PEPTIDES

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application c l aims the benefit of priority of United States Provisional Patent Application No. 61/789,307, filed March 15, 2013, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with United States government support awarded by the National Institutes of Health grant numbers .1 P30RRQ3 1.54 and P30 GM 103450. The United. States may have certain rights in. this invention,

SEQUENCE LISTING

This application is being tiled electronically via EFS-Web and includes an electronically submitted Sequence Listing in .txt format. The ,t t file contains a sequence listing entitled "2014- 03-l.4_5965-00039_ST25.txt" created on March 14, 201 and is 5782 bytes in size. The

Sequence Listing contained, in this .txt file is pari of the specification and is hereby incorporated, by reference herein in its entirety.

INTRODUCTION

Fungi have emerged worldwide as an increasingly frequent cause of opportunistic infections. A survey of the epidemiology of sepsis in the United States reveals that the incidence of fungal sepsis increased three- fold between 1979 and 2000, Candida and Aspergillus spp. are the most frequent, causes of invasive fungal infections and are frequently associated with high morbidity and mortality. The rate of invasive candidiasis is 7 to 15-fold, higher than aspergillosis, i fact, Candida is the fourth leading microorganism responsible for bloodstream infections i the United States, in recent decades, there has been a shift in the epidemiology of Candida infections, characterized by a progressive move from the predominance of Candida albicans toward non~albicam Candida spp. such as Candida giahrata and Candida krusesi. in tact, C. giahrata now accounts for 15% to 20% of Candida infections in most countries. in intensive care unit patients, the most common, types of Candida infections are bloodstream infections, catheter-related infections, intra-abdominal infections and urinary tract infections. Invasive candidiasis is a leading cause of morbidity and mortality in both

immunocompromised and immunocompetent critically ill patients with a mortality rate between 20% and 40%. The current antifungal chemotherapies are predominantly three major groups of compounds: the polyenes, the azotes, and the echinocandins. Some strains of Candida have acquired resistance lo the azo es while other Candida species, such as C. giabrat or C. k sei are not azole-sensitive at typical therapeutic concentrations. In contrast, the polyenes (e.g. amphotericin B) remain highly effective; however, drug toxicity has limited its usage in systemic infections. The echinocandins have pro ven useful, but resistance to these drugs is also observed via mutations in the FKSJ gene. Thus, there is an intensive effort to identify new antifungals that would be effective against a broad range of Candida -s e ie as well as other pathogenic fungi.

SUMMARY

Novel antimicrobial and in particular anti-fungal peptide compositions are provided herein. The compositions include a five amino acid peptide o SEQ ID NO: I fW/F/Y- - R- K- F/YAV). The peptides are antifungal when produced using either D or L amino acids and has similar antifungal activity when made in a retro or reverse form. Anti-fungal activity was increased when the peptide was dimerized via a cysteine added to either the or C terminal end of the peptide. The peptide ma be dimerized or circularized through disulpliide bonds between the cysteine residues. The peptides may also be circularized via other methods such as an amide bond between the N~ and C Terminal end. The N-termkal amino acid can be substituted with a methionine or a methionine residue can be added to the N-terminus to facilitate cireuiarizatton. The peptides may also be produced as a nine or ten amino acid duplicated peptide (i.e. F/Y - - - K~ F/Y- F Y- - R- K- F/Y (SEQ ID NO: 6) or F Y- K~ R~ K~ F Y- K- R- K- F/Y (SEQ ID NO: 7)).

The peptides have anii-fungat activity both m vitro and in vivo. In one aspect, the compositions may be used in methods of inhibiting microbial infections or microbial

contamination. The compositions comprising the peptides described herein may be administered to a subject in need of treatment for a microbial, infection, and may inhibit the growth, of the microbe, prevent further spread of the microbe or kill the microbe and cure or stop the infection. In another aspect, the compositions may be applied to a surface such, as a counter/top, a food item or a food preparation surface to reduce die chance of microbial or in particular fungal infection, in yet another aspect, the compositions may be added to a liquid such as a drink or media for growing cells to inhibit microbial growth, in particular to inhibit fungal growth,. In a still, further aspect, methods o inhibiting microbial growth by contacting cells with the compositions described herein to prevent microbial growth in, on or with the cells are provided.

BRIEF [DESCRIPTION OF THE DRAWINGS

Figure 1 is a. schematic depiction, of the sequence of the histatin 5 16mer derivative (W; SEQ ID NO: 8) and die retro-histatm 5 16 rae (R; SEQ ID NO; 9). The same peptides were generated using both L~ammo acids (W and R) and D-ammo acids (WD and RD), respectively.

Figure .2 is. a graph and table showing the antifungal activity of the hi statin 5 1 mer derivatives. Figure 2A shows the percent viability of Candida albicans SC53 14 following exposure to increasing concentrations of the four histatin 5 I6mer peptide derivatives. Figure 2B is a table summarizing the dose of peptide required to achieve 50% killing activity (LD¾ of C. albicans SC531.4 with each, of the peptides. The W, WD, and RD designates the wild-type (W) or retro- (R) histatin 5 16mer as shown in Figure I , with L- or D-amino acids, respectively.

Figure 3 is a set of graphs showing the fungal killing activity of histatin 5 16mer derivatives in. the absence of respiratory activity. Figure 3A shows the percent viability of Candida albicans SC5314 following exposure to the four histatin 5 peptide derivatives in the absence or presence of sodium azide (NaNj). As controls, samples were exposed to sodium, phosphate (NaPB) or sodium azide (NaN¾) alone; sodium chloride (NaC!) was used to balance the overall salt concentration in each reaction. Figure 3B shows the percent viability following exposure of Candida albicans SC53 I4 to the four histatin 5 peptide derivatives in the absence or presence of antimycin A. (AMA). As controls, cells were incubated in NaPB ethanol (EtOH) or NaPB + ethanol - AMA. Ethanol was the solvent tor AMA; hence, it was adjusted to the same concentration in all reactions. The W. WD, 1 and RD designates the wild-type (W) or retro- (R) histatin 5 I mer as shown in. Figure 1 , with L- or D-amino acids, respectively.

Figure 4 is a graph showing that the secondary structure of the wild-type and retro- histatin 5 16mer peptides are similar- CD spectra were obtained on the wild-type histatin 5 I6raer derivative (W) and the retro-bistat 5 derivative in the presence of increasing concentrations of trifluoroethanol (%TFE).

Figure 5 is a graph showing the results of liposome fluorescence leakage assays. The four hi statin 5 peptide derivatives at concentrations of ΙμΜ had comparable permeabilmng effects on artificial liposomes. The percentage of fluorescent leakage from liposomes in -seconds ($) is compared to total leakage obtained with Triton X-100 at time 800s. Maximal fluorescent intensities were in the range of 80,000-220,000 counts per second. As a control, liposomes were examined in sodium phosphate buffer (NaPB) only without peptide.

Figure 6 is a set of graphs showing the determination of the LD<o for KM-5. Figure 6A is a dose dependent assay. Candida albicans SC5314 ceils were exposed to increasing concentrations of K.M5 for 2 hours at 37 ^k! C. Cells were subsequently plated and grown on Sabouraud dextrose agar at 37°C and colony counts were subsequently performed and compared to ceils not exposed to the peptide. Figure 6B shows the LDs» calculated using a linear regression equation ,

Figure 7 is a graph showing the kinetics of antifungal activity with the KM-5 peptide. Time-dependent killing of Candida albicans SC5314 was evaluated by incubating cells with 10 μΜ peptide at 37°C for various periods of time. The percentage of viable cells was calculated as (viable colonies in the presence of peptide / viable colonies without peptide) x 100. Data represents three independent experiments and the error bar .represents the standard deviations.

Figure 8 is a. set of graphs showing a comparison of the activity of KM-6 and the hisiatin 5 1 mer peptides. In Figure 8A the killing activity of the histati 5 16mer peptide (C-l 6) versus KM5 and KM6 are shown using 25 μΜ of each peptide incubated with Candida albicans SC531.4 in 10 mM Sodium phosphate buffer for 2 hours at 37°C. The percentage of viable cells was calculated as (viable colonies in the presence of peptide / viable colonies without -peptide) ^· * 100. Data represents three independent experiments and the error bar represents the standard deviations. Figure SB shows a liposome leakage assay in whic ΙμΜ of each peptide was incubated with artificial liposome and release of acridine orange was monitored for 24 minutes, then Triton I 00 was added to achieve 100% liposome lysis. Liposomes were incubated with sodium phosphate (NaPB) alone as a control Figure 9 is a schematic diagram o the different KM5 derivativ peptides that, were synthesized and subsequently dimerixed via cysteine disulfide bonding. The disulfide bond is indicated with a thick line.

Figure 10 is a graph showing a direct comparison of th relative fungicidal activity of KM 12, KMI4, KM 16, and KM 17 against Candida albicans SC531.4. The peptides differ in structure as shown in Figure 9, yet all four peptides kill >90% of C, albicans at 5 μΜ concentration.

Figure 1.1 is a graph showing the toxicity of the KM! 2 peptide to N1H3T3 mammalian celts. Cells were incubated in culture with the KM 12 peptide at the concentrations indicated. After 48 hours, the WST1 cell proliferation assay was performed to evaluate cell viability. No loss in viability was noted. The assay was performed three times independently and the average is shown with error bars indicating the standard error.

Figure 12 is a graph showing hemolysis of red blood cells by KM5. Sheep red blood cells were incubated with the indicated concentrations of M5 or retro KMS (reverse sequence order) for 1 hour at 3?*C. The percentage of hemolysis was subsequently determined by the absorhance at 405 nm as outlined in the Methods section. The assay was performed three times independently and the average is show with the error bars indicating the standard error.

Figure 13 is a graph showing hemolysis of red blood cells by KM 12, KM 14, K 23, and KM29. Sheep red blood cells were incubated with the indicated concentrations of the peptides for 1 hour at 37 ^"a C. The percentage of hemolysis was subsequently determined by the absorhance at 405 nm as outlined in the Methods section. The assay was performed three times independently and the average is show wit the error bars indicating the standard error,

DETAILED DESCRIPTION

A novel, answe to the treatment of either, mucosal or systemic fungal infections can. be found in therapies thai involve the use of small peptides that display fungicidal activity.

Classically, the histatins are a family of naturally occurring peptides secreted into the oral cavity of primates, and some of the histatin peptides have antifungal activity. The predominant human, histatins are I: , 3, and 5, with histatin 5 being the most potent antifungal.

The Examples provided herein define the minimal functional region of histatin 5 that displays significant antifungal activity. Once that region was defined, the derived information was used to generate peptide variants to enhance th fungicidal activity of the peptide. The goal was to generate the smallest peptide that would display the most potent fungicidal activity; thereby .generating a potential antifungal therapeutic agent that would be economically feasible to produce. Through these studies, a five amino acid peptide is defined that maintains reasonable antifungal activity. Also provided are derivatives of the 5mer peptide. Some of these additional variants have significantly improved fungicidal activity. The activity of these peptides appears to have broad specificity among Candida species.

The invention features substantially pure peptides such as those provided i Table below. The peptides provided herein contain a core region of 5 amino acids designated 1-R2- R3-R4-R5 where Rl is phenylalanine, tryptophan or tyrosine, R2 is lysine, 3 is arginirse, R4 is lysine, and R5 is tyrosine, tryptophan or phenylalanine (SEQ I D NO; 1). The spatial, position of the amino acids relative to each other is critical, for antifungal activity with the .relative positioning R1-.R2-R3-R4-R5 (such as SEQ ID NO: 2); however, the reverse order R5-R4-R3- R2- 1 is equally effective with regards to its antifungal, activity, in addition., the D-amino acid enantioraers were equally active in antifungal activity (SEQ ID NO: 10). The peptides used herein are synthetically made, but those of skill, in the art will appreciaie that such peptides could be made using other means such as via genetic engineering.

Table 1: Summary of the antifungal peptides described in this application.

i pri. Se-iueuc- ^: i S ! o l i )

KM 5 F-K.-R-K-Y (SEQ ID NO; 2)

KM 6

K l ! C-F-K-R-K-Y (SEQ IP NO: 5)

KM 12 Y- -R-K-F-C— -C-F- ~R-K-Y (SEQ ID NO: 17)

KM 13 C-W-K-R-K-W (SEQ IP NO: 18)

KM 1 W-K-R-K-W-C C-W- -R-K-W (SEQ ID NO: 21 )

M: 15 F-K-C-R- -Y (SEQ ID NO: 22)

KM 16 F-K-C~R-K~Y (disulfide linked via Cys shown in hold)

E-K-C-R-K-Y (SEQ IP NO: 22)

KM 17 C-F-K-R-K-Y-C (disulfide linked via Cys shown to form a circular

peptide), (SEQ ID NO: 23) ^*

KM23 Y'-K-R-K-F-F-K-R-K-Y (SEQ ID NO: 3)

KM2 Y-K.-R-K-F-K-R-K-Y (SEQ ID NO: 4)

a The amino acid composition of each peptide is indicated by standard single letter designation.

h The inclusion of D-amin acids is indicated by a "d" presiding the amino acid single letter designation; otherwise they are assumed to be L-amino acids.

* Disulfide bonds are indicated by f— -) between cysteines. The addition, of an N-tennmal cysteine (designated C) to the 5mer peptide (i.e. C~ 1-R2~ R3-R4-.R5; such as shown in SEQ ID NO: 5, 1.6, 18, 20 and 23) does not negatively influence antifungal activity, A cysteine can also he added to the C-tenninus of the peptide (such as in SEQ ID NO: 15 and 1 ). The cysteine may be used to generate a 5mer peptide dimer (i.e. R5- R.4-R3-R2-R1 -C-C-R1 -R2-R3-R4-RS; such as SEQ ID NO: 17 or 21) that was shown to have substantially higher antifungal activity than the 5mer peptide. Repositioning of the cysteine residue (designated C) to the middle of the peptide (i.e. R1 ~R2-€-R3~R4-R5) to facilitate an alternative dimer structure maintained significant antifungal activity, in addition, cysteines (designated C) added to both the N - and C-iemiini (i.e. C- 1 -R2-R3-R4-R5-C; such as in SEQ ID NO; 23) of the peptide to facilitate circularization also maintained significant antifungal activity.

In the final versions of the peptides, the cysteine residues have been, removed and a IOmer peptide with the sequence R5-R4-R3-R2-RI -RI-R2-R3-R4-R5 (such as SEQ ID NO: 3 and 6) was generated. This peptide was as effective in fungal killing as the peptide dim.er.ized via cysteines. More importantly, the possible concern related to the stability of the cysteine disulfide bonds was eliminated. Since the R1 amino acid was repeated at the center of the peptide, one additional 9 amino acid peptide with the sequence R5-R4-R3-R2-R1 -R2-R3-R4-R5 (such as SEQ I ^' D NO: 4 and ?) was generated, This peptide was found to be equivalent to the IOmer peptide in terms of antifungal activity.

Suitably the peptides used in the compositions provided herein are 5, 6, 7, 8, 9, 10, ^" I 1 or even .12 amino acids long. As described above more than one peptide {the same peptide, different peptides or inverted peptides) may be joined together via di sulphide bonds between cysteine amino acid residues or via an amide linkage between the N- and C-temimus. A methionine can be added to the -terminus of each of the peptides described herein or can be used to replace the N-terminal amino acid of the peptides provided herein. Peptides may also be circularised or dmierized using any other means known to those of skill in the .art. Addition of a methionine to the N-termirius of the peptides provided herein can be used as a target to generate a circularized peptide using the method of Tam and X.u (Biopoiymers ( 1998) Methionine ligation strategy in the biomimetic synthesis of parathyroid hormones 46: 319-329). For example, a. methionine can be added to the N-teroiinus of a peptide such as SEQ D NO: 3, 4, 6, or 7. Such an addition may aid in expression, .modification or circularization of the peptide. Alternatively, a methionine can be used to replace the current N -terminal amino acid of one of the peptides provided herein, such as SEQ ID NO: 3, 4, 6 or 7, In addition, the C~termsnal amino acid can be replaced with, a threonine or a threonine could be added to the end of the peptides provided herein. The addition or substitution of a threonine at the C-termtnus of the peptides may allow for increased expression in a recombinant model, easier .modification, of the peptides or increased activity of the peptides or to allow circularization of the peptides. The peptides may include one or more non-natural amino acids. Suitably the peptide is not the native peptide of SEQ I D NO: 8 or 24.

The peptides may have substituents bonded to either terminus of the peptide. For example, the peptide may have an acetyl or a ca.rba.myl addition, at the N-tenninus, and or an amide addition at the C-terminus* In. addition, the peptides may be multimerized beyond a dimer. or circularized using standard chemistry to provide pharmacological, stability for antifungal treatment. The multirners may contain more than one copy of one of the peptides disclosed herein or may contain inverse copies of a single peptide or more than one of the peptides disclosed herein. Those of skill in the art will appreciate that various additional modifications of the peptides provided herein may be made to increase the stability or half-life of the peptides in culture or in the subject after administration. For example fatty acids or other modifications may be added to the N-tcrminus including but not limited to ormylation, myristoylation, or FBGylation. The peptide may be attached to a carrier protein to increase the stability of the peptide. The carrier proiein-peptide ma be a fusion protein and may be expressed as a recombinant protein using techniques available to those of skill in the art. The peptide bonds connecting the amino acids of the peptide may be altered or at least one peptide bond may be altered to make the peptides more resistant to degradation, for example a methyl group could be added. The amino acids could be replaced with functionally related non-natural amino acid that share similar side chains to the natural amino acid, such as replacement of the cysteine with homocysteine or a-raethyl-cysteine. Alternatively peptoids based on the peptides provided herein could be generated. These and other peptidomimetics are expected to have similar antifungal activity to the peptides described herein.

The peptides described herein have potent antifungal properties. Several different Candida species have been shown to be susceptible to these peptides. The toxicity of the peptides has been examined, both, in vitro and in vivo {mice). The immunogenicity of the peptides has also been examined in mice and the peptides were only mildly immunogenic. Several peptides were also shown to not induce hemolysis of red blood cells.

The peptides ma be used in methods for treating microbial, infections, suitably fungal infections and potentially also bacterial infections. The methods include administering an effective amount of a peptide containing composition such as those described herein to a subject. The administration of the composition is effective to limit the spread of the microbial infection, inhibit the growth of the microbe or kill the microbe. Suitably, the microbe is a fungus or yeast and includes but is not limited to Candida spp,, Aspergillus spp., EiUoplasma spp., and

Crypiococcw spp. in the Examples, the peptides are shown to have broad effectiveness against a variety of Candida species. Suitable subjects include humans, domesticated animals, and other non-human mammals. The compositions may he provided to subjects who are

immunocompromised and may be effective in such subjects.

The peptide compositions may be used to make pharmaceutical compositions.

Pharmaceutical compositions comprising the peptides described herein and a pharmaceutically acceptable carrier are provided. A pharmaceutically acceptable carrier is any carrier suitable for in vivo administration. Examples of pharmaceutically acceptable carriers suitable for use in the composition include, but are not limited to, water, buffered solutions, glucose solutions, oil- based or bacterial culture fluids. Additional components of the compositions may suitably include, for example, excipients such as stabilizers, preservatives, diluents, emulsifiers and lubricants. Examples of pharmaceutically acceptable carriers or diluents include stabilizers such as carbohydrates (e.g., sorbitol, mannitol, starch, sucrose, glucose, dextran), proteins such as albumin or casein, proiem-contamtng agents such as bovine serum or skimmed milk and buffers (e.g., phosphate buffer). Especially when such, stabilizers are added to the compositions, the composition is suitable for freexe-drying or spray-drying. The composition may also be emulsified.

The peptide compositions provided herein may be administered in conjunction with other anti-mierobiais to treat a subject. The compositions may be administered m any order, at the same time or as part of a unitary composition. The peptide compositions provided herein, may be administered with a second pharmaceutical such that one is administered before the other with a difference in administration time of 1. hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 4 days, 7 days, .2 weeks, 4 weeks or more. An effective amount or a therapeutically effective amount as used herein means the amoun of a composition thai, when administered to a subject for treating a disease or infection, is sufficient to effect a treatment (as defined above). The therapeutically effective amount will vary depending on the composition, formulation of the composition, the disease and its severity and the age, weight, physical condition and responsiveness of the subject to be treated. The administration of the compositions may be effective to limit the spread of the microbial infection, inhibit the growth of the microbe or even kill the microbe.

The compositions described herein may be administered by any means known to those skilled in the art, including, but not limited, to, oral, topical intranasal, intraperitoneal, parenteral, intravenous, intramuscular, subcutaneous, intrathecal, tran cutaneous, nasopharyngeal, or transmucosal absorption. Thus the compositions may be formulated as an mgestable, injectable, topical or suppository formulation. The compositions may also be delivered with in a liposomal, or time-release vehicle. Administration of the compositions to a subject in accordance with the invention appear to exhibit beneficial effects in a dose-dependent manner.. Thus, within broad limits, administration of larger quantities of the compositions is expected to achieve increased beneficial biological effects than administration of a smaller amount. Moreover, efficacy is also contemplated at dosages below the level at which toxicity is seen.

It will be appreciated that the specific dosage administered in any given ease will be adjusted in accordance with the compositions and formulations being administered, the route of administration, the disease to be treated or Inhibited, the condition of the subject, and other relevant medical factors thai may modify the activity of the composition or the response of the subject, as is well known by those skilled in the art. For example, the specific dose for a particular subject depends on age, body weight general state of health, diet, the timing and mode of administration, the rate of excretion, medicaments used in combination and the severity of the particular disorder to which the therapy is applied. Dosages for a given patient can. be determined using conventional considerations, e.g., by customar comparison of the differential activities of the compositions of the invention and. of a known agent such as a polyene or azole, such as by means of an appropriate conventional pharmacological or prophylactic protocol.

The maximal dosage for a subject is the highest dosage that does not cause undesirable or intolerable side effects. As shown in the examples KM 12 was well-tolerated in mice at dosages up to iOrog/kg when, injected via intravenous, intramuscular or intraperitoneal route. The number of variables in regard to an indi vidual treatment regimen is large, and a considerable range of doses i s expected. The route of administration will also impact the dosage

requirements. It is anticipated that dosages of the composition will, reduce symptoms of the infection at least 10% 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to pre- treatment symptoms or symptoms if left untreated, li is specifically contemplated that pharmaceutical preparations and compositions may palliate or alleviate symptoms of the disease without providing a cure, or, in some embodiments, may be used to cure the disease.

Suitable effective dosage amounts for administering the compositions may be determined by those of skill in the art, but typically range from about I microgram to about 100 milligrams per kilogram of body weight weekly, although they are typically about 1,000 micrograms or less per kilogram of body weight weekly. In some embodiments, the effective dosage amount ranges from about 10 to about 10,000 micrograms per kilogram of body weight weekly. In another embodiment, the effective dosage amount ranges from about 50 to about 5,000 micrograms per kilogram of body weight weekly . In another embodiment, the effecti ve dosage amount ranges from about 75 to about 1 ,000 micrograms per kilogram of body weight weekly. The effective dosage amounts described herein refer to total amounts administered, that is, if more than one composition or more than one dose per week is administered, the effective dosage amounts correspond to the total amount administered. The composition ca be administered as a single dose or as divided doses. For example, the composition may be administered two or more times separated, by 4 hours, 6 hours, 8 hours, 12 hours, a day, two days, three days, four days, one week, two weeks, or by three or more weeks.

Methods of inhibiting microbial growth and in particular fungal growth are also provided herein. The methods include applying the composition to an object, such as a food item, surface or a liquid, including a cell culture medium, in an amount effective to prevent or limit microbial growth or contamination. The methods may als include contacting cells or ceil culture fluid with the compositions provided herein. The method inhibits microbial or fungal growth in, on or with the cells or the cell culture fluid. The application or contact with the compositions provided herein may be effective to limit the spread of the microbe, inhibit continued growth of the microbe or even kill the microbe. Thus, the methods can be used in a preventative means or may be used to deal with and clean up an. active microbial contamination. The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The use of any and ail examples, or exemplary language (e.g., "such as" } provided herein, is intended mereiy to facilitate the disclosure and does not imply an limitation on the scope of the disclosure unless otherwise claimed.: No language in the specification, and no structures shown in the drawings, should be construed as indicatin that any non-claimed element is essential to the practice of the disclosed subject matter. The use of the terms "including," "comprising," or "having," and variations thereof, is meant, to encompass the elements listed thereafter and equivalents thereof, as well as additional elements. Embodiments recited as "including," "comprising," or "having" certain elements are also contemplated as "consisting essentially of and "consisting of those certain elements. The terms "a", "an" and "the" may mean one or more than one unless specifically delineated. Recitatio of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individuall recited herein.

The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims. All references, included patents, patent publications and non-patent literature, cited herein are hereby incorporated, by reference in their entirety. Any conflict between statements in references and those made herein should be resolved in favor of the statements contained herein.

EXAMPLES

Histatins are a family of structurally related histidine-rich peptides found in the oral cavity of humans and have a broad-range of antimicrobial activity. Of the histati isofonns, histatin 5 shows the most potent antifungal activity (Helmerhorst, et aL J Biol Chem 274 (1999) 7286-7291). We synthesized a 16 amino acid derivative of histatin 5 known to be as active as the full length 24 amino acid histatin 5 (Raj et aL J Bio! Chem 269 ( 1994} 9610-9619), In addition, the D amino acid enantiomer and the retro-hi stati 5 and retro-enantio-histatin 5 using D amino acids were synthesized (Figure 1 ). Following synthesis and purification by ilPLC, the four 16mer peptides were tested for antifungal activity against Candida albicans SC5314. Using a standard in vUro .ki.ll.ing assay (Edgerton et aL, J Biol Chem 273 ( 1.998) 20438-20447) it was discovered that all four peptides maintained equivalent antifungal activity (Figure 2 Although all four peptides maintained equivalent antifungal activity, the question remained as to whether they were working via the same mechanism. To address this question, we took advantage of the fact thai histatin 5 antifungal activity requires targe ceil respiratory activity (Gyurko et al,. Antimicrobial Agents and. Chemotherapy 44 (2000) 348-354), In other words, inhibiting cellular respiration rendered target fungal cells resistant to histatin 5 killing. Thus, we evaluated whether the four 16.mer peptides synthesized in our laboratory required fungal cell respiration for killing activity. Candida a!hiea SC53I4 was exposed to either sodium azide or aiitimycin A, two known respiratory inhibitors, and it was found that all four peptides lost antifungal activit in the presence of these inhibitors (Figure 3), consistent, with the presumption that all four Knner derivatives were killing by the same mechanism.

To evaluate whether the peptides adopt similar secondary structures, circular dichroism spectroscopy (CD) was performed on the wild-type and retro-histatin 5 1 fever peptides in the presence of increasing concentrations of trifluoroethanol (TFE), We observed that both of these peptides showed similar propensities for alpha-helical formation in the presence of TFE (Figure 4). The D-amino acid derivatives showed a similar profile since they would be the mirror .image of the L-amino acid peptides.

To quantify the ability of the four histatin 5 16mer peptide derivatives to lyse

membranes, artificial liposomes were prepared with phospholipid and ergosterol concentrations similar to that of Candida albicans. The vesicles were loaded with acridme orange and a fluorescence release assay performed to compare the lysis potential of the four histatin 5 16mer derivatives (Figure 5). It was determined that all four peptides showed comparable membrane lysis potential

The preliminary studies led us to the hypothesi that there must be a region within histatin 5 that displays a quasi-dyad of symmetry such that the killing activity of the peptide is maintained whether it is the ^■ normal , wild-type amino acid sequence or the retro sequence.

Moreover, the enantiomeric form with D-amino acids would likely function identically. Given this hypothesis, we examined the sequence of histatin 5 and identified a small region of histatin 5 containing the sequence Y-K-R-K-P (SEQ ID ^' NO: 24) (Figure 1 ) that would fit the quasi-dyad of symmetry model with an arginine in the middle flanked by two lysines that are then flanked by two aromatic amino acids. To evaluate whether this sequence has antifungal activity, the F- -R- -Y (SEQ ID NO: 24) retro~5mer peptide was synthesized (designated KM5; SEQ ID NO; 2) and. examined for antifungal activity in vilm. It was observed that KM5 displayed significant antifungal activity with an LDa> of 5 μΜ (Figure 6). Although KM5 was less potent as compared to the 16mer peptides (Figure 2), it had significant antifungal activity and the smaller size was more conductive for large scale production. The kinetics of the antifungal activity of MS was also found, to be time-dependent over a two hour period and the .maximum killing activity was reached following two hours of incubation (Figure 7).

We also synthesized the retro-Smer peptide with D-amino acids, M6 (SEQ ID NO: 1.0), and found equivalent antifungal activity and the equivalent ability to per eabilfce artificial liposomes in vitro (Figure 8). This was particularly important because the D-arnfno acid peptides are likely to be resistant to proteolysis and more stable for use in the treatment of fungal infections humans.

Thus, we focused on the further refinement of KM5 to obtain a more potent antifungal agent To this end, we sought to determine the importance of the various residues within KM 5 b synthesizing additional peptides thai altered the amino acids at various positions as shown in Table 2. These peptides were synthesized and subsequently purified by HPLC. The peptides were then evaluated in a standard killing assay with Candida albicans SC5314. These data, indicate that the argi.nine at position 3 is essential for killing activity (K .5 versus KM? or KM8). Moreover, the tyrosine at position 5 shows greater fungiddial activity than phenylalanine at the fifth position (KM5 versus KM9). Overall, these data indicate sequence specificity in the iiingicklial activity of M5,

Tabie 2: Relative activity of the peptides at 26 Μ

Statu net fggq IE o,

KM5 F RKY (L-amino acids; »3¾

S£Q ID NO: 2

KM6 FK KY !P-smlnq H®

SSQ ID HO' m)

Km mm (SEQ ID NO: 12)

KM? m

IMS FKRKFiSea iD NO: 13) 70%

m KFRYK {SEQ ID NQU4J

il CFKRKY (SSGt IP H0tS} % The addition of a cysteine to the peptide did. not alter its antifungal activity (KM 1 1) ( Table 2), yet permitted dimerization of the peptide via disulfide bonds, When the peptide (KM 1.2) was dii.nerized, we observed a dramatic enhancement in antifungal activity (LDs<rT 2 nM). In an effort to further exploit potential peptide-membrane interactions, we modified both the tyrosine and phenylalanine residues to tryptophan (EMI 4); however, this resulted in a slight loss of antifungal activity (LD¾r ^™ 528 nM). Nevertheless, KM 14 remained a. viable compound for animal testing. To evaluate whether the position, of the cysteine at the N- or C-terminus was critical for activity, it was moved to the center of the peptide (KM! 6) (Figure 9). In addition, we synthesized a 7mer peptide with cysteines at both the - and C-termini and circularized the peptide via disulfide bonding, .KM 17 (Figure 9). The dimerized KM5 peptides designated KM.! 6 and KM1 ? have very promising antifungal activity compatible with that of KM 12 and KM 14, and we are continuing to evaluate their toxicity to mammalian cells and .fungicidal activity.

As we evaluated these peptides for protein binding activity using bovine calf serum, it was discovered that the disulfide bond in KM 12 was readily reduced generating two 6mer peptides with significantly less antifungal activity. To alleviate that difficulty, two additional peptides were synthesized, KM23 and KM29. The KM23 peptide was identical in sequence to KMI 2 except the cysteine residues were removed resulting in a peptide with the sequence Y-K- - -F-F-K- -K-Y (SEQ ID NO; 3). Since there were two pheny lalanines at the center of this symmetrical peptide another peptide, designated KM29 was synthesized to yield the symmetric peptide Y-K-R-K-F-K-R-K-Y (SEQ ID NO: 4) with one phenylalanine at the center. Both of these peptides were evaluated for antifungal activity against Candida albicans SC5314 as well and found to have an LDsoof 62.5nM in killing assays.

To further compare the activity of KM ^' 12, KM23 and KM29 for antifungal activity, a minimum inhibitory concentration (MIC) assay was performed with several different species of Candida. Table 3 indicates that all three peptides show similar profiles of killing activity with Candida species including C albicans. C. kefyr, C. glabmm, C. krusei, C lucitaniae, and C tropic-alls. Each of the Candida species were clinical isolates obtained from the ATCC. We are continuing to evaluate the sensitivity of other Candida species to the peptides including C dubiinie is, C. p rapsihsis and other independent isolates of C. albicans. At the preliminary stage, we have observed a broad spectrum of activity against most Candida species with C glahra a showing the most resistance to KM 12, KM23, and KM.29, Table 3: MIC assays for KM12 (SEQ ID HO: 17), KM2 (SEQ

ID HQ: 3), and K 29 {SEQ ID HO: 4) ³

Candida spectes KM12

C nd da albicans SCS314 5,5-11 4,? 4.2-8.4

Candida kef r ATCC4135 .S~5 ,S 1.2-4.7 2.1-4.3

Candida giafemta ATCCS083O SB 75.4 67.5

Candida kernel ATCCS2S8 5.5-tl.G 4.7 4.2

Candida !ucitaniae ATCC200 S1 1.4-2.8 2.4 2.1-4.2

Candida tfoplcafls ATCC7S0 2,S—5 ,S 2.4-4.7 2.1-4.2

Values are presented in pg ml of each peptide. Each MIC assay represents three

independent experiments with the range indicated for each. MIC assays were performed in

Q.125X RPM! medium,

To evaluate whether these peptides cause toxicity to mammalian, cells, we examined toxicity using cell proliferation assays. We observed no significant mammalian ceil toxicity after 48 hours of incubation in the presence o f KM ! 2 at concentrations as high, as 100 μΜ (Figure 1. 1 ). in addition, mammalian, cells were also examined, by FACS analysis with a propidium iodide uptake assay to determine whether the peptide caused permeabilizadon of the mammalian ceil plasma membrane. No significant membrane pemieabilization was observed.

To evaluate whether the various peptides have hemolytic activity, we examined, the hemolysis of sheep red blood cells in the presence of KM 5 and retro KM5 (Figure 12) and the difiier peptides KM 12, KM 14, K 23, and KM29 (figure 13). No te of the aforementioned peptides displayed significant hemolysi activity (3% or less) at peptide concentrations up to 100 μΜ. Thus, the KM peptides did not result in significant red blood cell hemolysis in vitro,

To evaluate acute toxicity in animals,€D1 outbred mice were injected via tail vein wit!i either KM! 2 or KM 14 using a standard up-down concentration protocol and observed for any symptoms consistent with the toxicity of the peptides ( ^' fable 4). Mice were initially injected with KM 12 or KM 14 at a concentration of 8 mg/kg. KM 12 peptide showed no toxicity at this concentration with five different animals; however, KM 14 caused immediate death with two mice and the acute toxicity evaluation of this peptide was temiiuated. After examining a limited number of peptide concentrations and sol vent formulations it was determined that. KM 12 at lOmg/kg administered in 5% glucose either intravenously, intramuscularly or intraperitoneally caused no major toxicity. Thus the KM 12 peptide was. deemed safe for animals at concentrations up to 1.0 mg kg and the use of 5% glucose versus phosphate-buffered saline was deemed safer for administration to the animals. We will continue to evaluate acute toxicity with other KM peptides (i.e. M23 and KM29) that show excellent promise as antifungal compounds. in addition, we plan to evaluate these peptides fox treatment of superficial mucosal infections with.

Candida species.

Table 4: Acute toxicity testing of KM12 (SEQ ID NO: 17) and KM 14 (SEQ ID NO;

21) in CD1 mice

FgPTlgg VSgP* mm QWBftVBP 9N ΤΒ»ΤΒΡΑΝ) ΑϋΕ

peptides were dissolved in either phosphate-buffered saline or a solution containing only 5% glucose, rt indicate the number of mice used for each study.

dosage In mg of peptide par kg mouse weight. Route of adminssration includes intravenous (iV), intramuscular (IM), or intraperitoneal (fP),

Small peptides have the potential to induce an immune response when used for

therapeutic purposes. To examine this possibility, mice were injected once per mouth tor four months with !Omg/kg of the KM12 peptide either by intravenous, intramuscular, or

intraperitoneal route. After the .fifth month, the mice were euthanized, and the serum collected and tested by dot blot analysts for antibodies directed against the KM 12 peptide. For each route of injection, five CD I mice were used. It was observed that 1/5 mice showed a moderate

immune response to KM 12 following intravenous injection, 1/5 mice displayed a weak immune response following intramuscular injection, and 0/5 mice displayed an immune response after intraperitoneal injection. These data suggest that KM 1.2 is weakly immunogenic; however, the smaller peptides, KM23 and KM ^' 29, have not been evaluated. It is plausible that the smaller size of those peptides may not elicit an immune response, yet be effect as antifungals. Such studies are currently underway on. K.M23 and KM29.

Methods;

Peptide synthesis. N-Fmoc protected amino acids and Rink resin was purchased from NovaBiochem (San Diego, CA) and Advanced Chemtech (Louisville, Y). All KM peptides were synthesized with an acetylated N-terminus and amidated C-terminus. The peptides were synthesized on a Model 433A solid-phase peptide synthesizer (Applied Biosystems; Foster City, CA) using Rink, resin and Fmoe-protected amino acids (NovaBiochem). After synthesis, the peptides were deproteeted and cleaved from, the resin using a high TFA (frifluroacetic acid) cleavage cocktail consisting of 85% TFA, 5% dH20, 5% triisopropylsilan, and 5% Phenol The resin was subsequently mixed in the cocktail, solution at room temperature for 3 hours, after which peptide precipitated into 50mFs 1 :1 v/v methy!-t-hutyi ether/hexane.per ml of cleavage cocktail. ext, the peptides were dissolved in 1 :1 v/v acetonitrile/ddil20 and reeovered by lypholyzatton under high vacuum. Crude peptides were purified on Prp-3 reverse phase column (7 by 305 mm; Bio-Rad, Hercules, USA) on a Hitachi L7100 HPLC instrument using a linear gradient of 0 - 30 % aeetonitriie and water (both were contained 0.1% TFA), The purity of each peptide was evaluated by mass spectroscopy. The peptides concentrations were determined by the extinction coefficient .

Killing assays. The fungicidal activity of the peptides toward Candida albicans was examined by mierodilution plate assay as described, previously (13). Briefly, from a fresh overnight cultured plate, a single colony was inoculated and diluted in 1 ml of 10 mM sodium phosphate buffer at pH 7,4. Once cell counts had been confirmed by using a hemacytometer, the ceils diluted within the same buffer at 1.8 ^χ ΗΫ cells/ml. Cell suspensions of 20 μΙ were mixed with 20 μ! of peptide, which were dissolved in 10 mM sodium phosphate buffer at H 7,4, and then incubated for 2 hours at 37°C with shaking at speeds of 55Grpm. The reactions were stopped by the addition of 360 pi yeast itrogen base (Y B) and then 40 μΙ of cell suspension was spread onto plates and incubated for 24 hours at 37°C. Sabouraud dextrose agar plates were used for C alhiea ki lling assay . The number of colony-forming units ( CPUs) was counted and each assay was repeated in triplicate. Loss of viability was calculated as p .-(colonies from suspension with peptide/colonics from suspension with no peptide)) ^χ 100. For the respiratory activity experiments, Candida albicans was grown in the presence of sodium azide or antiraycm A (Sigma Aldrich) before the killing assay was performed.

Circular Dichroism Spectroscopy. CD measurements for the peptides were acquired on a Jasco-710 spectropolarimeter. The readings were done using a quartz cell of 0.1 em path length ai 25° C .Peptides was measured in increasing ^" concentrations of trifinoroeil. mol (TFE) (vol/vol). The spectra were recorded between i90nm and 250nm every 0.2 ran, with a 1 ,0 am bandwidth and a scan speed of 20 nm min. The background was subtracted from all spectra and

smoothened using the Fourier filters. The CD spectra are reported as the mean residue molar elUpticity ί θ ^' |) in degrees.em^d or .

Fluorescence Leakage Assay: Reagents included: 1, 2-dipalm.itoykv«-glycero~3- phosphocho!ine (DPPC, catalog # 850355), !,2-dipalmitoy w-glycert>--3«phosphoethanolainine f DPPE, catalog # 850705), Soy PI (catalo # 840044) and 1. pm polycarbonate membranes (catalog # 610010) purchased from Avanti Polar Lipids. Ergosterol was purchased from Avanii Polar Lipids, PD-.10 desalting columns (catalog # 17-08 1-01) were purchased from GE Health care Life Sciences, Brgosterol-contaimng liposomes were prepared from phospholipid and ergosterol dissolved in ao organic solvent containing chloroi >rm:nietha:nol:water at a volume ratio of 65:35:8. Lipid mixtures were prepared at. a concentration of 12 m.g per 1ml of organic solvent and had a weight atio of DPPC: DPPE: SoyPJ; ergosterol of 5:4: 1 ;2, This relative ratio mimics C. albicans yeast biomernbraiies (14, 1.5). Lipid, mixtures were thoroughly mixed, dried under a nitrogen stream for 20 to 30 minutes, nd then evaporated in a vacuum evaporator overnight. Lipid cakes were hydrated with 1ml of 1 lOmM ammonium sulfate for a period of 1 hour at 72 ^,> C in a water bath with vigorous shaking. Lipid suspensions were subjected to three freeze-thaw cycles and. extruded through slacked l um polycarbonate membranes for at least 17 cycles to yield large ^• unilamellar vesicles. The extrusion was performed at 72-75 ⁰ C using a thermo-controller. Buffer exchange was done after extrusion by ge! filtration as per protocol provided with PDI.O desalting columns (GE Health Care Life Sciences), The buffer exchange step replaced the external liposomal buffe of 1 10 mM amonium sulfate with 150 mM sodium chloride. The size distribution of unilamellar vesicles (liposomes) in the final, lipid suspensions was evaluated with a Zeta. Potential Analyzer Utilizing Phase Analysis l ight Scattering Machine (Zetapals, Brookhaven Instruments Corp.). Liposomes in 150niM sodium chloride were stored at 4°C until used- Lor fluorescent leakage assays, liposomes were loaded with ΙΟμΜ ac.rid.ine orange in lOrnM sodium phosphate buffer (pH 7.4) supplemented with 5% glucose. A 60μ! of liposome suspension was added into 1.940 μί of Ι μΜ acridine orange in l OroM sodium, phosphate buffer/ 5% glucose and kept at room temperature in the dark for 4 hours. Externa! acridine orange was removed by gel filtration with the use ofPDIO desalting columns. During this gel filtration step, the external-liposornal solution, of .150mM sodium chloride was replaced with 1 OmM sodium phosphate buffer pH 7.4/ 5% glucose. Acridine orange-loaded liposomes in Ι τηΜ sodium phosphate buffer/ 5% glucose were tested with the four 16-mer peptides in four- sided polystyrene cuvettes (Sarstedt). The fluorescent intensity of the 2 ml samples was monitored by Fluoromax 4P instrument. (Horiba Scientific). More specifically. 120 μί of peptides were added into 1880μΙ of liposome suspensions at 300 seconds to a final peptide concentration of 20μΜ and 10% Triton X-1.00 was added at 1500 seconds to a final Triton X-l 00 concentration of 0..1 %, For control samples, .120 μΐ of I O.mM sodium phosphate buffer was added into liposome suspensions instead of the peptides. The fluorescent intensity of the whole samples was monitored during a 30 minute period (excitation 4 0nm, emission 525nm) and plotted as percentage of acridine orange release compared to the total release obtained with Triton X 100. The formula used to calculate percentage of release is as followed:

Ft: fluorescent intensity at time t; Fo: fluorescent intensity at time 0 Fioial: fluorescent intensity obtained with triton X-l 00 at time 1800s

Minimum Inhibitory Concentration Assays: MIC assays were used to evaluate the minimum concentration of each antifungal peptide that would lead to 100% inhibition of growth. In the MIC assay, the eoiorimetrie indicator, resazurin, is used to evaluate cell growth.

Resaxuriti turns pink after reduction by living cells, indicating active cell growth. The unchanged blue color indicated no active cell growth. Co!orinietric MIC end-points are interpreted as the lowest drug concentration that remained blue. The lowest dilution that, changed from, blue to slightly purple is indicative of significant ceil growth inhibition; whereas a pink color Indicates no growth inhibition. Each individual Candida species was assayed a minimum of three times and the results represent the range of MIC values obtained. The MIC assay was performed as described by the National Committee for Clinical Laboratory Standards M27-A, except we include .resazurin as an indicator dye rather than, visual inspection and the R.PM11690 tissue culture medium was used at 0- 125X ctwcentratkm for our assays.

Antifungal peptide toxicity to .mammalian cells in vitro. To evaluate the toxicity of the peptide to mammalian cells, N1H3T3 cells were grown in cell culture using Dnlbeeco's modified essential medium (DMEM) containing 10% newborn calf serum (NCS), The KM 1.2 peptide was added at various concentration and incubated with the cells for up to 48 hrs. WST- .1 cell viability assa (Roche) was performed as described by the manufacturer after 48 hrs. to evaluate the loss of cell, viability.

Hemolysis Assays. Red blood cell hemolysis assays were performed using sheep red blood cells In a 96-wetl microliter plate fonnat The peptide dissolved in phosphate-buffered saline (pH 7.2) was prepared by two-fold serial dilutions in a 96-weii. titer plate in a final volume of 100 ttl. The maximum concentration of peptide was 100 μΜ. Positive control used Triton X- 1 0 at a final concentration of 1% to achieve maximum red blood cell lysis. The negative control contained only phosphate-buffered saline. For the assay, 100 μΐ of 1% sheep red blood cells in phosphate-buffered saline were added to wells prepared as described above and the cells were incubated at 37°C for one hr with shaking at 170 rpm. The plates were subsequently eentrifuged at 1000 X g for 5 min. and 100 μΐ of the supernatant per well wa s collected for the measurement ofabsorban.ee at 405 nm by a microliter plate reader (Bio-Tek Instruments. Inc. ELS0S). The percentage of hemolysis was calculated by the following equation:

Where:

Abs (sample) is the ahsorhance of supernatant obtained f om the samples treated with peptides

Abs (negative control) is the absorbance of supernatan obtained from the samples treated with phosphate buffered saline

Abs (positive control) is the absorbance of supernatant obtained from the samples treated with 1% Triton X- .100. Animal Studies: For the animal studies, CD-I mice of 18-25 grams were injected, with the antifungal peptides dissolved in either phosphate-buffered saline or 5% glucose. Peptide concentrations used for the injection of mice were chosen using a standard up-down protocol starling at 8 mg, kg. Peptides were injected into mice intravenously, intramuscularly, or intraperitoneal.iy as indicated. The mice were closely monitored for signs of distress after injection. After two hours of continuous monitoring, the mice were evaluated daily for two weeks and necropsies performed after two weeks. For evaluating the immunogentcUy of the peptides, mice were injected with peptides at a concentration of iOmg kg in 5% glucose. The routes of injection were intravenous, intramuscular, or intraperitoneal. Five mice were injected with the peptide per rout of injection. The mice were injected with the same dose of peptide once per month for four months. The mice were monitored for signs of distress after each injection. At the end of the fifth month, the mice were euthanized by an. overdose of anesthesia and the serum was collected from each mouse individually as assay for activity against the peptide using dot blots.