated from about 5-10 times. The composition is then sonicated for a convenient period of time, generally from about 1-10 sec. and may be further agitated by vortexing. The volume is then expanded by adding aqueous medium, generally increasing the volume by about from 1-2 fold, followed by shaking and cooling. This method allows for the incorporation into the lumen of high molecular weight molecules.

Formulations with Other Active Agents

[89] For use in the subject methods, retrocyclins may be formulated with other pharmaceutically active agents, particularly other antimicrobial agents. Other agents of interest include a wide variety of antibiotics, as known in the art. Classes of antibiotics include penicillins, e.g. penicillin G, penicillin V, methicillin, oxacillin, carbenicillin, nafcillin, ampicillin, etc.; penicillins in combination with β-lactamase inhibitors, cephalosporins, e.g. cefaclor, cefazolin, cefuroxime, moxalactam, etc.; carbapenems; monobactams; aminoglycosides; tetracyclines; macrolides; lincomycins; polymyxins; sulfonamides; quinolones; cloramphenical; metronidazole; spectinomycin; trimethoprim; vancomycin; etc.

[90] Cytokines may also be included in a retrocyclin formulation, e.g. interferon γ, tumor necrosis factor α, interleukin 12, etc.

[91] Antiviral agents, e.g. acyclovir, gancyclovir, etc., and other circular mini-defensins (theta defensins) may also be included in retrocyclin formulations.

EXPERIMENTAL

[92] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the subject invention, and are not intended to limit the scope of what is regarded as the invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g. amounts, temperature, concentrations, etc.) but some experimental errors and deviations should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees centigrade; and pressure is at or near atmospheric.

Example 1

[93] Table 1 provides a description of the peptides used in the examples, by showing their names and the linear sequences of their synthetic precursors. In a circular peptide, residue numbering is arbitrary; residue #1 has been assigned to the N-terminal glycine of the synthetic precursor. Table 1 Retrocyclins and rhesus theta defensins.

[94] These peptides by solid phase peptide synthesis and purified to >95% purity as previously described. Their concentrations were determined by amino acid analysis. RC112 is composed exclusively of D-amino acids. The others shown in Table 1 contained only L-amino acids.

[95] Antimicrobial activity is measured in many ways, including colony counting, broth micro- dilution assays, radial diffusion assays, and virtual colony counting. Two stage radial diffusion assays are precise, reproducible and consume <4 μg of peptide per assay. The results shown in Tables 2a and b were obtained in two-stage assays done as follows.

[96] Stage 1 : 1-4 x 10⁶ colony forming units (CFU) were dispersed in a thin 1% agarose ("underlay") gel containing 10 mM phosphate buffer (pH 7.4), 100 mM NaCI, and nutrients (a 1 :100 dilution of trypticase soy broth) sufficient for several doublings. A regularly spaced, 6 X 6 array of wells (3 mm diameter, capacity 9 μl) was punched, allowing us to assay up to 6 peptides on each plate. Each set of 6 wells received 8 μl_ of a serially ("half-log") diluted peptide solution containing 250, 79, 25, 7.9, 2.5 or 0.79 μg of peptide/ml. The plate was incubated for 3h to allow the peptides to diffuse into the underlay gel.

[97] Stage 2: A double-strength trypticase soy agar overlay gel was poured over the underlay gel, and the plate was incubated overnight to allow surviving bacteria to form micro- colonies. The clear zones around each well were measured 18-24h later. To determine the minimal effective concentration (MEC), we calculated the linear regression function relating the adjusted diameter (zone diameter minus the_; well diameter) to the log₁₀ peptide concentration. The X-intercept of the line defined by this function gives the MEC. Typically, the correlation coefficient (r²) of this line was >0.98. Figure 2a shows a detail from the assay plate shown in its entirety on the next page. The test organism was B. anthracis (Sterne). The wells that 8 μl of retrocyclin-2 (RC2) at the concentrations of 7.9, 2.5 and 0.79 μg/ml. The amount of RC2 delivered to each well is shown above the well. Table 2a compares the MEC of retrocyclins (RCs) 1 and 2, protegrin PG-1 and human α- defensin HNP-1 against B. anthracis (Sterne) and other Gram-positive bacteria.

[98] RC2 was very active (MEC < 1 μg/ml) against spores and vegetative cells of B. anthracis, B. subtilis, vancomycin-resistant enterococci (VRE)₁ Group B streptococcus, and S. epidermidis. RC1 was almost as active as RC2 against B. anthracis but was 3-5 fold less potent against other Gram-positive bacteria. HNP1 was also active against S. anthracis, but lacked activity against S. aureus, S. epidermidis, VREF and Group B streptococci. RC 1&2 were inactive against "wild-type" methicillin sensitive and methicillin-resistant strains of S. aureus (MSSA and MRSA strains). However, they cold kill isogenic mutants of the MSSA strain whose teichoic acids lacked D-alanine or whose membrane lipids lacked lysylphosphatidylglycerol. Table 2b shows the importance of arginine residues for the activity of retrocyclin-1 to kill B. anthracis (Sterne). R9G retrocyclin-1 (RC107G) was only about half as potent as RC1. R9G, R18G-retrocyclin-1 (RC107GG) was ~15% as potent, and when all four arginines in retrocyclin-1 were replaced, the resulting peptide (RC107G₂H₂) was <1% as active against B. anthracis.

Table 2b

RC1 RC2 RC107G RC107GG RC107G2H2 HNP1

B. anthracis 1.0 0.6 2.04 6.44 >79.1 8.7

[99] The four conserved arginines (arg 4,9, 13, and 18) found in all θ-defensins contribute individually and collectively to their activity against β. anthracis. Retrocyclin-2, which contains a fifth arginine, is more potent against B. anthracis and other gram-positive bacteria. Example 2

Θ-Defensins inhibit the enzymatic activity of Lethal Factor (LF)

[100] Lethal Factor is a zinc metalloprotease, whose enzymatic activity can be measured monitoring the cleavage of its specific substrate by fluorescence resonance energy transfer (FRET). We purchased recombinant LF and its substrate from Calbiochem (catalog numbers 176900 and 176902) and performed kinetic assays using a Molecular Dynamics instrument and a multi-well format. When LF cleaves the substrate (an internally-quenched, fluorogenic 19-mer peptide containing the MAPKK motif, (38)) between Pro⁸-lle⁹, increased fluorescence results. The assay medium contained 50 μM substrate, 150 mM NaCI, 10 mM phosphate and 1 mM Ca⁺⁺ (pH 7.4). LF was tested at 100 nM.

[101] To more precisely assess their potency as LF inhibitors, we determined the IC₅₀Of these and additional retrocyclin variants (Table 3). The IC₅₀ (the concentration that inhibits enzyme activity by 50%) of retrocyclins 1-3 and RTDs 1-3 ranged from 1.4 to 2.78 μM. RdOO(OX ), the β-hairpin precursor of retrocyclin-1 , was 14.7-fold less potent than the cyclic form, despite having all 3 SS, bonds, both β-sheets and one of the β-turns found in retrocyclin-1. Thus, the cyclic backbone of retrocyclins is needed for LF inhibition. However, reduced and alkylated forms of retrocyclin-1 and RTD-1 that remained cyclic but lacked SS bonds were unable to inhibit LF (Table 3). We conclude that the SS bonds and the cyclic backbone are both required for inhibition of anthrax LF.

[102] Role ofarginine residues. Replacing arg9 with glycine (RC107G) reduced relative molar potency by 55.6% and replacing arg 9 &18 with glycine reduced it by 64% (Table 3c). RC107G₂H_2- the RC1 variant in which arg 9 and 18 were replaced by glycines and arg 4 and 13 were replaced by histidines completely lost the ability to inhibit LF at pH 7.4, and at pH 7.06.5 or 6.0 as well. Thus, the ability of θ-defensins to inhibit LF requires the majority of their conserved structural features, namely: a cyclic backbone, a disulfide scaffold, and conserved arginine residues. These are precisely the same features that enable θ-defensins to kill β. anthracis (Table 2b).

[103] LF inhibition is noncompetitive. We measured the rate (V_max) of enzymatic activity using 9 μg/ml of LF (~100 nM), 5 μg/ml of retrocyclin (~ 2.5 μM) and various substrate concentrations ( 5 to 50 μM). Because the extent of inhibition was independent of substrate concentration, the inhibition of LF by retrocyclin was noncompetitive with respect to substrate. We also measured enzyme activity while keeping LF, RC100 and substrate concentrations constant, and varying Zn from 0 to 100 μM. If retrocyclins inhibited LF activity by competing with Zn⁺⁺ for binding to the glutamic acid in its HEXXH site, adding more zinc might reverse the inhibition. Instead, additional Zn enhanced the extent of inhibition. Since 100 μM Mg⁺⁺ neither enhanced nor reduced the inhibitory potency of retrocyclin-1 , this effect may be specific for Zn⁺⁺. [104] Molecular modeling. LF, like many other metalloproteases, contains a zinc-binding HEXXH motif and another acidic residue (glutamic acid). The structure formed by LF and the N terminus of MAPKK-2 has been resolved by X-ray crystallograpy (139), and the NMR structure of retrocyclin-2 was recently solved (2ATG, Protein Data Base). LF has four domains (Dm): Dm1 binds PA, the membrane-translocating component of anthrax toxin. Together, Dm 2-4 create a groove that holds the 16-aa N-terminai tail of MAPKK-2 before cleavage and Dm4 holds the catalytic center.

[105] The docking of Retrocyclin-2 to anthrax LF was modeled. Arg9 or Arg18 were well positioned to interact with the glutamic acid of the HEXXH zinc-binding motif. Such binding could allow retrocyclins to obstruct the access of MAPKK-2 to the groove that binds it in position for cleavage described above.

[106] NMR studies. Evidence has been found for concentration-dependent oligomerization of retrocyclins, resulting in the formation of dimers, trimers and higher order oligomers. Evidence for this came from three modalities: analytical ultracentrifugation, nanospray LC-MS, and PAGE.

As expected from earlier studies of RTD-1 (184), the β-turns of retrocyclin-2 monomers were flexible, The structures can be found in the Brookhaven Protein Database (PDB), with the identifier "2ATG". The cystine disulfides (yellow) form the interface between the three monomers (colored red, turquoise and purple). The formation of trimers in LF's substrate binding groove could further impede access of its specific MAPKK substrate. Although the arginine residues of θ-defensins are already flexible, the flexibility of the backbone should increase their ability to explore surrounding space. This could improve the ability of θ-defensins to fit key functional sites in other metalloproteases, whose binding residues are spaced differently from those in LF.

Binding studies.

[107] Anthrax Lethal Factor. We examined the binding of retrocyclins to LF by immobilizing recombinant LF on a CM5 biosensor chip Retrocyclin-1 and RC112 (identical sequence, but composed exclusively of D-amino acids) bound LF with equally high (Kd 115-130 nM) affinity and with almost identical rate constants (Table 4a). Since RC112 was 76.4% less potent than retrocyclin-1 in inhibiting LF's enzymatic activity (Table 3), there is not a 1 :1 concordance between binding to the LF holoproteins and inhibiting its enzymatic activity. Neither the β-hairpin precursor of retrocyclin-1 nor a cyclic analog whose SS bonds had been reduced and alkylated showed much binding, indicating the importance of its cyclic backbone and tri-disulfide ladder.

Table 4a

Table 4B

[108] The affinity constant (KD) is calculated from the formula: KD = (k_Off) /(k_on). Surface plasmon resonance provides the values for these binding and dissociation rates. These binding constants are informative. For example, they show that RC107G (R9G-retrocyclin-1 ), binds ALF less than half as rapidly as RC100. Thus, arg9 facilitates initial docking. They also show that RC107G/ALF complexes dissociate over twice as rapidly (3.12/1.39=2.24) than complexes between ALF and RC-100. Thus, arg9 also allows RC100 to form more stable complexes with ALF. Altering arg9 &18 to gly 9&18 had little additional effect on docking, but the RC107GG/ALF complexes dissociated a bit more rapidly than ALF/RC107G complexes. Replacing both remaining arginines of RC107GG reduced the binding rate (K_0n) by an additional 81.3%, without further impairing the stability (K_Off) of the complexes RC107G₂H₂.

[109] Binding of these peptides to anthrax edema factor followed a somewhat different pattern. Arg 9 and 18 played no roles in initial docking to EF, since RC1 , RC107G and RC107GG had similar "on rates". However, arg9 and arg18 helped to stabilize the complex, since replacing one or both with glycine increased the dissociation rate. From RC107G₂H₂, we learned that arg 4 and 13 are critical for docking, but not for stabilizing the complexes. The tri- disulfide ladder does stabilize, for without its presence there is minimal binding to LF, EF, or to bovine serum albumin. Similar results were obtained when we performed the binding studies at pH 6.5.

[110] Binding to Edema Factor (EF). EF is a calmodulin-activated adenylyl cyclase without significant homology to the mammalian enzyme. It has an N-terminal, PA-binding domain followed by a calmodulin-activated adenyl cyclase domain. The four discrete regions of EF form a surface that recognizes an extended conformation of calmodulin, whose binding induces allosteric changes that cause enzymatic activation. After PA transports EF into a host cell, calmodulin activates its cyclase activity and intracellular cAMP levels increase to detrimental levels. Shen proposed a two metal catalytic mechanism. One metal, Mg⁺⁺, is near the 3'OH of ATP, and is coordinated by asp-491 , asp-493, and his-577. Another Mg⁺⁺ ion is coordinated by asp-493 and the nonbridging oxygens of all 3 ATP phosphates. In SPR experiments, retrocyclin- 1 (RC100 ) bound EF as well as LF. RC100(ox), the noncyclic precursor of RC100, showed much less binding. Removing RC100's SS bonds by reduction and alkylation (r&a) abolished its binding completely (Figure 2a).

[111] As documented above, the structural attributes of retrocyclins that enable them to recognize anthrax toxins include their cyclic backbone, tri-disulfide ladder, and at least three of their four conserved arginines. Figure 2b shows that retrocyclins bind all three anthrax toxins far more extensively than BSA.

[112] Inhibition of other bacterial metalloproteases. The ability of α and θ-defensins to inhibit three other secreted metalloproteases (MPs) is shown in Table 5. The MPs were botulinum neurotoxins (BoNTs)- A and B, and thermolysin. The BoNT substrate, SNAPtide" (CalBiochem, Cat. 567333)", contained the native cleavage site of SNAP-25 and FRET (fluorescence resonance-energy-transfer) prosthetic groups. The assays were done on a microplate spectrofluorimeter (Molecular Devices), with excitation at 326 nm and emission at 423 nm. [113] The proteolytic activity of BoNT-A was inhibited more effectively by 7.5 μM HNP 1 -3 than by 1 1.2 μM AC-CRATKML-NH₂ its end-blocked, competitive inhibitor, θ-defensins (RTD3, RC107GG, RC101 ) not particularly effective against anthrax LF showed considerable ability to inactivate BoNT-A. In contrast, θ-defensins that were good inhibitors of LF ( retrocyclins 1 and 2 and RTD 1 and 2), lacked significant inhibitory effect on BoNT-A. In other experiments, we found that none of the α or θ-defensins inhibited trypsin nor chymotrypsin (both serine proteases), nor did they inhibit bovine pancreatic arboxypeptidase B, a zinc-metalloprotease structurally unrelated to anthrax LF. This exquisite selectivity indicates that the inhibitory effects of theta-defensins on these microbial toxins are highly specific.

[114] Inhibitory effects on botulinum neurotoxins and thermolysin Defensins were tested at 25 μg/ml. The BoNT substrate was"SNAPtide" (Calbiochem, Cat. 567333), which contained the native cleavage site of SNAP-25 and FRET fluorophors). Assays were done on a microplate spectrofluorimeter (Molecular Devices), with excitation at 326 nm and emission at 423 nm. Ac- CRATKML-N H2 is an end-protected heptapeptide that is a competitive inhibitor of the proteolytic activity of Type A botulinum neurotoxin. It was purchased from Calbiochem and used at 11.2 μM. Phosphoramidon (N-alpha-L-rhamnopyranosyloxy(hydroxyphosphinyl)-L-Leu-L-Trp) , a selective metalloprotease inhibitor, was used at 50 μg/ml (-86 μM). Phosphoramidon also inhibits P. aeruginosa elastase (a zinc metalloprotease) and metalloproteases secreted by L pneumophila, C. perfringens (l-toxin), V. vulnificus, and L. monocytogenes.

Table 5. Inhibitory effects on Botulinum neurotoxins and Thermolysin (TL).

Cell-based assays for antitoxin activity.

[115] Theta defensins protect RA W 264.7 from lethal toxin Anthrax LeTx treatment of murine RAW 264.7 macrophage-like cells results in a rapid lytic response that occurs within two hours. Thus, RAW 264.7 intoxication affords a convenient in vitro bioassay for toxin internalization and activity as well as for testing putative antitoxins.

[116] Using LeTx challenge of RAW 264.7 cells, we tested 18 defensin-like peptides for their ability to inhibit the intoxication process. RAW 264.7 cells were resuspended at 1 x 10⁵ cells/ml in Dulbecco's MEM (DMEM) containing 25 mM HEPES buffer, 10% fetal bovine serum (FBS) and supplemented with penicillin, streptomycin, and glutamine (ps/g), then dispensed into 384- well tissue culture plates at 40 μl per well. After overnight culture, the medium was replaced with DMEM supplemented with 1 % FBS and ps/g. In addition, some wells received defensins (0.3 - 30 μM), while others received an equivalent volume of buffer. Next, cells were exposed to 100 ng/ml PA and 100 ng/ml LF (final concentration) and incubated overnight at 37^°C, 5% CO2. The following day, medium was removed and cell viability was estimated by measuring total cellular ATP using the CellTiter-Glo Luminescent Cell Viability kit (Promega). Results from this assay were validated in parallel assays by measuring mitochondrial dehydrogenase activity (Wst-1 reagent, Roche) and by microscopic observation of cell number (data not shown). Interestingly, a number of the theta defensins provided substantial protection to the host cells and displayed no cytotoxicity at concentrations as high as 60 μg/ml as demonstrated by cell viability equivalent to the control. Consistent with a role for arginine residues, the RC107GG peptide showed poor protection at 20 μg/ml, and required higher concentrations to block LeTx-mediated cytotoxicity (60 μg/ml). RC107G₂H₂ was not yet available when these assays were performed. As a positive control we used a known antitoxin, the soluble ectodomain of ANTXR1 (sANTXRI ). Consistent with previous reports, full protection from LeTx was seen at the lowest sANTXRI concentration we tested, 740 ng/ml.

[117] To learn how effectively theta defensins protect against LeTx, we determined the IC₅₀ values for the most potent retrocyclin peptides in the presence of a constant (and high) amount of toxin. RAW 264.7 cells were seeded on 384-well plates as described above. Peptides were added at 6-9 different concentrations per peptide, followed by the toxin (100 ng/ml each PA and LF). As a positive control, we assayed the three alpha defensins (HNP 1-3) previously reported by Kim et a/ to show protection in a similar assay. Additionally, we assayed several other alpha- and beta-defensins. Theta defensins were found to protect RAW 264.7 cells with similar molar potencies as HNP 1-3 (Table 6). Interestingly, we observed protection with HNPs at concentrations equivalent to those reported by Kim and colleagues had used 16-fold and 4-fold more PA and LF respectively. A human beta-defensin (HBD3), human alpha-defensins HNP4, HD5 and HD6 and a conotoxin (a mollusk-derived defensin-like peptide) showed no protective effects, supporting the conclusion that antitoxin activity does not occur simply as a result of nonspecific charge or peptide concentration effects (Kim et al).

[118] Having determined that retrocyclins, like HNP's, protect RAW 264.7 cells from cytolysis, we next wished to determine which intoxication step(s) were blocked by these defensins. In order to cause cytolysis, PA must first bind to cell-surface exposed ANTXRs, be processed by a furin-like protease and oligomerize into a heptamer. This PA heptamer must then bind LF, re- localize to lipid rafts and be endocytosed.

Table 6 IC IC

[119] Trafficking of the toxin complex to an acidic endosome triggers a conformational change in the PA heptamer that results in formation of a pore in the endosomal membrane. Finally, LF must translocate through this PA-pore and attack its cytosolic targets, which include MEK/MKK molecules. Conveniently, assays exist for each of the steps described above, thereby allowing for identification of specific processes affected by antitoxins. Because retrocyclins bind both PA and LF, several intoxication steps may be targeted simultaneously. However, we predicted that the earliest block to intoxication would contribute significantly to protection under standard conditions.

[120] Because defensins bind PA, we hypothesized that they might protect cells from LeTx in part by preventing the initial binding of toxin to cells. To test this, RAW 264.7 cells were incubated with a fluorescently-labeled version of PA in which amino acid 733 was mutated to cysteine and subsequently labeled with AlexaFluor 647- maleimide (PA-E733C-AF647) (7). PA binding assays were performed in the presence or absence of defined retrocyclins or alpha defensins. As a control, cells from a mutant cell line derived from RAW 264.7 that lacks ANTXR expression (R3D line) were incubated with PA-E733C-AF647. PA-E733C-AF647 binding was then assessed by flow cytometry. Surprisingly, the presence of retrocyclins resulted in an increase of PA bound to both WT RAW 264.7 and mutant R3D cells as determined by an increase in the geometric mean fluorescence associated with both cell types (Figure 3). This increase in cell surface binding was receptor independent, as a similar fluorescence value was seen with WT and R3D cells (a value ~10-fold higher than that for receptor-mediated binding to WT RAW 264.7 cells).

[121] Theta defensins block anthrax toxin entry. Defensins may misdirect PA to bind in a nonproductive manner to cell surface components other than its receptors. We recently reported that a single retrocylin molecule has several carbohydrate binding sites, and show above how this intrinsic multivalency might be smplified by their ability to form oligomers. Attaching PA to cell surface acceptor molecules that do not allow its entry, might afford partial protection by reducing the amount of free PA available to bind to its specific receptors. We performed binding assays using SPR to see if retrocyclins blocked specific PA-ANTXR1 interactions. In these experiments (Figure 3c), soluble versions of the ANTXR-1 or -2 ectodomains were biotinylated and bound to a streptavidin-coated sensor chip (Biacore). RC100 (1 μg/ml) and/or PA (100 μg/ml) were then introduced into the flow cells and the change in response units was recorded. The results show that combined binding was additive, suggesting that retrocyclins do not block the binding of PA to its specific receptors. Whether retrocyclins affect subsequent transfer of receptor-bound PA to lipid rafts or prevent internalization from these rafts will be examined by confocal microscopy, using BODIPY-labeled retrocyclin and Alexafluor labeled PA.

[122] We wished to determine if the increased PA on cell surfaces following coincubation with defensins was proteolytically-processed to form PA₆₃ and, if so, if it oligomerized appropriately. To test this, we incubated RAW 264.7 cells with 500 ng/ml PA and 20 μg/ml defensin for one hour at 4^°C to allow for binding, then for 1h at 37°C to allow for oligomerization and internalization. The conformational transition associated with forming a membrane-inserted pore forms a heptamerthat resists solubilization by SDS. Therefore, cells were lysed and equivalent amounts of total protein from each lysate were incubated for 20 min in SDS-containing buffer to dissociate the pre-pore form of PA heptamer. The samples were analyzed by SDS-PAGE₁ and by Western blot using a polyclonal antiserum to PA. Neither α nor θ- defensins blocked PA₆3 formation completely, indicating that at least some cell surface-PA had been correctly processed by a furin-like enzyme. However, co-incubation with θ-defensins decreased the amount of SDS- resistant PA heptamer present, indicating that these defensins may block toxin entry at or before pore formation. In contrast, co-incubation with α-defensins had no effect on the amount of PA- heptamer, indicating that α-defensins block LeTx at a different step. Several other host-defense properties of α-and θ-defensins also differ. For example, whereas α-defensins act after viral entry, θ-defensins prevent it. These mechanistic distinctions imply that combining α- and θ- defensins could provide more protection against LeTx than using either alone.

Example 3

How retrocyclins kill B. anthracis

[123] Binding to Bacillus spores and vegetative bacteria. We used ¹⁴C-labeled retrocycIin-2 (RC-2) to examine binding to B. anthracis (Sterne). The bacteria were grown overnight at 37⁰C in trypticase soy broth (TSB), then diluted 1 :100 into fresh TSB and subcultured for 3h at 37 oC to mid-log phase. The bacteria were washed twice with PBS (10 mM NaPB, 100 mM NaCI, pH 7.4). Then, 10⁷ cfu in 100 μL of PBS were placed in a conical microcentrifuge tube and incubated with 1 μg/ml ¹⁴C RC2 at 4°C for 30 minutes. Next, 900 μL of cold incubation buffer was added, followed by 200 μl of a 2:1 mixture of dibutylphthalate and diisodecyl-phthalate esters (Pflatz & Bauer Chemicals). The mixture had a density of 1.02, was immiscible with water, and formed a cushion below the aqueous layer. Centrifugation at 2940 x g deposited the bacteria beneath the phthalate cushion. After removing the supernatant and some of the phthalate ester layer, the bottom of the tube was severed, placed into a counting vial of Ready SafeTM liquid scintillation fluid (Beckman Coulter ) and counted in a liquid scintillation spectrometer. From these counts and the specific activity of the 14C-RC2, we found that each B. anthracis CFU bound, on average, 166,000 molecules of RC-2. In a similar study with mid-log Bacillus atrophaeus, on average, each CFU bound 61 ,200 molecules of RC-2. When we exposed 10⁷ spores of S. atrophaeus (also called B. subtilis var. niger) to 1 μg/ml of RC-2, each spore bound, on average, 23, 100 RC2 molecules. The B. atrophaeus spores used in this experiment were purchased from NAMSA (Northwood, OH) as a SPORTROL® spore suspension. B. anthracis spores have not yet been tested.

[124] Killing kinetics. Whereas exposure to 2.5 μg/ml PG-1 kills >99% of B. anthracis (Sterne) within 30 seconds, RC2 kills B. anthracis more gradually, in a concentration-dependent manner. In this (and many other) respects, PG-1 behaved more like rabbit defensins NP-1&2, while retrocyclins behave more like rabbit defensin NP-5 and human defensins HNP 1-3. Thus, although PG-1 and RC-2 showed similar potency in our radial diffusion assays, their functional convergence masked significant kinetic differences. This provides strong justification for using multiple techniques to determine how retrocyclins kill vegetative B. anthracis and how they prevent successful germination of their spores.

Table 7

Inhibition of Anthrax Lethal Factor (ALF) by retrocyclins. All peptides were tested at 5μg/ml

(around 2.5 μM) .

[125] Recombinant Anthrax Lethal Factor (ALF) and its substrate, a fluorogenic 19-mer peptide containing the MAPKK motif was purchased from Calbiochem (Cat# 176900 and #176902). ALF and theta-defensins were preincubated at room temperature for 30 minutes. Assay was carried out in 10 mM HEPES, 150 mM NaCI, 1 mM Ca2+, pH 7.4, with substrate added last. Fluorescence was monitored on a fmax microplate reader (Molecular Device), with excitation at 355 nm and emission at 460 nm. The final concentration of ALF was 100 nM, and the the substrated was 50μM. Vmax was calculated from the first 10 minutes, and used to calculated % inhibition. Table 8.

[126] Peptides were tested at 25 μg/ml, which corresponds to -12.5 μM for theta defensins and -7-8 μM for α-defensins. Clostridium botulinum neurotoxin A (BoNT A) was purchased from List Biological Laboratories, Inc. Clostridium botulinum neurotoxin B (BoNT B) and fluorogenic SNAPtide substrate were purchased from Calbiochem. Peptide and Botox was preincubated at room temperature for 30 min. Substrate was added at last. The assay was done on an fmax microplate reader (Molecular Devices), with excitation at 326 nm and emission at 423 nm. The final concentration of Toxin was1 nM/ml, and substrate was 40 nM/ml. The BoTox inhibitor is a cysteine containing, end-protected heptapeptide (Ac-Cys-Arg-Ala-Thr-Lys-Met-Leu-NH2) described as a selective, high-affinity (Ki = 2 μM), competitive inhibitor of the proteolytic activity of Type A botulinum neurotoxin. The inhibitor was purchased from Calbiochem and used at a final concentration of 11.6 μM.

Example 4

[127] To study the protective effects of HNP 1 -3, briefly, 7 to 8-week-old female BALB/c mice receive an IV injection of LeTx (50 μg LF+ 50 μg PA in 0.2 ml PBS) directly into a tail vein. Immediately thereafter, some LeTx-treated mice receive IV doses of the "test article" (initially, retrocyclin-2), others receive IV injections of HNP1-3 (positive controls) or PBS (untreated controls). An additional group of mice receive α- and θ-defensins without prior injection of LeTx (toxicity controls). 5 experimental groups, each consisting of 6 mice, 30 mice are required to test each peptide concentration. Before testing any peptide in this assay, we perform a dose- escalation study on no more than 10 mice (2 mice/dose), starting with a dose of 5 mg/kg and doubling this concentration sequentially until dose limiting toxicity is observed. Once the efficacy of retrocyclin-2 is established, it is used as the positive control and compared to other promising peptides as they are identified. Mouse survival is monitored for 10 days after toxin treatment.

Surviving animals are euthanized and autopsied for evidence of gross or microscopic lesions.

Spore-challenge model: Whereas the toxin-challenge model provides important information about the ability of defensins to inactivate LeTx in vivo, this model is artificial in that it requires simultaneous bolus injections of toxin and antitoxin. During an infection, toxin is constantly produced by S. anthracis, which itself is multiplying in the mouse. Defensins are unique among existing antitoxins or antibiotics in that a single defensin simultaneously targets both the bacilli that produce toxin and the toxin itself. In order to determine the efficacy of the overall protective effect of retrocylins (a combination of their antimicrobial and antitoxin activities), retrocyclins are tested in a spore-challenge model using the A/J strain of mice.

[128] Initial assays are performed using a toxigenic strain of B. anthracis, Sterne 7702, because: a) 7702 efficiently kills A/J mice; b) killing of mice by 7702 requires toxin production

(144). However, depending on the activity of defensins against capsulated strains, experiments may also be performed with a fully virulent, toxin- and capsule-producing strain such as Ames.

[129] Infections are performed using intraperitoneal (i.p.) injections of spores. Spores will be prepared using standard procedures and heat-treated to ensure the preparations are free from vegetative bacilli. Initial experiments will be performed to identify the LD₅₀ values of the system and assay endpoint (moribund state). Subsequent experiments to determine efficacy of retrocyclins are performed at 10 x LD₅₀ to ensure complete killing of control mice receiving no defensin. In these assays, groups of 6-10 mice receive spores followed immediately by injection of various concentrations of defensin via the tail vein. The range of defensin concentrations is based on results from toxicity studies and in vitro (RAW 264.7 protection) assays, and several concentrations per retrocyclins are tested. Mice are monitored for time to moribund state and/or percent survival.

Example 5

[130] Table 9 provides a description of the peptides used in the examples, by showing their names and the linear sequences of their synthetic precursors. In a circular peptide, residue numbering is arbitrary; residue #1 has been assigned to the N-terminal glycine of the synthetic precursor.

Table 9 Retrocyclins and rhesus theta defensins.

RC101 R9K-retrocyclin-1 GICRC ICGKG ICRCI CGR

RC107G R9G-retrocyclin-1 GICRC ICGGG ICGCI CGR

RC107GG R9,i8G-retrocyclin-1 GICRC ICGGG ICRCI CGG

RC107G₂H₂ R9.18G, R4.13H GICRC ICGGG ICRCI CGG

RCIOOb retrocyclin-2 GICRC ICGRR ICRCI CGR

RTD-1 rhesus θ-defensin-1 GFCRC LCRRG VCRCI CTR

RTD-2 rhesus θ-defensin-2 GVCRC LCRRG VCRCI CRR

RTD-3 rhesus θ-defensin-3 GFCRC ICTRG FCRCI CTR

[131] These peptides by solid phase peptide synthesis and purified to >95% purity as previously described. Their concentrations were determined by amino acid analysis. RC112 is composed exclusively of D-amino acids. The others shown in Table C1 contained only L-amino acids.

[132] Antimicrobial activity is measured in many ways, including colony counting, broth micro- dilution assays, radial diffusion assays, and virtual colony counting. Two stage radial diffusion assays are precise, reproducible and consume <4 μg of peptide per assay. The results shown in Figure 1 were obtained in two-stage assays done as follows.

[133] Stage 1 : 1-4 x 10⁶ colony forming units (CFU) were dispersed in a thin 1% agarose ("underlay") gel containing 10 mM phosphate buffer (pH 7.4), 100 mM NaCI, and nutrients (a 1 :100 dilution of trypticase soy broth) sufficient for several doublings. A regularly spaced, 6 X 6 array of wells (3 mm diameter, capacity 9 μl) was punched, allowing us to assay up to 6 peptides on each plate. Each set of 6 wells received 8 μl_ of a serially ("half-log") diluted peptide solution containing 250, 79, 25, 7.9, 2.5 or 0.79 μg of peptide/ml. The plate was incubated for 3h to allow the peptides to diffuse into the underlay gel.

H 341 Stage 2: A double-strength trypticase soy agar overlay gel was poured over the underlay gel, and the plate was incubated overnight to allow surviving bacteria to form micro- colonies. The clear zones around each well were measured 18-24h later. To determine the minimal effective concentration (MEC), we calculated the linear regression function relating the adjusted diameter (zone diameter minus the well diameter) to the log₁₀ peptide concentration. The X-intercept of the line defined by this function gives the MEC. Typically, the correlation coefficient (r²) of this line was >0.98. Figure C2a shows a detail from the assay plate shown in its entirety on the next page. The test organism was as labeled in Figure 4.

[135] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[136] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. The use of a retrocyclin or analog thereof in the treatment or prevention of anthrax infection or infection by a vancomycin resistant enterecoccus.

2. The use of a retrocyclin or analog thereof in the neutralization of an anthrax toxin.

3. The use according to Claim 1 or Claim 2, wherein said retrocyclin comprises a circular polypeptide sequence as set forth in the present application.

4. The use according to any one of Claims 1 -3, wherein said retrocyclin is a circular polypeptide comprising two independently selected nonamers as set forth in the consensus sequence:

5. The use according any one of Claims 1 -3, wherein said retrocyclin is two linked nonapeptides, wherein each nonapeptide sequence is independently selected from the group consisting of SEQ ID NO:19 to SEQ ID NO:64; SEQ ID NO:74 to SEQ ID NO:119; or SEQ ID NO:136.

6. The use according any one of Claims 4-5, wherein at least one of said nonamers comprises an arginine to lysine or lysine analog substitution at residue 6.