WO2016205902A2 - Compositions and methods for treating biofilms - Google Patents

Abstract

The present invention relates to a novel composition for use in inhibiting or treating microbial biofilm formation, said composition comprising a plant defensin or a peptide derivative thereof and an antifungal compound, preferably caspofungin, anidulafungin, micafungin, or amphotericin B. Accordingly, the present invention relates to use of said composition for the inhibition or treatment of biofilms, particularly fungal biofilms, such as Candida biofilms, in a subject or on a surface or other medium susceptible to biofilm formation.

Description

COMPOSITIONS AND METHODS FOR TREATING BIOFILMS

TECHNICAL FIELD

The present invention relates to novel compositions and methods for the treatment or prevention of microbial biofilms, preferably a fungal biofilm, more preferably a Candida biofilm. More in particular, said compositions and methods comprise combining an antifungal agent, with a potentiating compound, preferably a plant defensin or peptide derivatives thereof, for increasing the antibiofilm activity of the antifungal agent, and for reducing, eradicating, inhibiting or preventing fungal biofilms or fungal biofilm formation in a subject and/or on a surface or other medium susceptible to biofilm formation.

BACKGROUND

Biofilms of microbial pathogens consist of dense layers of microorganisms surrounded by an extracellular polymeric matrix, adherent to a surface, thereby protecting the microbes from the action of antimicrobial agents, and are believed to be involved in at least 80% of human microbial infections. Nowadays, it becomes more and more clear that biofilms are critical to the development of clinical infections in general. Due to the increasing number of immunocompromised patients, combined with the advances in medical technology, fungi have emerged as a major cause of infectious disease, with Candida sp., particularly C. albicans being the major pathogen.

Apart from their existence under free-living or planktonic form, Candida sp. are known to form biofilms upon contact with various surfaces. C. albicans cells are able to colonize and subsequently form biofilms on both host cutaneous or mucosal surfaces and on indwelling medical implants and devices, such as (dental) implants, intravascular and urinary catheters, (voice) prostheses and heart valves. C. albicans is an opportunistic human fungal pathogen, causing not only superficial infections, but also life-threatening systemic diseases. C. albicans is now recognized as the fourth most common cause of bloodstream infections in the United States, with a high attributable mortality rate, namely 20-40%.

Candida sp. also play a predominant role in mixed-species fungal biofilms. Such biofilm cells are tolerant towards most conventional antimycotics and there are only few novel agents that can be used to treat biofilm-related infections. To date, only miconazole, caspofungin, anidulafungin and liposomal formulations of amphotericin B are used to effectively treat these infections, and hence, there is a need to identify novel antibiofilm compounds. All the currently marketed antifungal drugs have major drawbacks, including no broad- spectrum activity, no per oral absorption, side-effects, low antifungal activity, no fungicidal activity, drug-drug interactions and/or high costs. In the case of biofilm treatments or of treatments of sessile cells, these drawbacks become prohibiting. Also, antibiotics (antimycotics) that are active against microbial biofilms often result in only partial killing of the biofilm cells, even when applied at high doses, leaving a subpopulation of the biofilm cells alive, the so called persisters. Persisters are antibiotic-tolerant cells that survive treatments with high antibiotic concentrations. Because they start growing again when the antibiotic pressure drops, persisters are considered as one of the most important reasons for the recurrence of biofilm-associated infections.

Plant defensins are present in all plant families, including the Brassicaceae, Fabaceae and Solanaceae. Plant defensins are small, basic, Cysteine-rich peptides with a length of approximately 45-54 amino acids. Their structure typically comprises a conserved structure known as a Cysteine-stabilized αβ-motif with a prominent a-helix and a triple-stranded antiparallel β-sheet that is stabilized by four disulphide bridges. Plant defensins exhibit antimicrobial activity against a broad range of microorganisms, whereas they are in general non-toxic to human cells. To date, there has been a particular focus on their antifungal activity and several fungal targets have been identified, including membrane sphingolipids and phospholipids (1). Upon interaction with the fungal membrane, plant defensins are either internalized into the cell and interact with cytosolic or nuclear proteins, or they remain localized at the cell wall or membrane of the fungus. The mechanisms by which plant defensins induce fungal cell death are diverse, but common aspects are observed. These include the production of reactive oxygen species (ROS) and the induction of apoptosis.

Despite the fact that their mechanisms of antifungal action have been studied extensively, no reports exist about the activity of plant defensins against fungal biofilms.

HsAFPI is a plant defensin found in coral bells ("Heuchera sanguinea"), which was previously characterized by Osborn and colleagues (2). HsAFPI inhibits the growth of various plant pathogenic fungi, including Botrytis cinerea, Verticillium albo-atrum and Fusarium culmorum, and causes swelling of germ tubes and hyphae in the latter (2). In addition, it was reported that HsAFPI shows antifungal activity against Saccharomyces cerevisiae and the human pathogen C. albicans, and induces apoptosis in the latter (3). Furthermore, it was shown that HsAFPI has a low in vitro frequency of resistance occurrence in planktonic C. albicans cultures (i.e. less than 1 in 2,000,000 mutants) (4). In an attempt to unravel HsAFPI 's mode of antifungal activity, this defensin was tested against the complete S. cerevisiae deletion mutant library for identification of yeast mutants with altered HsAFPI sensitivity (3). In this study, 84 yeast genes were identified that were found to be implicated in governing HsAFPI tolerance or sensitivity of yeast (3).

However, these publications do not show an antibiofilm effect of hsAFPI , nor a combinatory effect of plant defensins with known antifungal drugs.

Accordingly, there is still a clear need for effective strategies to prevent and to eliminate deleterious biofilms, in particular fungal biofilms associated with the surface of body surfaces such as skin, or medical devices, such as implants.

SUMMARY OF THE INVENTION

The present invention addresses the increasing problems of biofilms, in particular of fungal biofilms on different surfaces including host tissue and medical devices, outside or within the human body and which escape conventional antifungal treatment. The present invention provides novel compositions and methods with improved antibiofilm properties by enhancing the efficacy of antifungal drugs, such as by increasing the susceptibility and sensitivity of biofilms, particularly of fungal biofilms to said drugs and/or by a continued, highly localised treatment with said drugs.

The present invention is based on the surprising finding by the inventors that plant defensin HsAFPI (SEQ ID No. 1 ), and/or peptide derivatives (SEQ ID Nos. 2 to 17) thereof, are biofilm inhibiting agents and potentiating compounds, which increase the antibiofilm inhibiting and eradicating activity of antifungal agents, preferably of the echinocandin or polyene class, more preferably caspofungin, anidulafungin, micafungin, or amphotericin B, against Candida biofilms, preferably against C. albicans biofilms.

Thus, the present invention relates to compounds of a peptidic nature which inhibit microbial biofilm formation and development, for use in suppressing, reducing, inhibiting, controlling, treating or preventing microbial biofilm formation, and therefore can be formulated into antibiofilm compositions for administration to humans and animals and for application to inert surfaces susceptible to infection by microbial biofilms. The present invention also provides a method for suppressing, reducing, inhibiting, controlling, treating or preventing the development of a microbial biofilm on a biotic or abiotic surface or in a subject, which comprises the step of exposure or administration of such a peptide or composition on said surface or to the subject.

1. An isolated peptide wherein said peptide comprises an amino acid sequence with at least 70% sequence identity to amino acid sequences GAXHYQFPSVKX (SEQ ID No. 8), QQXKDREHFAYG (SEQ ID No. 9) or WSGHXGSSSKXS (SEQ ID No. 10) or a derivative thereof wherein X stands for Cystein or a-aminobutyric acid, wherein said peptide consists of 12 to 44 amino acids, and wherein said isolated peptide inhibits microbial biofilm formation and development and/or potentiates the effect of an antifungal agent.

The isolated peptide according to statement 1 wherein said peptide comprises a sequence with at least 70% sequence identity to the amino acid sequences GAXHYQFPSVKX (SEQ ID No. 8), GAXHYQFPSVKXFXKR (SEQ ID No. 11 ) or AYGGAXHYQFPSVKX (SEQ ID No. 12), or a derivative thereof and wherein said peptide consists of 12 to 24 amino acids.

The isolated peptide according to statement 1 or 2 wherein said peptide comprises a sequence with at least 70% sequence identity to the amino acid sequences selected from the group consisting of EHFAYGGAXHYQFPSVKXFXKRQ (SEQ ID No. 13), AYGGAXHYQFPSVKXFXKRQX (SEQ ID No. 14), YGGAXHYQFPSVKXFXKRQX (SEQ ID No. 15), HFAYGGAXHYQFPSVKXFXK (SEQ ID No. 16), or

FAYGGAXHYQFPSVKXFXK (SEQ ID No. 17), or a derivative thereof.

The isolated peptide according to any one of the statements 1 to 3 wherein said peptide is selected from the group consisting of EHFAYGGAXHYQFPSVKXFXKRQ (SEQ ID No. 13), AYGGAXHYQFPSVKXFXKRQX (SEQ ID No. 14), YGGAXHYQFPSVKXFXKRQX (SEQ ID No. 15), HFAYGGAXHYQFPSVKXFXK

(SEQ ID No. 16), FAYGGAXHYQFPSVKXFXK (SEQ ID No. 17), or FAYGGAXHYQFPAVKXFXK (SEQ ID No. 18), or a derivative thereof.

The isolated peptide according to any one of statements 1 to 4 wherein said peptide comprises a sequence with at least 70% sequence identity to the amino acid sequences selected from the group consisting of

DGVKLXDVPSGTWSGHXGSSSKXS (SEQ ID No. 2),

DVPSGTWSGHXGSSSKXSQQXKDR (SEQ ID No. 3),

WSGHXGSSSKXSQQXKDREHFAYG (SEQ ID No. 4),

SSSKXSQQXKDREHFAYGGAXHYQ (SEQ ID No. 5),

QQXKDREHFAYGGAXHYQFPSVKX (SEQ ID No. 6), or

EH FAYGGAXH YQFPSVKXFXKRQX (SEQ ID No. 7), or a derivative thereof.

The isolated peptide according to any one of the statements 1 to5 wherein said peptide is selected from the group consisting of DGVKLXDVPSGTWSGHXGSSSKXS (SEQ ID No. 2), DVPSGTWSGHXGSSSKXSQQXKDR (SEQ ID No. 3),

WSGHXGSSSKXSQQXKDREHFAYG (SEQ ID No. 4),

SSSKXSQQXKDREHFAYGGAXHYQ (SEQ ID No. 5),

QQXKDREHFAYGGAXHYQFPSVKX (SEQ ID No. 6), EHFAYGGAXHYQFPSVKXFXKRQX (SEQ ID No. 7), GAXHYQFPSVKX (SEQ ID No. 8), QQXKDREHFAYG (SEQ ID No. 9) or WSGHXGSSSKXS (SEQ ID No. 10), or a derivative thereof.

7. The isolated peptide according to any one of the statements 1 to 6, or according to SEQ ID No. 1 for use in the treatment or prevention of a microbial biofilm associated condition or infection in a human or animal subject.

8. The isolated peptide according to any one of the statements 1 to 6, or according to SEQ ID No. 1 for use in a treatment according to claim 7, wherein said microbial biofilm or biofilm associated condition or infection comprises Candida cells.

9. A composition for use in treatment of prevention of a fungal biofilm associated condition or infection in a human or animal subject, said composition comprising at least one peptide according to any one of the statements 1 to 6, or according to SEQ ID No. 1 and at least one antifungal agent.

10. The composition of statement 9, wherein said at least one antifungal agent is selected from the group of echinocandins or polyenes.

1 1. The composition of statement 9, wherein said at least one antifungal agent is caspofungin, anidulafungin, micafungin or amphotericin B.

12. The composition of any one of the statements 9 to 11 , further comprising one or more pharmaceutically acceptable compounds, carries and/or adjuvants.

13. The composition of any one of the statements 9 to 12, wherein said subject has been implanted with a medical device, which is infected or at risk of being infected with a fungal biofilm.

14. The composition of statement 13 wherein said medical device is selected from the group consisting of catheters, stents, surgical plates, prostheses, valves or pins, artificial joints, pacemakers, contact lenses and bio-implants.

15. The composition of any one of the statements 9 to 14, wherein said fungal biofilm is a Candida biofilm.

16. A method for reducing, eradicating, inhibiting or preventing fungal biofilms or fungal biofilm formation, characterized in that a surface or medium outside the body of a human or animal subject carrying said fungal biofilm or susceptible to said fungal biofilm formation, is exposed to at least one peptide according to any one of the statements 1 to 6, or according to SEQ ID No. 1.

17. The method according to statement 16, wherein said surface or medium is exposed to at least one peptide according to any one of the statements 1 to 6, or according to SEQ ID No. 1 and at least one antifungal agent.

18. The method of statement 17, wherein said at least one antifungal agent is selected from the group of echinocandins or polyenes. 19. The method of statement 17, wherein said at least one antifungal agent is caspofungin, anidulafungin, micafungin or amphotericin B.

20. The method of any one of the statements 16 to 19 wherein said biofilm is exposed to said at least one peptide before, after or concurrent with exposing said biofilm to said antifungal agent.

21. The method of any one of the statements 16 to 20 wherein said fungal biofilm is a Candida biofilm.

22. A method for the treatment or prevention of a condition or infection associated with fungal biofilm development in a human or animal subject, said method comprising administering to said human or animal subject a composition comprising at least one peptide according to any one of the statements 1 to 6, or according to SEQ ID No. 1 and at least one antifungal agent.

23. The method of statement 22, wherein said at least one antifungal agent is selected from the group consisting of echinocandins or polyenes.

24. The method of statement 22, wherein said at least one antifungal agent is caspofungin, anidulafungin, micafungin or amphotericin B.

25. The method of any one of the statements 22 to 24, wherein said fungal biofilm is a Candida biofilm.

DETAILED DESCRIPTION OF THE INVENTION LEGENDS TO FIGURES

Figure 1. shows the sequence alignment of HsAFPI with other plant defensins. (A) Amino acid sequence alignment of NaD1 , Psd1 , MtDef4, RsAFPI , RsAFP2 and HsAFPI , matching their Cysteine residues (numbered l-VIII). Multiple alignment was performed using the COBALT alignment tool. Cysteine-pairing is shown at the top of the figure. Highly conserved residues are shown in vertical boxes; (-) denote gaps in the alignment. Horizontal boxes represent peptide fragments that exhibit antifungal activity similar to the parental peptide, and hence, are important for antifungal activity. The large box indicates the position of the v- core. (B) Amino acid sequence alignment of HsAFPI and the HsAFPI linear peptide fragments (HsLin01-HsLin06). Multiple alignment was performed using the COBALT alignment tool. Highly conserved residues are shown in in vertical boxes; (-) denote gaps in the alignment. The large box indicates the position of the γ-core.

Fig. 2 shows the secondary shift analysis of rHsAFPI , pH 4.0 at 298 K. Regions of a-helix and β-strand are indicated at the top of the figure. Fig. 3 shows the three-dimensional structure of rHsAFPI . (A) A family of 20 lowest energy structures superimposed over all backbone heavy atoms; (B) A ribbon representation with disulfide bonds shown by arrows. The termini are labeled as N and C. Diagrams were generated using MOLMOL. Fig. 4 shows scanning electron microscopy images of 4 hours-old biofilms, grown in the presence or absence (untreated) of 11.8 μΜ rHsAFPI . Images at multiple magnifications (500x, 1000x and 2000x) are presented.

Fig. 5 shows synergy between rHsAFPI and caspofungin or amphotericin B, for (A) biofilm inhibition, as determined by CTB assay; (B) biofilm eradication, as determined by CTB assay; and (C) growth inhibition of planktonic cultures. Growth was analysed by measuring the OD490. Sigmoidal curves were generated using data of at least three independent experiments (n > 3), using the model Y=Bottom+(Top-Bottom)/(1+10^A((LoglC50- X)*HillSlope)) in GraphPad Prism. Dose response curves of caspofungin in the presence of synergistic concentrations of rHsAFPI are presented. Black arrows represent synergy. The lines and corresponding numbers in the graphs indicate the different rHsAFPI doses for each curve (μΜ).

Fig. 6 shows cell viability and cell proliferation of HepG2 cells treated with rHsAFPI . HepG2 cells were treated with water (control treatment) or rHsAFPI (0.01 μΜ - 42 μΜ) for 24 hours. Cell viability and cell proliferation were determined by XTT staining and BrdU staining, respectively, and results were expressed relative to cells receiving control treatment. Mean and SEM of three experiments in quadruplicate is shown(Unpaired Student t-test; P<0.05 was defined as statistically significant).

Fig. 7 shows representation of the HsLin peptides imposed on the rHsAFPI structure, according to the amino acid sequence. HsLin peptides are shown as a thick line in the same orientation as rHsAFPI ; other residues of rHsAFPI , not present in the HsLin peptide, are shown as a thin line. Note that (i) the Cysteine residues are replaced by a-aminobutyric acid to avoid formation of disulfide bonds and that (ii) the CSa3 scaffold is not present in the HsLin peptides, and therefore, the peptides do not adopt the same conformation as the mature rHsAFPI .

Fig. 8 shows synergy between caspofungin and HsLinOI (A), HsLin02 (B), HsLin03 (C), HsLin04 (D) and HsLin05 (E) for biofilm inhibition. Metabolic activity is measured using CTB. Sigmoidal curves were generated using data of at least three independent experiments (n≥ 3), using the model Y=Bottom+(Top-Bottom)/(1 +10^A((LoglC50-X)*HillSlope)) in GraphPad Prism. Dose response curves of caspofungin in the presence of synergistic concentrations of HsLin are presented. Black arrows represent synergy. The lines and corresponding numbers in the.graphs indicate the different HsLin doses for each curve (μΜ).

Fig. 9 shows synergy between caspofungin and HsLin06 for biofilm inhibition. Metabolic activity is measured using CTB. Sigmoidal curves were generated using data of at least three independent experiments (n > 3), using the model Y=Bottom+(Top- Bottom)/(1+10^A((LoglC50-X)^*HillSlope)) in GraphPad Prism. Dose response curves of caspofungin in the presence of synergistic concentrations of HsLin06 are presented. Black arrows represent synergy. The lines and corresponding numbers in the graphs indicate the different HsLin doses for each curve (μΜ).

Fig. 10 shows HsLin06_18 potentiation of (A) caspofungin, (B) micafungin and (C) anidulafungin for C. albicans biofilm formation inhibition, using microtiter plates. Dose- response curves of the echinocandin (caspofungin, micafungin and anidulafungin) with a concentration series of HsLin06_18 were presented respectively in A, B and C, with the different connecting lines corresponding to the HsLin06_18 concentration. Black arrows represent HsLin06_18's potentiation effect. Means ± SEM of triplicates are represented.

Fig. 11 shows HsLin06_18 potentiation of caspofungin for C. albicans biofilm formation inhibition, using cathethers. Survival of the viable biofilm-associated cells on the catheters was determined via CFU counting. CASPO: caspofungin. Data are means ± SEM for n = 3 independent experiments. Significant differences between all treatments were determined via one-way ANOVA followed by Tuckey multiple comparison, with * representing P < 0.05.

Fig. 12 shows HsLin06_18 does not affect HepG2 cell viability. HepG2 cells were treated with water (control treatment) or HsLin06_18 (0.09 μΜ - 93 μΜ) for 24 hours. Cell viability was determined by MTT staining, and results were expressed relative to cells receiving the control treatment. Mean and SEM of two experiments in quadruplicate is shown. No statistically significant differences were found in cell viability between untreated (control treatment) and HsLin06_18-treated cells up to the highest tested HsLin06_18 concentration (i.e. 93 μΜ) (One way ANOVA-Tukey test; P<0.05 was defined as statistically significant).

Fig. 13 shows potentiation of caspofungin with (A) HsLin06, (B) HsLin06_18 and (C) HsLin06_18_13 for C. albicans biofilm formation inhibition. Dose response curves of caspofungin in the presence of subinhibitory concentrations of HsLin06, HsLin06_18 or HsLin06_18_13 are represented. Sigmoidal curves were generated in GraphPad Prism using data from at least three (n ≥ 3) independent experiments and the fitting model Y = Bottom+(Top-Botom)/(1+10((LoglC50-X)*HillSlope)). Data points are means ± SEMs. The lines and corresponding numbers in the graphs indicate the different HsLin doses for each curve (μΜ). DETAILED DESCRIPTION

The inventors have shown that the plant defensin HsAFPI (SEQ ID No. 1 ) or peptide derivatives (SEQ ID Nos. 2 to 17) thereof show inhibitory activity against microbial biofilms and that said plant defensin and peptide derivatives thereof are potentiating compounds which act synergistically with antifungal agents, such as caspofungin, anidulafungin, micafungin and amphotericin B, against microbial biofilms and/or planktonic cells, increasing the susceptibility and sensitivity of microbial biofilms and/or of planktonic cells, in particular fungal biofilms or cells, such as Candida biofilms, to said antifungal drugs.

Accordingly, the present invention provides an isolated peptide or derivatives thereof, or a composition comprising said isolated peptide, and at least one antifungal agent, preferably an echinocandin or a polyene, more preferably caspofungin, anidulafungin, micafungin, or amphotericin B, for use in the treatment or prevention of fungal biofilms or fungal biofilms associated infections or diseases, particularly Candida biofilms, more particularly C. albicans biofilms. Typically the compositions provide means for carrying out the methods of the invention.

The present invention therefore overcomes the increasing problems of biofilms, in particular of fungal biofilms on different biotic and abiotic surfaces such as on medical devices outside or within the human body and which escape conventional antifungal treatment.

The scope of the applicability of the present invention will become apparent from the detailed description and drawings provided below. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

DEFINITIONS

Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances, of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to one of ordinary skill in the art from the present invention, in one or more embodiments. Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated.

Similarly, it should be appreciated that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the present invention and aiding in the understanding of one or more of the various inventive aspects.

As used herein the terms "reducing", "suppressing", "inhibiting", "eradicating" or the like in reference to microorganisms or a biofilm or biofilm formation means complete or partial inhibition (more than 50%, preferably more than 90%, still more preferably more than 95% or even more than 99%) of microorganisms or biofilm formation (in the term of number of remaining cells) and/or development and also includes within its scope the reversal of microorganisms or biofilm development or processes associated with microorganisms or biofilm formation and/or development. Further, inhibition may be permanent or temporary. In terms of temporary inhibition, microorganisms or biofilm formation and/or development may be inhibited for a time sufficient to produce the desired effect (for instance at least 5 days, preferably at least 10 days). Preferably, the inhibition of microorganisms or a biofilm is complete and/or permanent (no persisters) ("eradicating").

As used herein, "preventing" or the like in reference to microorganisms or a biofilm or biofilm formation means complete or partial prevention (more than 50%, preferably more than 90%, still more preferably more than 95% or even more than 99%) of microorganisms or biofilm formation (in the term of number of remaining cells) and also includes within its scope processes associated with microorganisms or biofilm formation. Further, prevention may be permanent or temporary. In terms of temporary prevention, microorganisms or biofilm formation may be inhibited for a time sufficient to produce the desired effect (for instance at least 5 days, preferably at least 10 days). Preferably, the prevention of microorganisms or biofilm is complete and/or permanent.

As used herein the term "exposing" means administering to, or otherwise bringing into contact with. A microorganism or biofilm may be exposed to an active agent directly or indirectly. Typically direct exposure refers to administration of the agent to the microorganism or biofilm to be treated or otherwise bringing the microorganism or biofilm into contact with the agent itself. Typically indirect exposure refers to the administration of a precursor of the active agent or a compound or molecule capable of generating, either solely or in reaction with other compounds or molecules, the active agent to the microorganism or biofilm or otherwise bringing the microorganism or biofilm into contact therewith. Similarly, the terms "treat" and "treating" and variations thereof as used herein mean administering to, or otherwise bringing into contact with.

A "microorganism", sometimes referred to as a microbe, is any organism too small to be visible to the naked eye. Bacteria, viruses, protozoans, fungi and some algae are microorganisms.

The term "antimicrobial" as used herein means that the composition of the present invention reduces, eradicates, inhibits or prevents the growth or proliferation of, a microbe or microorganism. The term "antimicrobial" may hence refer to antibacterial, antibiofilm, antiviral, antiprotozoan and/or antifungal activity. Similarly, the term "antifungal" means that a compound or composition of the present invention reduces, eradicates, inhibits or prevents the growth or proliferation of a fungus.

As used herein the term "biofilm" refers to a mode of microbial growth comprising sessile cells, usually within a complex and highly heterogeneous matrix of extracellular polymers, and characterized by a reduced sensitivity to antifungal agents.

In the context of the present invention, "biofilms" can contain single species (e.g. a fungi/yeast such as C. albicans) or multiple species microorganisms (such as C. albicans, C. glabrata and other microorganisms, preferably yeasts and/or fungi or even prokaryotes). In a preferred embodiment said biofilm is a fungal biofilm, more preferably a Candida species biofilm, comprising C. albicans, C. glabrata, and/or C. /cruse/, an Aspergillus species (e.g. A. flavus, A. fumigatus, A. clavatus) biofilm or a Fusarium species (e.g. F. oxysporum, F. culmorum) biofilm, most preferably a Candida albicans biofilm. Accordingly, the term "fungal biofilm" refers to a biofilm comprising fungal species, and the term "Candida biofilm" refers to a biofilm comprising Candida species.

The term "consisting essentially of C. albicans" refers to a percentage (number of C. albicans celhtotal cell) in the biofilm. Preferably the percentage is above 50%, more preferably above 75%, still more preferably above 90% and/or this term refers to the fact that C. albicans is present in a concentration sufficient to provoke the biofilm.

Preferably, the biofilms are associated with microbial infection (e.g., burns, wounds or skin ulcers) or a disease condition including, without limitation, dental caries, periodontal disease, prostatitis, osteomyelitis, septic arthritis, and cystic fibrosis.

Typically, biofilms, preferably a fungal biofilms, are associated with a microbial (fungal) infection on medical devices like indwelling intravascular catheters and in the oral cavity (e.g. on dental implants). Biofilms may be associated with a surface, e.g., a solid support surface. Such surface can be the surface of any structure in animals or humans. For example, such surface can be any epithelial surface, mucosal surface, or any host surface associated with microbial infection, e.g. persistent and chronic microbial infections. The surface can also include any surface of a bio-device in an animal or human, including without limitation, bio- implants such as dental implants, bone prostheses, heart valves, pacemakers and indwelling catheters. The microbial or fungal biofilm can also be associated with the oral cavity, including the surface of dental implants or speech prostheses.

In addition to surfaces associated with biofilm formation in a biological environment, the surfaces can also be any surface associated with industrial biofilm formation. For example, the surfaces being treated can be any surface associated with biofouling of pipelines, heat exchangers, air filtering devices, or contamination of computer chips or water-lines in surgical units like those associated with dental hand-pieces.

The term "controlled release" as used herein refers to a relatively slow or delayed or prolonged release of a bio-active compound from a device in its environment. Particularly, an 80% release of the bio-active compound into an aqueous fluid at a pH between 1.0 and 8.0 is only obtained when a period of time of at least 30 minutes, preferably at least 60 min, at least 24 hours, or at least 48 hours has passed, even more preferably when a period of time lasting several hours, days, weeks or even months has passed (i.e. 20% (or more) of the bio- active compound remains in the device after at least 30, 60 min, 24 h, 48 h or even several days).

As used herein the term "effective amount" or "effective concentration" includes within its meaning a non-toxic but sufficient amount or concentration of an agent to provide the desired effect. The exact amount/concentration required will vary depending on factors such as the species of microorganism(s) being treated, the extent, severity and/or age of a biofilm being treated, whether the biofilm is surface-associated or suspended, the particular agent(s) being administered and the mode of administration and so forth. Thus, it is not possible to specify an exact "effective amount". However, for any given case, an appropriate "effective amount" may be determined by one of ordinary skill in the art using only routine experimentation. "Plant defensin" as used herein refers to small, cationic peptides with a length of approximately 45-54 amino acids. Their structure typically comprises a conserved structure known as a Cysteine-stabilized αβ-motif (CSaP) with a prominent a-helix and a triple- stranded antiparallel β-sheet that is stabilized by four disulphide bridges. Although the tertiary structure of some plant defensins is known, the structure of many defensins is yet to be determined. Plant defensins are present in all plant families, including the Brassicaceae, Fabaceae and Solanaceae. These peptides were primarily found in the seeds, but leaves and flowers are also common sources. They are either constitutively expressed in storage and reproductive organs or produced upon pathogenic attack or injury as part of a systemic defence response. In addition, production of plant defensin is also induced in response to environmental stress, such as drought, and signaling molecules, including methyl jasmonate, ethylene and salicylic acid (1 ). Plant defensins exhibit antimicrobial activity against a broad range of microorganisms, whereas they are in general non-toxic to human cells. Their activity is primarily directed against fungi, but bactericidal and insecticidal actions have also been reported. To date, several fungal targets have been identified, including membrane sphingolipids and phospholipids. Upon interaction with the fungal membrane, plant defensins are either internalized into the cell and interact with cytosolic or nuclear proteins, or they remain localized at the cell wall or membrane of the fungus. The mechanisms by which plant defensins induce fungal cell death are diverse, but common aspects are observed. These include the production of reactive oxygen species and the induction of apoptosis.

"HsAFPI" as used herein refers to a plant defensin from coral bells {"Heuchera sanguinea") with SEQ ID No. 1 which was previously characterized by Osborn and colleagues (2).

The present invention also includes analogs and derivatives of the plant defensins of the present invention, provided that the analog or derivative has a detectable antimicrobial, more preferably a detectable antifungal activity. It is not necessary that the analog or derivative has activity identical to the activity of the plant defensin from which the analog or derivative variant is derived.

The term "potentiating compounds" as used herein in reference to microorganisms or biofilms or biofilm formation, refers to compounds that increase the susceptibility and sensitivity of biofilms (and/or sessile cells) of microorganisms, in particular of fungal species, to antifungal drugs, even in situations where the potentiating compounds alone, or the antifungal drugs alone, do not reduce, eradicate, inhibit or prevent biofilm formation and/or development.

In contrast to the potentiating compounds alone, the combination of both the potentiating compound and an antimicrobial agent, preferably an antifungal agent, synergizes against microorganisms in the form of biofilms (and/or of sessile cells) in reducing the number of microbial cells, in particular in reducing the number of fungal species, preferably Candida species, more preferably Candida albicans, or of the frequency of infections.

In addition, because of the sensitizing effect of the potentiating compound, combination therapy comprising one or more potentiating compounds and one or more antimicrobial agents, preferably antifungal agents, preferably antifungal agents selected from the group consisting of echinocandins or polyenes, more preferably caspofungin, anidulafungin, micafungin or amphotericin B, allows reducing the antifungal dose of the antifungal agent, or even applying an antifungal concentration of the antifungal agent which would be sub-lethal for a microorganism in the form of a biofilm and/or of sessile cells in the absence of the potentiating compound.

Also, because of the sensitizing effect of the potentiating compound, combination therapy comprising one or more potentiating compounds and one or more antimicrobial agents, preferably antifungal agents, allows reducing the antifungal dose of the antifungal agent, thereby also lowering the potential side effects of the antifungal agents, preferably antifungal agents selected from the group consisting of echinocandins or polyenes, more preferably caspofungin, anidulafungin, micafungin or amphotericin B.

Particularly, the activity of said antifungal agents, particularly echinocandins, such as caspofungin, anidulafungin or micafungin, or polyenes, such as amphothericin B, against a microorganism, in particular a fungal species, such as Candida species, in the form of a biofilm and/or sessile cells, can be improved by combining said antifungal agent(s) with a potentiating compound, and thus concomitantly resulting in a reduction of its minimal inhibitory concentration (MIC) against a microorganism infection, especially a fungal infection and/or Candida (C. albicans) in the form of a biofilm.

The potentiating compound may also be used in combination with one or more antifungal agent(s) against a fungal species, and/or yeast, such as Candida infection in the form of a biofilm at a reduced amount, which is the amount effective against this (fungal) infection in planktonic form (non sessile, non forming a biofilm).

The terms "peptide" or "polypeptide" as used herein refer to a polymer of amino acid residues, including D-amino acids, typically L-amino acids, and to variants and synthetic analogues of the same, encompassing native peptides (including synthetically synthesized or recombinant peptides), modified peptides and peptidomimetics (typically, synthetically synthesized peptides and peptide analogues). Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. A peptide of the present invention may also be produced by recombinant expression in prokaryotic and eukaryotic engineered cells other than plant cells, such as bacteria, fungi, or animal cells. Suitable expression systems are known to those skilled in the art. By "recombinant (poly)peptide" is meant a (poly)peptide made using recombinant techniques, i.e., through the expression of a recombinant or synthetic polynucleotide. Suitable expression protocols and strategies are known to the skilled person and can be retrieved e.g. from Sambrook, 2001. When the polypeptide is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the peptide preparation.

The term "peptide" as used herein also refers to modified peptides wherein the modifications render the peptides even more stable e.g. while in a body. Such modifications include, but are not limited to N-terminus modification, C-terminus modification, peptide bond modification, including, but not limited to, CH₂-NH, CH₂-S, CH₂-S=0, 0=C-NH, CH2-O, CH₂- CH₂, S=C-NH, CH=CH or CF=CH, backbone modifications, cyclisation (including by coupling the C-terminus to the N-terminus of the peptide or by Cys-Cys disulphide linkages) and residue modification. Other modified peptides include peptides comprising D-amino acids, retro modified peptides, inverso modified peptides, retro-inverso modified peptides or cyclic peptides. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C.A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992).

The term "derivative(s) of a peptide" or "derivative" as used herein refers to peptides or polypeptides which, compared to the amino acid of a peptide according to this invention, may comprise: (i) substitutions, deletions or additions of naturally and non-naturally occurring amino acid residues (including D-amino acids); (ii) amino acid residues that are substituted by corresponding naturally or non-naturally altered amino acids; (iii) naturally occurring altered, (such as glycosylated, acylated, myristoylated or phosphorylated amino acids) or non-naturally occurring amino acid residues (such as biotinylated amino acids, or amino acids modified after CNBr treatment); (iv) peptides carrying post-translational modifications. A derivative may also be a retro, inverso or retro-inverso modified form of any of the peptides according to this invention. A derivative may also comprise one or more non-amino acid substituents compared to the amino acid from which it is derived, for example a reporter molecule or other ligand, covalently or non-covalently bound to the amino acid such as, for example, a reporter molecule which is bound to facilitate its detection. Preferably, amino acid substitutions comprise conservative amino acid substitutions. One or more amino acid residues may be introduced into a predetermined site in said peptide of the present invention. Insertions can comprise amino-terminal and/or carboxy-terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Examples of amino- or carboxy- terminal fusion proteins or peptides include the binding domain or activation domain of a transcriptional activator as used in the yeast two-hybrid system, phage coat proteins, (histidine)6-tag, glutathione S-transferase-tag, protein A, maltose-binding protein, dihydrofolate reductase, Tag* 00 epitope, c-myc epitope, FLAG®-epitope, lacZ, CMP (calmodulin-binding peptide), HA epitope, protein C epitope and VSV epitope.

A "retro modified" peptide is a peptide that is made up of amino acids in which the amino acid residues are assembled in opposite direction to the native peptide with respect to which it is retro modified. Where the native peptide comprises L-amino acids, the "retro modified" peptide will also comprise L-amino acids. However, where the native peptide comprises D- amino acids, the "retro modified" peptide will comprise D-amino acids. An "inverso modified" peptide is a peptide in which the amino acid residues are assembled in the same direction as the native peptide with respect to which it is inverso modified, but the chirality of the amino acids is inverted. Thus, where the native peptide comprises L-amino acids, the "inverso modified" peptide will comprise D-amino acids. Where the native peptide comprises D-amino acids, the "inverso modified" peptide will comprise L-amino acids. A "retro-inverso modified" peptide refers to a peptide that is made up of amino acid residues which are assembled in the opposite direction and which have inverted chirality with respect to the native peptide to which it is retro-inverso modified. A retro-inverso analogue has reversed termini and reversed direction of peptide bonds while approximately maintaining the topology of the side chains as in the native peptide sequence. Such retro-inverso peptidomimetics may be made using methods known in the art, for example such as those described in Meziere et al (1997) J. Immunol. 159, 3230-3237, incorporated herein by reference. Partial retro-inverso peptide analogues are polypeptides in which only part of the sequence is reversed and replaced with enantiomeric amino acid residues. Processes for making such analogues are described in Pessi, A., Pinori, M., Verdini, A. S. & Viscomi, G. C. (1987) "Totally solid phase synthesis of peptide(s)- containing retro-inverted peptide bond, using crosslinked sarcosinyl copolymer as support", European Patent 97994-B.

The term "non-natural amino acid" refers to a non coded, non proteinogenic amino acid, or a post-translationally modified variant thereof that is not naturally encoded or found in the genetic code of any organisms. In particular, the term refers to an amino acid that is not one of the 20 common amino acids or pyrrolysine, selenocysteine or N-formylmethionine, or post- translationally modified variants thereof.

The term "sequence identity" as used herein refers to the extent that sequences are identical on an amino acid-by-amino acid basis over a window of comparison. Sequence identity is generally determined by aligning the residues of the two sequences to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical residues, although the amino acids in each sequence must nonetheless remain in their proper order. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met or a non-naturally altered amino acid) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Preferably, sequence identity between two amino acid sequences is determined by comparing said sequences using the Blastp program, available at http://blast.ncbi.nlm.nih.gov/Blast.cgi. Preferably, the default values for all BLAST 2 search parameters are used, including in the case of Blastp: matrix = BLOSUM62; open gap penalty = 11 , extension gap penalty = 1 , gap x-dropoff = 5 0, expect = 10, wordsize = 3, and filter on. "Similarity" refers to the percentage number of amino acids that are identical or constitute conservative substitutions.

The phrase "pharmaceutically acceptable carrier" as used herein refers to any material, substance, or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound with which the active ingredient is formulated in order to facilitate its application or dissemination to the locus to be treated, for instance by dissolving, dispersing or diffusing the said composition, and/or to facilitate its storage, transport or handling without impairing its effectiveness. An adjuvant is included under these phrases. The term "excipient" as used herein refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition (herein incorporated by reference). Suitable pharmaceutical carriers for use in the said pharmaceutical compositions and their formulation are well known to those skilled in the art, and there is no particular restriction to their selection within the present invention. They may also include additives such as wetting agents, dispersing agents, stickers, adhesives, emulsifying agents, solvents, coatings, antibacterial and antifungal agents (for example phenol, sorbic acid, chlorobutanol), isotonic agents (such as sugars or sodium chloride) and the like, provided the same are consistent with pharmaceutical practice, i.e. carriers and additives which do not create permanent damage to mammals. The pharmaceutical compositions of the present invention may be prepared in any known manner, for instance by homogeneously mixing, coating and/or grinding the active ingredients, in a one-step or multi- steps procedure, with the selected carrier material and, where appropriate, the other additives.

Peptide for inhibiting biofilm formation

In a first object the present invention presents an isolated peptide (also herein referred to as "peptide of the present invention") comprising an amino acid sequence which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, 97%, 99% or 100% identical to the amino acid sequence GAXHYQFPSVKX (SEQ ID No. 8), QQXKDREHFAYG (SEQ ID No. 9) or WSGHXGSSSKXS (SEQ ID No. 10) or a derivative thereof, wherein X stands for Cystein or a-aminobutyric acid, and wherein said isolated peptide has microbial antibiofilm activity, particularly wherein said isolated peptide inhibits microbial biofilm formation and development and/or potentiates the effect of an antifungal agent. Preferably, said isolated peptide potentiates the effect of an antifungal agent on a fungal biofilm.

Preferably, said isolated peptide consists of 12 to 54 amino acids. Preferably, a peptide of the present invention consists of 12 to 44 amino acids. More preferably, a peptide of the present invention comprises at least 12 amino acids, for instance at least 14, 16, 18, 20, 22, 24 or at least 28 amino acids. It is further preferred that a peptide of the present invention does not comprise more than 54 amino acids, for instance not more than 24, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 or 52 amino acids. Preferably, said isolated peptide does not comprise more than 44 amino acids. More preferably, said isolated peptide consists of 24 amino acids. More preferably, said isolated peptide consists of 19, 20, 21 , 22 or 23 amino acids.

In a preferred embodiment of the present invention, said isolated peptide comprises an amino acid sequence which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, 97%, 99% or 100% identical to the amino acid sequences selected from the group consisting of GAXHYQFPSVKX (SEQ ID No. 8), GAXHYQFPSVKXFXKR (SEQ ID No. 1 1 ) or AYGGAXHYQFPSVKX (SEQ ID No. 12), or a derivative thereof wherein X stands for Cystein or a-aminobutyric acid, and wherein said isolated peptide has microbial antibiofilm activity, particularly wherein said isolated peptide inhibits microbial biofilm formation and development and/or potentiates the effect of an antifungal agent. Preferably, said isolated peptide potentiates the effect of an antifungal agent on a fungal biofilm. In another preferred embodiment of the present invention, said isolated peptide comprises an amino acid sequence which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, 97%, 99% or 100% identical to the amino acid sequences selected from the group consisting of EHFAYGGAXHYQFPSVKXFXKRQ (SEQ ID No. 13), AYGGAXHYQFPSVKXFXKRQX (SEQ ID No. 14), YGGAXHYQFPSVKXFXKRQX (SEQ ID No. 15), HFAYGGAXHYQFPSVKXFXK (SEQ ID No. 16), or FAYGGAXHYQFPSVKXFXK (SEQ ID No. 17), or a derivative thereof wherein X stands for Cystein or a-aminobutyric acid, and wherein said isolated peptide has microbial antibiofilm activity, particularly wherein said isolated peptide inhibits microbial biofilm formation and development and/or potentiates the effect of an antifungal agent. Preferably, said isolated peptide potentiates the effect of an antifungal agent on a fungal biofilm. Preferably, said isolated peptide comprises an amino acid sequence which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, 97%, 99% or 100% identical to the amino acid sequences selected from the group consisting of EHFAYGGAXHYQFPSVKXFXKRQ (SEQ ID No. 13), YGGAXHYQFPSVKXFXKRQX (SEQ ID No. 15), or FAYGGAXHYQFPSVKXFXK (SEQ ID No. 17). More preferably, said isolated peptide comprises an amino acid sequence which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, 97%, 99% or 100% identical to the amino acid sequence FAYGGAXHYQFPSVKXFXK (SEQ ID No. 17).

In another preferred embodiment, said isolated peptide is selected from the group consisting of EHFAYGGAXHYQFPSVKXFXKRQ (SEQ ID No. 13), AYGGAXHYQFPSVKXFXKRQX (SEQ ID No. 14), YGGAXHYQFPSVKXFXKRQX (SEQ ID No. 15), HFAYGGAXHYQFPSVKXFXK (SEQ ID No. 16), FAYGGAXHYQFPSVKXFXK (SEQ ID No. 17), or FAYGGAXHYQFPAVKXFXK (SEQ ID No. 18), or a derivative thereof wherein X stands for Cystein or a-aminobutyric acid, and wherein said isolated peptide has microbial antibiofilm activity, particularly wherein said isolated peptide inhibits microbial biofilm formation and development and/or potentiates the effect of an antifungal agent. Preferably, said isolated peptide potentiates the effect of an antifungal agent on a fungal biofilm. Preferably, said isolated peptide potentiates the effect of an antifungal agent on a fungal biofilm. Preferably, said isolated peptide is selected from the group consisting of EHFAYGGAXHYQFPSVKXFXKRQ (SEQ ID No. 13), YGGAXHYQFPSVKXFXKRQX (SEQ ID No. 15), FAYGGAXHYQFPSVKXFXK (SEQ ID No. 17), or FAYGGAXHYQFPAVKXFXK (SEQ ID No. 18). More preferably, said isolated peptide is FAYGGAXHYQFPSVKXFXK (SEQ ID No. 17) or FAYGGAXHYQFPAVKXFXK (SEQ ID No. 18).

In another preferred embodiment of the present invention, said isolated peptide comprises an amino acid sequence which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, 97%, 99% or 100% identical to the amino acid sequences selected from the group consisting of DGVKLXDVPSGTWSGHXGSSSKXS (SEQ ID No. 2), DVPSGTWSGHXGSSSKXSQQXKDR (SEQ ID No. 3), WSGHXGSSSKXSQQXKDREHFAYG (SEQ ID No. 4),

SSSKXSQQXKDREHFAYGGAXHYQ (SEQ ID No. 5), QQXKDREHFAYGGAXHYQFPSVKX (SEQ ID No. 6), or EHFAYGGAXHYQFPSVKXFXKRQX (SEQ ID No. 7), or a derivative thereof, wherein X stands for Cystein or a-aminobutyric acid, and wherein said isolated peptide has microbial antibiofilm activity, particularly wherein said isolated peptide inhibits microbial biofilm formation and development and/or potentiates the effect of an antifungal agent. Preferably, said isolated peptide potentiates the effect of an antifungal agent on a fungal biofilm.

In another preferred embodiment, said isolated peptide is selected from the group consisting of DGVKLCDVPSGTWSGHCGSSSKCSQQCKDREHFAYGGACHYQFPSVKCFCKRQC (SEQ ID No. 1 ), DGVKLXDVPSGTWSGHXGSSSKXS (SEQ ID No. 2), DVPSGTWSGHXGSSSKXSQQXKDR (SEQ ID No. 3), WSGHXGSSSKXSQQXKDREHFAYG (SEQ ID No. 4),

SSSKXSQQXKDREHFAYGGAXHYQ (SEQ ID No. 5), QQXKDREHFAYGGAXHYQFPSVKX (SEQ ID No. 6), EHFAYGGAXHYQFPSVKXFXKRQX (SEQ ID No. 7), GAXHYQFPSVKX (SEQ ID No. 8), QQXKDREHFAYG (SEQ ID No. 9) or WSGHXGSSSKXS (SEQ ID No. 10), or a derivative thereof, wherein X stands for Cystein or a-aminobutyric acid, and wherein said isolated peptide has microbial antibiofilm activity, particularly wherein said isolated peptide inhibits microbial biofilm formation and development and/or potentiates the effect of an antifungal agent. Preferably, said isolated peptide potentiates the effect of an antifungal agent on a fungal biofilm.

Typically, the peptides of the present invention as described herein are used in the treatment or prevention of a microbial biofilm associated condition or infection in a human or animal subject. Preferably, said microbial biofilm associated condition or infection is a fungal or yeast biofilm associated condition or infection, more preferably said biofilm comprises Candida species, even more preferably said biofilm comprises Candida albicans, even more preferably said biofilm consists essentially of Candida albicans.

Antibiofilm Composition

In a second object the present invention presents an antibiofilm composition (also herein referred to as "composition of the present invention") comprising an isolated peptide of the present invention comprising an amino acid sequence which is at least 70% or 80%, more preferably at least 90%, more preferably at least 95%, 97%, 99% or 100% identical to amino acid sequence GAXHYQFPSVKX (SEQ ID No. 8), QQXKDREHFAYG (SEQ ID No. 9) or WSGHXGSSSKXS (SEQ ID No. 10) or a derivative thereof, wherein X stands for Cystein or a-aminobutyric acid, and wherein said isolated peptide has microbial antibiofilm activity, particularly wherein said isolated peptide inhibits microbial biofilm formation and development and/or potentiates the effect of an antifungal agent. Preferably, said isolated peptide potentiates the effect of an antifungal agent on a fungal biofilm.

Preferably, said isolated peptide consists of 12 to 54 amino acids. Preferably, a peptide comprised in the composition of the second object of the present invention consists of 12 to 44 amino acids. More preferably, said peptide comprises at least 12 amino acids, for instance at least 14, 16, 18, 20, 22, 24 or at least 28 amino acids. It is further preferred that a peptide of the present invention does not comprise more than 54 amino acids, for instance not more than 24, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 or 52 amino acids. Preferably, said isolated peptide does not comprise more than 44 amino acids. More preferably, said isolated peptide consists of 24 amino acids. More preferably, said isolated peptide consists of 19, 20, 21, 22 or 23 amino acids.

In a preferred embodiment of the second object of the present invention, said composition comprises an isolated peptide comprising an amino acid sequence which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, 97%, 99% or 100% identical to the amino acid sequences selected from the group consisting of GAXHYQFPSVKX (SEQ ID No. 8), G AXH YQ FPS VKXFXKR (SEQ ID No. 11) or AYGGAXHYQFPSVKX (SEQ ID No. 12), or a derivative thereof wherein X stands for Cystein or a-aminobutyric acid, and wherein said isolated peptide has microbial antibiofilm activity, particularly wherein said isolated peptide inhibits microbial biofilm formation and development and/or potentiates the effect of an antifungal agent. Preferably, said isolated peptide potentiates the effect of an antifungal agent on a fungal biofilm.

In another preferred embodiment of the second object of the present invention, said composition comprises an isolated peptide comprising an amino acid sequence which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, 97%, 99% or 100% identical to the amino acid sequences selected from the group consisting of EHFAYGGAXHYQFPSVKXFXKRQ (SEQ ID No. 13), AYGGAXHYQFPSVKXFXKRQX (SEQ ID No. 14), YGGAXHYQFPSVKXFXKRQX (SEQ ID No. 15), HFAYGGAXHYQFPSVKXFXK (SEQ ID No. 16), or FAYGGAXHYQFPSVKXFXK (SEQ ID No. 17), or a derivative thereof wherein X stands for Cystein or α-aminobutyric acid, and wherein said isolated peptide has microbial antibiofilm activity, particularly wherein said isolated peptide inhibits microbial biofilm formation and development and/or potentiates the effect of an antifungal agent. Preferably, said isolated peptide potentiates the effect of an antifungal agent on a fungal biofilm. Preferably, said isolated peptide comprises an amino acid sequence which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, 97%, 99% or 100% identical to the amino acid sequences selected from the group consisting of EHFAYGGAXHYQFPSVKXFXKRQ (SEQ ID No. 13), YGGAXHYQFPSVKXFXKRQX (SEQ ID No. 15) or FAYGGAXHYQFPSVKXFXK (SEQ ID No. 17). More preferably, said isolated peptide comprises an amino acid sequence which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, 97%, 99% or 100% identical to the amino acid sequence FAYGGAXHYQFPSVKXFXK (SEQ ID No. 17).

In another preferred embodiment of the second object of the present invention, said composition comprises an isolated peptide selected from the group consisting of EHFAYGGAXHYQFPSVKXFXKRQ (SEQ ID No. 13), AYGGAXHYQFPSVKXFXKRQX (SEQ ID No. 14), YGGAXHYQFPSVKXFXKRQX (SEQ ID No. 15), HFAYGGAXHYQFPSVKXFXK (SEQ ID No. 16), FAYGGAXHYQFPSVKXFXK (SEQ ID No. 17), or FAYGGAXHYQFPAVKXFXK (SEQ ID No. 18), or a derivative thereof wherein X stands for Cystein or ot-aminobutyric acid, and wherein said isolated peptide has microbial antibiofilm activity, particularly wherein said isolated peptide inhibits microbial biofilm formation and development and/or potentiates the effect of an antifungal agent. Preferably, said isolated peptide potentiates the effect of an antifungal agent on a fungal biofilm. Preferably, said isolated peptide is selected from the group consisting of EHFAYGGAXHYQFPSVKXFXKRQ (SEQ ID No. 13), YGGAXHYQFPSVKXFXKRQX (SEQ ID No. 15), FAYGGAXHYQFPSVKXFXK (SEQ ID No. 17) or FAYGGAXHYQFPAVKXFXK (SEQ ID No. 18). More preferably, said isolated peptide is FAYGGAXHYQFPSVKXFXK (SEQ ID No. 17) or FAYGGAXHYQFPAVKXFXK (SEQ ID No. 18).

In another preferred embodiment of the second object of the present invention, said composition comprises an isolated peptide comprising an amino acid sequence which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, 97%, 99% or 100% identical to the amino acid sequences selected from the group consisting of DGVKLXDVPSGTWSGHXGSSSKXS (SEQ ID No. 2), DVPSGTWSGHXGSSSKXSQQXKDR (SEQ ID No. 3),

WSGHXGSSSKXSQQXKDREHFAYG (SEQ ID No. 4),

SSSKXSQQXKDREHFAYGGAXHYQ (SEQ ID No. 5), QQXKDREHFAYGGAXHYQFPSVKX (SEQ ID No. 6), or EHFA YGGAXHYQFPSVKXFXKRQX (SEQ ID No. 7), or a derivative thereof, wherein X stands for Cystein or a-aminobutyric acid, and wherein said isolated peptide has microbial antibiofilm activity, particularly wherein said isolated peptide inhibits microbial biofilm formation and development and/or potentiates the effect of an antifungal. Preferably, said isolated peptide potentiates the effect of an antifungal agent on a fungal biofilm.

In another preferred embodiment of the second object of the present invention, said composition comprises an isolated peptide selected from the group consisting of DGVKLCDVPSGT SGHCGSSSKCSQQCKDREHFAYGGACHYQFPSVKCFCKRQC (SEQ ID No. 1 ), DGVKLXDVPSGTWSGHXGSSSKXS (SEQ ID No. 2), DVPSGTWSGHXGSSSKXSQQXKDR (SEQ ID No. 3),

WSGHXGSSSKXSQQXKDREH FAYG (SEQ ID No. 4),

SSSKXSQQXKDREHFAYGGAXHYQ (SEQ ID No. 5), QQXKDREHFAYGGAXHYQFPSVKX (SEQ ID No. 6), EHFAYGGAXHYQFPSVKXFXKRQX (SEQ ID No. 7), GAXHYQFPSVKX (SEQ ID No. 8), QQXKDREHFAYG (SEQ ID No. 9) or WSGHXGSSSKXS (SEQ ID No. 10), or a derivative thereof, wherein X stands for Cystein or a-aminobutyric acid, and wherein said isolated peptide has microbial antibiofilm activity, particularly wherein said isolated peptide inhibits microbial biofilm formation and development and/or potentiates the effect of an antifungal agent. Preferably, said isolated peptide potentiates the effect of an antifungal agent on a fungal biofilm.

Typically, the compositions according to the present invention comprise an isolated peptide of the present invention, and at least one antimicrobial (such as an antibacterial, antiprotozoal or antifungal) agent, preferably in an effective amount thereof. Preferably, said at least one antimicrobial agent is selected from the following compounds: antifungal agents, such as polyenes (e.g. amphotericin B, nystatin, natamycin); azoles (e.g. miconazole, fluconazole, itraconazole, voriconazole); allylamines (e.g. terbinafine); echinocandins (e.g. caspofungin, anidulafungin, micafungin); gentamycin, ofloxacin, ciprofloxacin; piperazine-1- carboxamidine derivatives (W 02010068296); antifungal peptides with antibiofilm activity; other antimycotic substances such as cetyltrimethylammonium bromide and the like. Preferably, said antimicrobial agent is an antifungal agent, more preferable said antimicrobial agent is an antifungal agent selected from the group of echinocandins or polyenes, even more preferably said antifungal agent is caspofungin, anidulafungin, micafungin or amphotericin B. Even more preferably, said antifungal agent is caspofungin.

Typically, the compositions according to the present invention are used in the treatment or prevention of a microbial biofilm associated condition or infection in a human or animal subject. Preferably, said microbial biofilm associated condition or infection is a fungal or yeast biofilm associated condition or infection, more preferably said biofilm comprises Candida species, even more preferably said biofilm comprises Candida albicans, even more preferably said biofilm consists essentially of Candida albicans. Typically, the compositions according to the present invention are used for treatment or prevention of a fungal biofilm associated condition or disease in a human or animal subject, wherein said subject has been implanted with a medical device, which is infected or at risk of being infected with a fungal biofilm. Preferably, said medical device is selected from the group consisting of catheters, stents, surgical plates, prostheses, valves or pins, artificial joints, pacemakers, contact lenses and bio-implants.

The composition of the present invention may, depending upon the desired mode of administration or application, be formulated in very different forms such as, but not limited to, liquids, gels, foams, semi-solids and solids. Said antibiofilm composition may further comprise one or more non-active pharmaceutically acceptable carriers, adjuvants, excipients and/or diluents (i.e. ingredients that do not interfere with the antibiofilm function of the active peptide). A pharmaceutically acceptable carrier is a nontoxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Pharmaceutically acceptable excipients, ingredients, adjuvants and carriers are well known, and one skilled in the pharmaceutical art can easily select them for any particular route of administration (Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985).

The composition of the present invention may be formulated for topical administration (e.g., as a lotion, cream, spray, gel, or ointment). Such topical formulations are useful in treating or inhibiting microbial and/or fungal and/or biofilm presence or infections on the eye, skin, and mucous membranes such as mouth, vagina and the like. Examples of formulations in the market place include topical lotions, creams, soaps, wipes, and the like. It may be formulated into liposomes to reduce toxicity or increase bioavailability. Other methods of administration will be known to those skilled in the art.

Preparations for parenteral administration of a composition of the present invention include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of nonaqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters such as ethyl oleate. Examples of aqueous carriers include water, saline, and buffered media, alcoholic/aqueous solutions, and emulsions or suspensions. Examples of parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives such as, other antimicrobials, anti-oxidants, cheating agents, inert gases and the like also can be included.

The composition of the present invention may also be in the form of a kit wherein each agent is kept separate until effective use. Methods for inhibiting biofilm formation

In a third object the present invention presents methods for reducing, eradicating, inhibiting or preventing microbial biofilms or biofilm formation, preferably fungal biofilms or biofilm formation, on a solid support surface or other medium susceptible to biofilm formation, preferably on a surface or medium outside the body of a human or animal subject, wherein said surface or medium carrying said biofilm or susceptible to said biofilm formation is exposed to at least one isolated peptide of the present invention or a derivative thereof, or to a composition of the present invention. Preferably said composition comprises at least one isolated peptide of the present invention or a derivative thereof, and at least one antimicrobial agent. Preferably said at least one antimicrobial agent is an antifungal agent, more preferably said antifungal agent is selected from the groups consisting of echinocandins or polyenes, even more preferably said at least one antifungal agent is caspofungin, anidulafungin, micafungin, or amphotericin B, even more preferably said at least one antifungal agent is caspofungin. Preferably, said biofilm is a Candida biofilm.

Optionally, said method further comprises the step of exposing the surface or medium carrying said biofilm or susceptible to said biofilm formation to at least one antimicrobial agent before, after or concurrent with the isolated peptide of the present invention or a derivative thereof.

The present invention also relates to a method for inhibiting biofilm formation and/or development, wherein said surface or medium susceptible to biofilm formation is treated with at least one peptide of the present invention or a derivative thereof, or a with a composition of the present invention, or wherein said peptide of the present invention is incorporated in said surface or medium susceptible to biofilm formation. Preferably said composition comprises at least one isolated peptide of the present invention or a derivative thereof, and at least one antimicrobial agent. Preferably said at least one antimicrobial agent is an antifungal agent, more preferably said antifungal agent is selected from the groups consisting of echinocandins or polyenes, even more preferably said at least one antifungal agent is caspofungin, anidulafungin, micafungin or amphotericin B, even more preferably said at least one antifungal agent is caspofungin. Preferably, said biofilm is a Candida biofilm.

The present invention also relates to a method for the treatment or prevention of a condition or infection associated with microbial biofilm development, preferably fungal biofilm development, in a human or animal subject comprising administering to said subject an effective amount of at least one peptide of the present invention or of the composition of the present invention. Preferably said composition comprises at least one isolated peptide of the present invention or a derivative thereof, and at least one antimicrobial agent. Preferably said at least one antimicrobial agent is an antifungal agent, more preferably said antifungal agent is selected from the groups consisting of echinocandins or polyenes, even more preferably said at least one antifungal agent is caspofungin, anidulafungin, micafungin or amphotericin B, even more preferably said at least one antifungal agent is caspofungin. Preferably, said fungal biofilm is a Candida biofilm.

It will be readily appreciated by those skilled in the art that according to the methods of the invention each component of the composition of the present invention may be administered at the same time, or sequentially in any order, or at different times, so as to provide the desired effect. Alternatively, the components may be formulated together in a single dosage unit as a combination product.

Another related aspect of the present invention is the combination of a medical device, particularly an implantable medical device, and a composition of the present invention comprising at least one peptide of the present invention or potentiating compound, medicament or bio-active agents for preventing or suppressing microbial biofilms in a patient, preferably a human or animal subject. Preferably said microbial biofilm is a fungal biofilm, more preferably said fungal biofilm is a Candida biofilm.

Another related aspect relates to the preparation (e.g. by coating, incorporation) of a solid support surface, such as a medical device, like implants, plastics or (subcutaneous) catheters, with the addition of a sufficient concentration (or dose) of an isolated peptide of the present invention upon this solid support surface (or inside the solid support), for reducing, eradicating, inhibiting or preventing microbial biofilms, preferably fungal biofilms, more preferably Candida biofilms, in combination with at least one antifungal agent, preferably said antifungal agent is an echinocandin or a polyene, more preferably said antifungal agent is caspofungin, anidulafungin, micafungin or amphotericin B, even more preferably said antifungal agent is caspofungin.

Typically, the methods and compositions of the present invention find application in a wide range of environments and circumstances. The composition may be an anti-fouling composition, incorporated in a medical device or component thereof, a coating for a medical device or a pharmaceutical composition. Preferably, the composition of the present invention or one or more components thereof, is used in coating medical devices, including implantable medical devices, including but not limited to venous catheters, urinary catheters, stents, prostheses such as artificial joints, hearts, heart valves or other organs, pacemakers, dental implants, surgical plates and pins, dialysis equipment and contact lenses. Alternatively, said implantable medical devices may have the capacity to release the composition on the surface of such medical device, thus imparting a highly localised treatment with said composition.

Methods and compositions of the invention also find application in the management of infectious diseases. For example, a variety of infections associated with (fungal) biofilm formation may be treated with methods and compositions of the invention, such as urinary tract infections, pulmonary infections, dental plaque, dental caries and infections associated with surgical procedures or burns.

When using the methods, means and compositions of the present invention, dosages of antifungal agents may be reduced but remain effective in reducing, eradicating, inhibiting, or preventing biofilms. Particularly, such treatment may be continued during a defined period of treatment, at a preferably constant and/or localised dose, without antifungal pressure drops so that development of resistance, persisters and recurrence of biofilm associated conditions or infections can be prevented. The antifungal agents used in the composition of the present invention are preferably used at a concentration effective against the planktonic form of the cells, which is a concentration too low to be effective against microorganisms in biofilms (in sessile form).

EXAMPLES

The present invention is based on the surprising finding by the inventors that plant defensins such as HsAFPI and synthetic peptides derived from HsAFPI are potentiating compounds which increase the antibiofilm activity of antifungal drugs such as caspofungin, anidulafungin, micafungin or amphotericin B against fungal biofilms, preferably against Candida biofilms. Particularly, the present invention shows that the antibiofilm activity of caspofungin, anidulafungin, micafungin or amphotericin B, can be improved by combining the antifungal compound with said plant defensin HsAFPI and/or peptide derivatives thereof.

In this study, the plant defensin from coral bells, HsAFPI , was used which was previously characterized by Osborn and colleagues (2), and its potential antibiofilm activity was assessed. Since HsAFPI has a potent antifungal activity towards C. albicans, its potential activity towards C. albicans biofilms was further analyzed. To this end, HsAFPI was heterologously expressed using the yeast Pichia pastoris and the solution structure of recombinant (r) rHsAFPI was determined by NMR analysis. Subsequently, the activity of the plant defensin was tested alone and in combination with conventional antimycotics against C. albicans biofilms. In view of the latter, a multi-drug approach in which multiple compounds are administered and a synergistic effect is observed, can be effectively used to combat biofilm-related infections (5). Finally, a structure-function study was conducted, using 24-mer synthetic peptides spanning the entire HsAFPI region. The HsAFPI derivatives were tested against C. albicans planktonic cultures and biofilms, and their potential to synergistically enhance the activity of caspofungin was analysed.

MATERIAL & METHODS

Strains and reagents. Pichia pastoris strain X33 was used for heterologous production of HsAFPI . Fusarium culmorum strain K0311 was used to evaluate the antifungal activity of the recombinant peptide and to compare it with that of native HsAFPI purified from seeds, in a fungal growth inhibitory assay (2). C. albicans strain SC5314 was used in all biofilm experiments. rHsAFPI toxicity testing was performed on HepG2, human hepatoma cells, purchased from ATCC (catalogue number HB-8065; Rockville, MD, USA).

HsLin06_18 cytotoxicity tests were performed on HepG2, human hepatoma cells (29), purchased from ATCC (HB-8065; USA).

All culture media were purchased from LabM (UK), unless stated otherwise. For heterologous production, P. pastoris was cultured in YPD (1% yeast extract, 2% peptone and 2% glucose), BMGY (buffered complex glycerol medium; 1 % yeast extract, 2% peptone, 1.34% yeast nitrogen base w/o amino acids (Becton Dickinson, UK), 1% glycerol, 100 mM K3P04 pH 6, 4 x 10-5% biotin) or BMMY (buffered complex methanol medium; 1% yeast extract, 2% peptone, 1.34% yeast nitrogen base w/o amino acids (Becton Dickinson, UK), 0.5% methanol, 100 mM K3P04 pH 6, 4 x 10-5% biotin). F. culmorum was grown in half strength PDB (1.2% potato dextrose broth). C. albicans was grown overnight in YPD (1% yeast extract, 2% peptone and 2% glucose), with all compounds purchased from LabM (UK).

Biofilm experiments were performed in RPMI-1640 medium (Roswell Park Memorial Institute- 1640 medium; pH 7) with L-glutamine and without sodium bicarbonate (purchased from Sigma Aldrich, St.-Louis, MO, USA), buffered with MOPS (Sigma Aldrich, St.-Louis, MO, USA). Amphotericin B and caspofungin (Cancidas) were purchased from Sigma Aldrich (St. Louis, MO, USA) and Merck (Beeston Nottingham, UK), respectively. HepG2 cells were grown in MEM (Minimal Essential Medium, Gibco, Invitrogen; CA, USA), supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/mL penicillin and 100 pg/mL streptomycin, and cultured using standard cell culture conditions (37°C, 5% C02, 95% humidity). The Cell Proliferation Kit II (XTT) and Cell Proliferation ELISA BrdU (colorimetric) kit were purchased from Roche Diagnostics (Mannheim, Germany).

Caspofungin (cancidas), micafungin (mycamine) and anidulafungin (ecalta) were purchased from Merck (USA), Astellas Pharma Europe (The Netherlands) and Pfizer (UK) respectively. Polyurethane triple-lumen intravenous catheters (2.4 mm diameter) and Fetal Bovine Serum (FBS) were respectively purchased from Arrow International (USA) and Thermo Fisher Scientific (USA).

Production and purification of recombinant (r) rHsAFPI. The PCR fragment encoding mature HsAFPI was cloned in frame with the a-factor secretion signal present in the pPICZaA transfer vector, after which the plasmid was integrated into the genome of Pichia pastoris X33 strain via double homologous recombination. This transgenic P. pastoris strain was grown in YPD overnight at 30°C and 250 rpm. BMGY medium was inoculated with the overnight culture to an optical density at 600 nm (OD600nm) of 0.5 and grown for 24 hours at 30°C and 200 rpm. Cells were pelleted by sterile centrifugation at 8000 rpm for 10 minutes at room temperature and re-suspended in BMMY medium, thereby concentrating the culture 4- fold and inducing gene expression. The culture was grown for 96 hours at 25°C, and 2.5% methanol (v/v%) was added to the culture every 24 hours to maintain induction of gene expression. After induction, cells were pelleted at 8000 rpm for 10 minutes at 4°C and the cleared supernatant, containing the peptides of interest, was filter sterilized through a Steritop-GP 0.22 μιη Express PLUS membrane Bottle-top filter (EMD Millipore, MA, USA). The filtered supernatant was then subjected to automated tangential flow filtration using an automated peristaltic pump (Spectrum Laboratories, CA, USA) and a hollow fiber module with 1 kDa cut-off mPES membranes (Spectrum Laboratories, CA, USA). During the ultrafiltration, the sample was concentrated a 15-fold and subsequently dialyzed against 50 mM sodium acetate pH 5.

rHsAFPI was purified by cation exchange chromatography, using 75 mL SP sepharose High Performance resin (GE Healthcare, UK) packed in a XK26/20 column (GE Healthcare) and 50 mM sodium acetate buffers at pH 5. The flow rate was maintained at 5 mUmin. Elution of the peptides was carried out by a washing step with 10% (v/v%) elution buffer (50 mM sodium acetate, 1 M sodium chloride, pH 5) for 10 column volumes (CV), followed by a linear gradient to 50% (v/v%) elution buffer in 15 CV, resulting in a peak at approximately 29% (v/v%) elution buffer. The eluted fraction was further purified by reversed phase chromatography employing a Gemini C18 250x10 column (Phenomenex, CA, USA) and acetonitrile (ACN) for elution of the bound peptides. The flow rate was maintained at 4.6 mL/min. Elution of the peptides was carried out by a washing step at 15% (v/v%) ACN for 1.9 CV, followed by a linear gradient to 35% (v/v%) ACN in 2.3 CV. Elution of rHsAFPI occurred at 28%. The eluted fraction was vacuum dried by centrifugal evaporation (SpeedVac Savant, Thermo Fisher Scientific, MA, USA), re-dissolved in MilliQ water and subjected to a micro bicinchoninic acid assay (Pierce, Thermo Scientific, USA) according to the manufacturer's instructions, to determine the protein concentration. Bovine serum albumin served as a reference protein. At least 40 mg/L of culture of purified rHsAFPI was obtained.

Characterization of rHsAFPI by NMR. Dry powder (1 mg) of rHsAFPI was dissolved in 500 μΙ_ of 10% D2O/90% H20 (~pH 4) for NMR experiments. Spectra were recorded at 298 K on a Bruker Avance-600 spectrometer. Two-dimensional NMR experiments included total correlation spectroscopy (TOCSY (6)) using a MLEV-17 spin lock sequence (7) with a mixing time of 80 ms; nuclear Overhauser effect spectroscopy (NOESY (8)) with a mixing time of 150, 200, or 300 ms; exclusive correlation spectroscopy (ECOSY (9)); and 13C and 15N heteronuclear single-quantum coherence (HSQC (10)). Solvent suppression was achieved using excitation sculpting with gradients (11 ). Spectra were acquired with 4096 complex data points in F2 and 512 increments in the F1 dimension. Slowly exchanging amide protons were identified by spectra also recorded in 100% D20.

Spectra were processed using TopSpin (Bruker) software. The t1 dimension was zero-filled to 1024 real data points, and 90° phase-shifted sine bell window functions were applied prior to Fourier transformation. Chemical shifts were referenced to internal 2,2-dimethyl-2- silapentane-5-sulfonate (DSS). Processed spectra were analysed and assigned using CcpNmr Alalysis (12). Spectra were assigned using the sequential assignment protocol (13).

Structure calculations. Structure calculations were based on distance restraints derived from NOESY spectra recorded in both 10% and 100% D20. Initial structures were generated using the program CYANA (14), followed by addition of restraints for the disulfide bonds, hydrogen bonds as indicated by slow D20 exchange and sensitivity of amide proton chemical shift to temperature, chil restraints from ECOSY and NOESY data, and backbone phi and psi dihedral angles restraints generated using the program TALOS+ (15). The structural family was generated using torsion angle dynamics, refinement and energy minimization in explicit solvent and protocols as developed for the RECOORD database (16) within the program CNS (17). A family of structures consistent with the experimental restraints was then visualized using MOLMOL (18) and assessed for stereochemical quality using MolProbity (19). Coordinates and NMR chemical shift assignments have been submitted (PDB ID: 2n2q; BMRB ID: 25605).

Antifungal activity assays. To test whether rHsAFPI is as potent as HsAFPI purified from the seeds of coral bells, we analysed the antifungal activity of both peptides against F. culmorum, following the standard CLSI protocol M28-A2 (20), with minor modifications as previously described by Osborn and colleagues (2): an inoculum of approximately 104 spores/mL of F. culmorum was suspended in half strength PDB and added to a two-fold dilution series of rHsAFPI in water. Seed-derived HsAFPI was purified according to the protocol as previously described by Osborn and colleagues (2). The IC50 value, which is the concentration required for 50% growth inhibition as compared to control treatment, was determined by measuring the optical density at 490 nm (OD490nm) after 48 hours of incubation and was confirmed microscopically. The antifungal activity of rHsAFPI against C. albicans was subsequently analysed according to the standard CLSI protocol M27-A3 (21 ) with minor modifications: an inoculum of approximately 106 cells/mL was suspended in RPMI-1640 medium and added to a two-fold dilution series of rHsAFPI in water. The DMSO concentration was similar to that in the biofilm assays, i.e. 0.5% DMSO. The MIC50 value, i.e. the minimum concentration required to reduce planktonic growth by 50% as compared to control treatment, was determined by measuring the OD490nm after 24 hours of incubation.

Antibiofilm activity assays:

Biofilm inhibition assay. The Biofilm Inhibitory Concentration 50 value (BIC50; the minimum concentration required to reduce biofilm formation by 50% as compared to control treatment) of rHsAFPI or HsLin peptides was determined using the following antibiofilm assay: a C. albicans SC5314 overnight culture, grown in YPD, was diluted to an optical density (600 nm) of 0.1 in RPMI 1640 medium and 100 μΙ_ of this suspension was added to the wells of a round-bottomed microtitre plate (TPP, Tradingen, Switzerland). After 1 h of adhesion at 37°C, the medium was aspirated and the biofilms were washed with 100 μΙ_ phosphate- buffered saline (PBS) to remove non-adherent cells. Fresh RPMI 1640 medium, followed by an rHsAFPI concentration series was added to the biofilms. The DMSO concentration was similar to that in the checkerboard assays, i.e. 0.5%. Biofilms were allowed to grow for 24 h at 37°C and were subsequently washed with PBS and quantified with CellTiter-Blue (CTB; Promega, Wl, USA)) (22) by adding 100 μΐ of CTB diluted 1/10 in PBS to each well. After 1 h of incubation in the dark at 37°C, the fluorescence was measured with a fluorescence spectrometer (AEx/AEm: 535/590 nm). The fluorescence values of the samples were corrected by subtracting the average fluorescence value of the CTB of uninoculated wells (blank). The percentage of surviving biofilm cells was calculated relative to the control treatment (0.5% DMSO).

Biofilm eradication assay. The Biofilm Eradicating Concentration 50 value (BEC50; the minimum concentration required to reduce the viability of the cells in a pre-grown biofilm by 50% as compared to control treatment) of rHsAFPI was determined using the BEC50 determination assay as described by De Cremer and co-workers (23). Briefly, a C. albicans SC5314 overnight culture, grown in YPD, was diluted to an optical density (600 nm) of 0.1 in RPMI 1640 medium and 100 μΙ_ of this suspension was added to the wells of a round- bottomed microtitre plate (TPP, Tradingen, Switzerland). After 1 h of adhesion, the biofilms were washed with 100 μΙ_ PBS to remove non-adherent cells, followed by addition of 100 μΙ_ RPMI 1640 medium. The biofilms were allowed to grow for 24 h at 37°C. Next, an rHsAFPI concentration series in RPMI was added to the biofilms. The DMSO concentration was similar to that in the checkerboard assays, i.e. 0.5%. The biofilms were incubated for another 24 h at 37°C, after which they were washed and quantified with CTB as described above. Checkerboard assay. C. albicans biofilms or C. albicans planktonic cultures were grown as described above. A combination of rHsAFPI or HsLin peptides and antimycotic (caspofungin, anidulafungin, micafungin or amphotericin B), two-fold diluted across the columns and rows of a 96-well plate, respectively, was added to the planktonic culture or to the biofilms. Biofilms were treated either after 1 hour or 24 hours starting from the adhesion phase to analyse biofilm inhibition or biofilm eradication, respectively. After 24 hours incubation at 37°C, the MIC50 values were determined by measuring the OD490nm, whereas BIC50 and BEC50 values were determined using CTB as described above. In all experiments, the DMSO concentration was kept at 0.5%. Synergy was determined by calculating the Fractional Inhibitory Concentration Index (FICI) (24, 25).

Scanning electron microscopy (SEM). Qualitative analysis of samples was performed using scanning electron microscopy (XL30-FEG, FEI). Samples were prepared using a protocol previously described (26). Briefly, the biofilm-containing titanium discs were rinsed in PBS and fixed in gluteraldehyde (2.5% v/v in a cacodylate buffer). Samples were rinsed three times in PBS, and subsequently dehydrated in a series of ethanol/H20 solutions with increasing alcohol content, followed by air drying. Finally, a thin conductive Au-Pd film was sputtered (Edwards S150) on the samples and SEM was operated at standard high-vacuum settings and using 10 mm working distance and 20 keV accelerating voltage.

rHsAFPI toxicity in HeoG2 cells. HepG2 cells were seeded at 10.000 cells/well in 96 well- plates and incubated for 24 hours. Subsequently, cells were treated with water (untreated) or rHsAFPI (0.01 μΜ - 40 μΜ) for 24 hours after which cell viability or cell proliferation was determined using the "Cell Proliferation Kit II (XTT)", as described previously (27), or the "Cell Proliferation ELISA BrdU (colorimetric) kit", according to the manufacturer's instructions, respectively.

Structure-function analysis of HsAFPI. Synthesis and purification of the 24-mer peptides (HsLin01-HsLin06) spanning the HsAFPI amino acid sequence was performed as described previously (28). Cysteine residues were replaced by a-aminobutyric acid to avoid formation of disulfide bonds.

In vitro biofilm inhibition assay - catheters. C. albicans biofilms were grown on catheters as described previously (30). After an adhesion phase of 90 minutes, C. albicans biofilms (on catheters) were treated with caspofungin, caspofungin + HsLin06_18 or control treatment (0.5% DMSO +0.5% MQ water) in RPMI for 24 hours, after which biofilm-associated cells were determined via Colony Forming Units (CFU's) counts (30). Averages of 4 technical replicates were used to calculate the CFU/catheter for each treatment in one experiment. Next, data were normalized to the CFU/catheter corresponding to the control treatment.

HsLin06 18 toxicity assays. Human hepatoma HepG2 cells were incubated for 24 hours in 96 well-plates at 10 000 cells/well. Subsequently, cells were treated with water (control treatment) or HsLin06_18 (0.09 μΜ - 93 μΜ). After 24 hours of treatment, cell viability was determined via the cell proliferation kit II (MTT), as described by van Malenstein and coworkers (27).

Data analysis. Data were analysed with GraphPad Prism (GraphPad Software, Inc., CA, USA). For dose-response data, sigmoidal curves were generated using nonlinear regression. The concentration required to cause 50% planktonic growth inhibition (IC50 or MIC50), reduction of biofilm formation (BIC50) and biofilm eradication (BEC50) as compared to control treatment was derived from the whole dose-response curves. In all experiments, mean ± standard error of the mean (SEM) for n≥ 3 is presented. Unpaired Student t-tests were performed to analyse significant differences between the IC50 value of native HsAFPI and that of recombinant HsAFPI , and between the MIC50, BIC50 and BEC50 of caspofungin or amphotericin B alone and the combination of these compounds with rHsAFPI or its derivatives in the checkerboard assays. To analyse significant differences in cell viability or cell proliferation between untreated and rHsAFPI -treated HepG2 cells in the rHsAFPI toxicity assays, unpaired Student t-tests were performed. In all cases, P<0.05 was defined as statistically significant.

RESULTS EXAMPLE 1 - rHsAFPI shows potent antifungal activity against filamentous fungi.

rHsAFPI was produced in Pichia pastoris and subsequently purified using cation exchange and reversed phase chromatography. A yield of at least 40 mg/L of culture of purified rHsAFPI was obtained. The antifungal activity of HsAFPI against a broad range of fungi, including the fungus Fusarium culmorum, has been reported previously (2). In this respect, Osborn and colleagues showed that native HsAFPI can inhibit growth of F. culmorum with an IC50 value of 1 pg/mL (2). Hence, to assess the potency of rHsAFPI , we tested the antifungal activity of rHsAFPI and native HsAFPI against F. culmorum according to the method of Osborn (2). We found the IC50 values of the recombinant and native peptide against F. culmorum not to be significantly different, i.e. 0.45 ± 0.13 μΜ and 0.23 ± 0.02 μΜ respectively, with a P-value of 0.1707, and hence, rHsAFPI seems as potent as native HsAFPL

EXAMPLE 2: Characterization ofrHsAFPI by NMR.

The solution structure of rHsAFPI was solved via NMR analysis, a technique that has been previously used to characterize the structures of other plant defensins, including RsAFPI , MtDef4, Psd1 and NaD1. A sequence alignment of HsAFPI with these peptides and RsAFP2 is presented in Fig. 1 A, showing the disulfide bond pattern common for plant defensins.

The NMR spectra of rHsAFPI showed the sample to be of high purity and good dispersion in the amide region was indicative of a highly structured peptide. Two-dimensional spectra were recorded at several temperatures in the range 283 to 303 K to obtain full proton assignments (data not shown). Secondary chemical shift analysis was then used to locate elements of secondary structure. Ha secondary shifts are calculated by subtracting the chemical shift of the alpha proton from "random coil" values. Deviations greater than 0.1 ppm from random coil are indicative of structured peptides, with positive values present for beta type structures and negative values for helical structures. The secondary Ha shifts of rHsAFPI are shown in Fig. 2 and indicate that the solution structure of rHsAFPI consists of both a-helix and β- strand elements.

The three-dimensional structure of rHsAFPI was calculated from 614 distance restraints, 15 hydrogen bond pairs, and a total of 90 dihedral angle restraints (data not shown). The disulfide connectivities (l-VIII, ll-V, lll-VI, IV-VII) were fully consistent with the NOE data and were included as restraints in the structure calculations. Similarly to RsAFPI , one proline (Pro9) is present in the trans configuration and the second (Pro44) has a cis peptide bond. Fig. 3A shows the ensemble of structures superimposed over the backbone heavy-atoms of all residues (rmsd 1.16 ± 0.40 A). A ribbon representation of the lowest energy structure is shown in Fig. 3B. Analysis of the structures shows that 96% of residues fall in the most favored regions of the Ramachandran plot and a mean MolProbity score of 1.8 indicates good structural quality. rHsAFPI forms a compact globular fold with a three turn a-helix spanning residues Ser20-Arg30 and a triple-stranded anti-parallel β-sheet (β1 = Leu5-Pro9; β2 = Ala38-His40; β3 = Lys47-Gln53) forming another element of secondary structure. The four disulfide bonds are arranged in a typical Cysteine-stabilized αβ motif in that the a-helix is tethered to the β-sheet by two disulfide bonds to the central strand (Cys23-Cys39 and Cys27-Cys50). There are three loops present in the molecule that link β-strand 1 with the helix, the helix to β-strand 2, and the β-strands 2 and 3. These loops are reasonably well- defined although the loop that incorporates a β-turn between strand 2 and 3 is apparently more flexible as judged by greater disorder in the structural ensemble in this region.

EXAMPLE 3: rHsAFPI prevents C. albicans biofilm formation.

At first, we assessed the antifungal activity of rHsAFPI against planktonic C. albicans cells. rHsAFPI showed antifungal activity against planktonic C. albicans cultures, with a MIC50 value of 18.00 ± 4.60 μΜ. Subsequently, we investigated the ability of rHsAFPI to prevent or eradicate C. albicans biofilms. rHsAFPI inhibited C. albicans biofilm formation, resulting in a BIC50 value of 11.00 ± 1.70 μΜ. Fifty percent eradication of C. albicans biofilms by this peptide, as compared to control treatment, was not observed at the highest tested concentration, i.e. 109.00 μΜ (i.e. BEC50 of rHsAFPI is > 109.00 μΜ) (data not shown).

In order to investigate the effect of rHsAFPI on the growth of C. albicans biofilms, SEM images of biofilms grown for 4 hours in the presence or absence of rHsAFPI (1 1.8 μΜ) were taken. As shown in Figure 4, cells in the untreated biofilms were able to form a dens hyphal network, covering the titanium discs. In contrast, no true biofilm was formed in the presence of 1 1.8 μΜ rHsAFPI , as in this case, biofilms mainly consisted of cells attached to the titanium disc without formation of a hyphal network.

EXAMPLE 4: rHsAFPI acts synergistically with caspofungin or amphotericin B against C. albicans.

As rHsAFPI prevented C. albicans biofilm formation, we further investigated the effect of rHsAFPI on the biofilm inhibitory and eradicating activity of conventional antimycotics, such as caspofungin and amphotericin B. To this end, checkerboard assays were performed and the corresponding FICI values for each combination were calculated to determine whether rHsAFPI acts synergistically with these compounds against C. albicans biofilms (Figure 5 and Tables 1 and 2).

Table 1 Synergistic activity of rHsAFPI with caspofungin or amphotericin B against C. albicans SC5314 biofilms, resulting in biofilm formation inhibition*

^*BIC50 values were determined by CTB assay; mean ± SEM for n≥ 3 independent experiments is presented; BIC50, minimum inhibitory concentration that is required to inhibit biofilm formation by 50% as compared to control treatment; FICI, Fractional Inhibitory Concentration Index, FICI≤ 0.5 indicates synergy between two compounds; NA, not applicable. Values in bold represent synergistic effects between two compounds. Unpaired Student t-tests were performed in case FICI did not indicate synergy to analyse significant differences between the effect of the compound alone and the combination of compound and rHsAFPI ; the significance level is presented (^*, ^** and *** represent P<0.05, P<0.01 and P<0.001 , respectively; NS, no significant difference). Table 2 Synergistic activity of rHsAFPI with caspofungin or amphotericin B against C. albicans SC5314 biofilms, resulting in eradication of C. albicans biofilm cells*

*BEC50 values were determined by CTB assay; mean ± SEM for n≥ 3 independent experiments is presented; BEC50, minimum concentration that is required to reduce viability of 24 hours-old biofilm cells by 50% as compared to control treatment; FICl, Fractional Inhibitory Concentration Index, FICl≤ 0.5 indicates synergy between two compounds; NA, not applicable. Values in bold represent synergistic effects between two compounds.

Unpaired Student t-tests were performed in case FICl did not indicate synergy to analyse significant differences between the effect of the compound alone and the combination of compound and rHsAFPI ; the significance level is presented (*, ** and *** represent P<0.05, P<0.01 and P<0.001 , respectively; NS, no significant difference).

In the biofilm inhibition assays (Table 1 ), synergistic effects (FICl < 0.5) were observed between rHsAFPI and caspofungin: rHsAFPI increased the activity of caspofungin at concentrations of 1.05 μΜ and 2.1 μΜ, resulting in a 2.5-fold and 3.7-fold reduction of the caspofungin BIC50, respectively. Although not synergistic, 0.53 μΜ, 4.2 μΜ and 8.4 μΜ rHsAFPI also reduced the BIC50 of caspofungin significantly (P<0.05). No synergistic effects were observed between rHsAFPI and amphotericin B in the biofilm inhibition assays, however, a range of 1.05 μΜ to 8.4 μΜ rHsAFPI significantly reduced the amphotericin B BIC50. Moreover, we also found that rHsAFPI acted synergistically with caspofungin or amphotericin B in the eradication of C. albicans biofilms (Table 2): all rHsAFPI concentrations tested (i.e. a range from 0.53 μΜ to 16.8 μΜ rHsAFPI ) increased the biofilm eradicating capacity of caspofungin and although only 8.4 μΜ rHsAFPI displayed synergy with amphotericin B, multiple concentrations significantly reduced the BEC50 of amphotericin B.

To assess whether the synergistic effects observed between rHsAFPI and amphotericin B or caspofungin against C. albicans biofilms were biofilm-specific, a similar checkerboard assay was performed on planktonic C. albicans cells (Table 3). Synergistic effects were observed between rHsAFPI and caspofungin or amphotericin B against planktonic C. albicans cells and hence, synergy between rHsAFPI and these compounds seems not biofilm-specific. Synergy between rHsAFPI and amphotericin B was observed at lower rHsAFPI concentrations as compared to those observed between rHsAFPI and caspofungin. Interestingly, the concentration range of rHsAFPI that acted synergistically with caspofungin against planktonic C. albicans cells was more restricted as compared to a C. albicans biofilm setup: all rHsAFPI concentrations tested (i.e. 0.53 μΜ to 16.8 μΜ) increased caspofungin activity against C. albicans biofilms in the biofilm eradication assays, whereas only 2.1 μΜ and 4.2 μΜ rHsAFPI acted synergistically with caspofungin against planktonic C. albicans cells. In addition, only 1.05 μΜ and 2.1 μΜ rHsAFPI enhanced caspofungin activity against C. albicans biofilms in the biofilm inhibition assays. This indicates that synergy between caspofungin and rHsAFPI is more evident in the eradication of C. albicans biofilms. In contrast, synergy between amphotericin B and rHsAFPI was more pronounced against planktonic C. albicans cultures, as various rHsAFPI concentrations (i.e. 0.53 μΜ to 2.1 μΜ) acted synergistically with amphotericin B against planktonic C. albicans cells and only 8.4 μΜ rHsAFPI increased amphotericin B activity against C. albicans biofilms in the biofilm eradication assay. No synergistic effects between amphotericin B and rHsAFPI were observed in the biofilm inhibition assays.

Table 3 Synergistic activity of rHsAFPI with caspofungin or amphotericin B against C. albicans SC5314 planktonic cultures*

*MIC50 values were determined by measuring the OD at 490 nm; mean ± SEM for n > 3 independent experiments is presented; MIC50, minimum inhibitory concentration that is required to reduce planktonic growth by 50% as compared to control treatment; FICI, Fractional Inhibitory Concentration Index, FICI≤ 0.5 indicates synergy between two compounds; NA, not applicable. Values in bold represent synergistic effects between two compounds. Unpaired Student t-tests were performed in case FICI did not indicate synergy to analyse significant differences between the effect of the compound alone and the combination of compound and rHsAFPI ; the significance level is presented (*, ** and *** represent P<0.05, P<0.01 and P<0.001 , respectively; NS, no significant difference).

EXAMPLE 5: rHsAFPI does not affect HepG2 cell viability and proliferation.

Various plant defensins are reported to be non-toxic to human cells due to their fungal membrane-specific interactions (11 ). As no records exist yet on potential toxicity of HsAFPI , we analysed the effect of rHsAFPI on human hepatoma cells (HepG2) and found that rHsAFPI did not affect HepG2 cell viability nor cell proliferation up to 40 μΜ, the highest rHsAFPI concentration tested in this setup. No statistically significant differences were found in cell viability and proliferation between untreated and rHsAFPI -treated cells (Fig.6).

EXAMPLE 6: The γ-core and adjacent regions are important for rHsAFPI antibiofilm activity.

In order to gain insights in the structure-function relationship of HsAFPI against C. albicans planktonic and biofilm cells, we conducted a structure-function relationship study using HsAFPI -derived linear fragments. The selection of fragments was based on the procedure used by Schaaper et al. (29). We synthesized 24-mer peptides with an 18-mer overlap, spanning the entire HsAFPI amino acid sequence and analysed these peptides for their activity towards F. and C. albicans planktonic cultures and biofilms. The sequences of the linear fragments (HsLin01 -HsLin06) are presented in Fig.l B. Figure 7 shows a diagram in which the HsLin peptides are imposed on the rHsAFPI structure, according to their amino acid sequence. Note that (i) the Cysteine residues are replaced by a-aminobutyric acid to avoid formation of disulfide bonds and that (ii) the CSa scaffold is not present in the HsLin peptides, and therefore, the peptides do not adopt the same conformation as the mature rHsAFPI .

None of the linear HsAFPI -derived fragments inhibited the growth of F. culmorum up to the highest tested concentration, 1.5 μΜ, whereas rHsAFPI inhibited growth of this fungus with an IC50 value of 0.45 ± 0.13 μΜ. In addition, these truncated peptides did not inhibit the growth of C. albicans in contrast to full-length rHsAFPI . Hundred percent growth inhibition of C. albicans planktonic cells was observed at 70 μΜ for rHsAFPI , whereas concentrations up to 350 μΜ of the peptides were not sufficient to cause 100% growth inhibition. Furthermore, only HsLin06 inhibited C. albicans biofilm formation to the same extent as rHsAFPI : the BIC50 values of HsLin06 and rHsAFPI were 10.80 ± 3.59 μΜ and 11.00 ± 1.70 μΜ, respectively (Table 4), suggesting that the sequence comprising HsLin06 is important for antibiofilm activity. HsLin03 and HsLin05 showed antibiofilm activity as well, however, with a 10- to 15-fold higher BIC50 value than that of rHsAFPI or HsLin06. Other fragments did not inhibit biofilm formation up to 175 μΜ, the highest tested concentration. We further analysed the potential of the peptides to increase the activity of caspofungin to prevent biofilm formation. We found that HsLin06, but also HsLinOI and HsLin05, acted synergistically with caspofungin to inhibit C. albicans biofilm formation in a range of 0.75 μΜ to 1.5 μΜ (Fig. 9 and Table 5 for HsLin06 and Fig. 8 for the other HsLin). We did not observe synergistic effects between the other linear fragments and caspofungin for preventing biofilm formation (Fig. 8).

Table 4 Structure-function relationship study of HsAFPI -derived fragment against C. albicans planktonic cultures and biofilms*

Peptide BIC50 (μΜ) ± SEM Significance level

rHsAFPI 11.00 ± 1.70

HsLinOI >175 ***

HsLin02 >175 ***

HsUn03 96.78 ± 15.90 **

HsLin04 >175 _***

HsLin05 160.00 ± 33.36 *

HsLin06 10.80 ± 3.59 NS

*BIC50 values were determined by CTB assay; mean ± SEM for n ≥ 3 independent experiments is presented; BIC50, minimum inhibitory concentration that is required to inhibit biofilm formation by 50% as compared to control treatment. Unpaired Student t-tests were performed to analyse significant differences between the effect of the linear fragments and rHsAFPI ; the significance level is presented (*, ** and *** represent P<0.05, P<0.01 and P<0.001 , respectively; NS, no significant difference).

Table 5 Synergistic activity of Hsl_in06 with caspofungin against C. albicans SC5314 biofilms, resulting in biofilm formation inhibition*

^*BIC50 values were determined by CTB assay; mean ± SEM for n ≥ 3 independent experiments is presented; BIC50, minimum inhibitory concentration that is required to inhibit biofilm formation by 50% as compared to control treatment; FICI, Fractional Inhibitory Concentration Index, FICI < 0.5 indicates synergy between two compounds; NA, not applicable. Values in bold represent synergistic effects between two compounds. Unpaired Student t-tests were performed in case FICI did not indicate synergy to analyse significant differences between the effect of the compound alone and the combination of compound and rHsAFPI ; the significance level is presented (*, ** and ^*** represent P<0.05, P<0.01 and P<0.001 , respectively; NS, no significant difference).

EXAMPLE 7. Biofilm inhibitory activity of HsLin06-variants 01-44 in a microtiter plate assay

As the C-terminal part of HsAFPI (corresponding to HsLin06, 24 amino acids) was identified as an important region for antibiofilm and caspofungin's synergistic activity, we tested a series of 44 HsLin06-variants (HsLin06_01-44) with N- or C-terminal truncations of HsLin06's sequence, ranging from 16-23 amino acids (Table 6). Their antibiofilm activity in microtiter plates was evaluated by determining BIC50 values. Caspofungin potentiating activity of the HsLin06-variants against C. albicans biofilms was evaluated by comparing the reduction of BIC50 of caspofungin in the presence and absence of the HsL^'in06-variant, represented as fold change values (shown in Table 6). The higher the fold change, the better the potentiating effect of HsLin to caspofungin's antibiofilm activity.

Similar antibiofilm activity as compared to the antibiofilm activity of Hsl_in06 was found for 17 HsLin06-variants (HsLin06_01-05;07;08;10-12;15;16;22;29;30;36;38) as calculated by their BIC50 values (0.5 * BIC50(Hsl_in06) < BIC50(HsLin) < 2 * BIC50(HsLin06)) (Table 6). Interestingly, peptides HsLin06_02 and Hsl_in06_10 had both a pronounced caspofungin potentiating and antibiofilm effect. In addition, we found that 11 HsLin06-variants were able to potentiate caspofungin's antibiofilm activity at least 5.2-fold (i.e. > 2x F.C. HsLin06), irrespective of their BIC50 values. Five of them, namely HsLin06_02;06;10;13 and HsLin06_18 (Table 6), reduced the BIC50 of caspofungin by at least 10-fold. HsLin06_18, being the shortest peptide (containing 19 amino acids) was selected for further in vitro and in vivo experiments.

Based on all the data in Table 6, we delineated the core sequence of HsAFPI for both antibiofilm activity and caspofungin potentiating activity as GACYQFPSVKCFCKR and AYGGACHYQFPSVKCFC respectively, with cysteines replaced by X=a-ABA in all HsLin's. Both core sequences are partially overlapping (GACHYQFPSVKCFC).

Table 6. C. albicans biofilm formation inhibition of HsLin06-variants alone or in combination with caspofungin. BIC50 values, i.e. minimum inhibitory concentration resulting in 50% biofilm inhibition compared to control treatment. HsLin06_18 is the smallest Hsl_in06-variant with fold change value > 10 and is therefore marked in bolt.

HsLin alone HsLin + Caspofungin

Name #aa Sequence BIC50 HsLin | M) Fold change

HsLinOe 24 EHFAYGGAXHYQFPSVKXFXKRQX 0,53 2,56

HsLin06_01 23 HFAYGGAXHYQFPSVKXFXKRQX 0,71 4,98

HsLin06 02 23 EHFAYGGAXHYQFPSVKXFXKRQ 0,56 10,62

HsLin06_03 22 FAYGGAXHYQFPSV XFXKRQX 0,56 2,52

HsLin06_04 22 HFAYGGAXHYQFPSVKXFXKRQ 0,38 1,01

HsLin06 OS 22 EHFAYGGAXHYQFPSVKXFXKR 0,46 2,47

HsLin06_06 21 AYGGAXHYQFPSVKXFXKRQX 1,16 11,14

HsLin06_07 21 FAYGGAXHYQFPSVKXFX RQ 0,50 1,16

HsLln06_08 21 HFAYGGAXHYQFPSVKXFXKR 0,48 1,22

HsLinOe 09 21 EHFAYGGAXHYQFPSV XFXK >2 7,99

HsLin06_10 20 YGGAXHYQFPSVKXFXKRQX 0,84 12,49

HsLin06_ll 20 AYGGAXHYQFPSV XFX RQ 0,78 1,09

HsLin06_12 20 FAYGGAXHYQFPSVKXFXKR 0,71 1,17

HsLin06_13 20 HFAYGGAXHYQFPSVKXFXK >2 12,15

HsLinOe 14 20 EHFAYGGAXHYQFPSV FX >2 3,65

HsLin06_15 19 GGAXHYQFPSVKXFXKRQX 0,90 1,04

HsLin06_16 19 YGGAXHYQFPSVKXFXKRQ 1,03 1,72

HsLin06_17 19 AYGGAXHYQFPSVKXFXKR >2 1,49

HsLin06_18 19 FAYGGAXHYQFPSVKXFX >2 10,42

HsLin06_19 19 HFAYGGAXHYQFPSVKXFX >2 3,15

HsLin06 20 19 EHFAYGGAXHYQFPSVKXF >2 1,44

HsLin06_21 18 GAXHYQFPSVKXFXKRQX >2 1,49

HsLin06_22 18 GGAXHYQFPSVKXFXKRQ 0,63 4,37

HsLin06_23 18 YGGAXHYQFPSVKXFXKR 1,11 2,06

HsLin06_24 18 AYGGAXHYQFPSVKXFXK >2 5,36

EXAMPLE 8. HsLin06_18 potentiates different echinocandins

Besides caspofungin, other echinocandins such as micafungin and anidulafungin are used in the clinic to treat patients suffering from invasive fungal infections (31 ). Therefore, we examined the effect of HsLin06_18 on caspofungin, anidulafungin and micafungin's antibiofilm activity. We found that HsLin06_18 could potentiate caspofungin's antibiofilm activity, as well as that of anidulafungin (Table 7). Further, some HsLin06_18's potentiation effect on micafungin's antifbiofilm activity could be observed in Figure 10B.

Table 7. Potentiation activity of Hsl_in06_18 with different echinocandins: caspofungin (CASPO), micafungin (MICA) and anidulafungin (ANIDULA) in an in vitro microtiter plate biofilm inhibition test. BIC50 values, i.e. minimum inhibitory concentration resulting in 50% biofilm inhibition compared to control treatment. Unpaired student t- test were performed to determine significant differences between the single treatment with an echinocandin and the combinational treatment with both the echinocandin and Hsl_in06_18. The significance levels *, *** represents respectively P < 0.05 and P < 0.001; NS, not significant.

Compound(s) BIC50 (echinocandin) (μΜ) ± SEM P-value Significance level

CASPO 0.92 ± 0.040

CASPO + 5μΜ HsLin06_18 0.11 ± 0.045 0.0002 ***

MICA 0.40 ± 0.088

MICA + 5μΜ HsLin06_18 0.17 ± 0.035 0.0721 NS

ANIDULA 1.10 ± 0.120

ANIDULA + 5μΜ HsLin06_18 0.47 ± 0.161 0.0349 *

EXAMPLE 9. Caspofungin's potentiating activity ofHsLin06_18 in an in vitro catheter assay The potentiating effect of HsLin06_18 on caspofungin's antibiofilm activity was investigated in an in vitro catheter assay. Therefore, biofilms, adhering on serum pre-incubated catheters, were treated with caspofungin, caspofungin with Hsl_in06_18, HsLin06_18 alone or DMSO (control treatment) after which the survival of the biofilm cells was determined via plating assays. We found that neither caspofungin nor HsLin06_18 was able to inhibit C. albicans biofilm formation when administrated alone, while the combination of caspofungin and HsLin06_18 resulted in a significant (98%) reduction of cell survival, compared to the control treatment (Fig. 1 1 ). These data confirm the superior activity of a caspofungin-HsLin06_18 combination treatment of C. albicans biofilms over treatment with the single compounds in a setup mimicking in vivo conditions using serum pre-incubated catheters.

EXAMPLE 10. Toxicity of HsLin06_18

We investigated the potential toxicity of HsLin06_18 on human HepG2 cells. Hsl_in06_18 was found not cytotoxic up to 200pg/ml, the highest tested concentration, as presented in Figure 12.

EXAMPLE 11. Structure activity relationship study of HsLin06_18

To get more insight in the structure activity relationship of HsLin06_18 for its caspofungin potentiating activity, we performed an alanine scan in which every amino acid of HsLin06_18 was replaced by alanine, resulting in 19 HsLin06_18-derived peptides (HsLin06_18_01-19), which were subsequently tested for their ability to potentiate caspofungin's antibiofilm activity. By comparing fold change values (Table ), the amino acids important for HsLin06_18 caspofungin's potentiating activity can be identified. Surprisingly, none of the amino acid replacements in HsLin06_18 resulted in the abolishment of its caspofungin potentiating activity. Moreover, the fold change values of HsLin06_18 and HsLin06_18_01-19 were in the same range, indicating that the amino acid sequence of HsLin06_18 can be substituted at different positions without losing its caspofungin potentiating effect. Interestingly, in this experiment, HsLin06_18_13 potentiated caspofungin more than HsLin06_18 (Table and Fout! Verwijzingsbron niet gevonden.).

Table 8: Antibiofilm activity and caspofungin's potentiating activity of HsLin06_:18- derived peptide fragments, resulting in £ albicans biofilm formation inhibition. Amino acid replacements in HsLin06_18_01-19 are marked in bolt. HsLin + caspofungin

Name Sequence Fold change

HsLin06 18 FAYGGAXHYQFPSVKXFXK 7,49

HsLin06. _18_01 AAYGGAXHYQFPSVKXFXK 5,91

HsLin06. .18_02 FGYGGAXHYQFPSVKXFX 8,99

HsLin06. .18-03 FAAGGAXHYQFPSVKXFXK 6,63

HsLin06. .18_04 FAYAGAXHYQFPSVKXFXK 7,66

HsLin06. .18_05 FAYGAAXHYQFPSVKXFXK 5,54

HsLin06. _18_06 FAYGGGXHYQFPSV XFXK 7,70

HsLin06_ .18_07 FAYGGAAHYQFPSVKXFXK 7,65

HsLin06. _18_08 FAYGGAXAYQFPSVKXFXK 5,03

HsLin06. _18_09 FAYGGAXHAQFPSV XFX 5,66

HsLin06. .18_10 FAYGGAXHYAFPSVKXFXK 8,18

HsLin06. _18_11 FAYGGAXHYQAPSVKXFXK 4,90

HsLin06. _18_12 FAYGGAXHYQFASVKXFXK 4,75

HsLin06. _18_13 FAYGGAXHYQFPAV XFXK 11,72

HsLin06_ _18_14 FAYGGAXHYQFPSA XFXK 5,89

HsLin06. .18_15 FAYGGAXHYQFPSVAXFX 7,07

HsLin06. .18_16 FAYGGAXHYQFPSV AFXK 6,17

HsLin06. _18_17 FAYGGAXHYQFPSVKXAXK 7,22

HsLin06. _18_18 FAYGGAXHYQFPSV XFAK 6,31

HsLin06 18 19 FAYGGAXHYQFPSVKXFXA 6,65

*#aa: number of amino acids; mean for n≥ 2 experiment are represented; Fold change, i.e. the reduction of the caspofungin dose needed for 50% biofilm formation inhibition by a combinational treatment of HsLin with caspofungin compared to a treatment with caspofungin alone.

This study is the first to report the activity of plant defensins towards fungal biofilms and indicates, the relevance of using defensins as an approach to combat fungal biofilm- associated infections. We showed that rHsAFPI inhibited C. albicans planktonic growth and biofilm formation, and did not affect the viability and proliferation of human HepG2 cells in vitro. The latter indicates that HsAFPI does not exhibit a general cytotoxicity, which is supported by its lack of inhibitory activity to bacteria. It was already shown that the plant defensin RsAFP2 is prophylactically effective against murine candidiasis, pointing to the in vivo potential of plant defensins. Moreover, we showed that rHsAFPI acted synergistically with caspofungin against C. albicans biofilms and planktonic cells. In addition, we found that certain linear HsAFPI -derived fragments also increased the activity of caspofungin to prevent biofilm formation. A combinatorial approach to combat fungal infections is often more effective and decreases the chance of resistance occurrence. SEQUENCE LISTING

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Claims

1. An isolated peptide wherein said peptide comprises an amino acid sequence with at least 70% sequence identity to amino acid sequences GAXHYQFPSVKX (SEQ ID No. 8), QQXKDREHFAYG (SEQ ID No. 9) or WSGHXGSSSKXS (SEQ ID No. 10) or a derivative thereof wherein X stands for Cystein or a-aminobutyric acid, wherein said peptide consists of 12 to 44 amino acids, and wherein said isolated peptide inhibits microbial biofilm formation and development and/or potentiates the effect of an antifungal agent.

2. The isolated peptide according to claim 1 wherein said peptide comprises a sequence with at least 70% sequence identity to the amino acid sequences

GAXHYQFPSVKX (SEQ ID No. 8), GAXHYQFPSVKXFXKR (SEQ ID No. 11) or AYGGAXHYQFPSVKX (SEQ ID No. 12), or a derivative thereof and wherein said peptide consists of 12 to 24 amino acids.

3. The isolated peptide according to claim 1 or 2 wherein said peptide comprises a sequence with at least 70% sequence identity to the amino acid sequences selected from the group consisting of EHFAYGGAXHYQFPSVKXFXKRQ (SEQ ID No. 13), AYGGAXHYQFPSVKXFXKRQX (SEQ ID No. 14), YGGAXHYQFPSVKXFXKRQX (SEQ ID No. 15), HFAYGGAXHYQFPSVKXFXK (SEQ ID No. 16), or FAYGGAXHYQFPSVKXFXK (SEQ ID No. 17), or a derivative thereof.

4. The isolated peptide according to any one of claim 1 to 3 wherein said peptide is selected from the group consisting of EHFAYGGAXHYQFPSVKXFXKRQ (SEQ ID No. 13), AYGGAXHYQFPSVKXFXKRQX (SEQ ID No. 14), YGGAXHYQFPSVKXFXKRQX (SEQ ID No. 15), HFAYGGAXHYQFPSVKXFXK (SEQ ID No. 16), FAYGGAXHYQFPSVKXFXK (SEQ ID No. 17), or FAYGGAXHYQFPAVKXFXK (SEQ ID No. 18), or a derivative thereof.

5. The isolated peptide according to any one of the claims 1 to 4 wherein said peptide comprises a sequence with at least 70% sequence identity to the amino acid sequences selected from the group consisting of

DGVKLXDVPSGTWSGHXGSSSKXS (SEQ ID No. 2),

DVPSGTWSGHXGSSSKXSQQXKDR (SEQ ID No. 3),

WSGHXGSSSKXSQQXKDREHFAYG (SEQ ID No. 4),

SSSKXSQQXKDREHFAYGGAXHYQ (SEQ ID No. 5),

QQXKDREHFAYGGAXHYQFPSVKX (SEQ ID No. 6), or

EHFA YGGAXHYQFPSVKXFXKRQX (SEQ ID No. 7), or a derivative thereof.

6. The isolated peptide according to any one of the claims 1 to 5 wherein said peptide is selected from the group consisting of DGVKLXDVPSGTWSGHXGSSSKXS (SEQ ID No. 2), DVPSGTWSGHXGSSSKXSQQXKDR (SEQ ID No. 3),

WSGHXGSSSKXSQQXKDREHFAYG (SEQ ID No. 4),

SSSKXSQQXKDREHFAYGGAXHYQ (SEQ ID No. 5),

QQXKDREHFAYGGAXHYQFPSVKX (SEQ ID No. 6),

EHFAYGGAXHYQFPSVKXFXKRQX (SEQ ID No. 7), GAXHYQFPSVKX (SEQ ID

No. 8), QQXKDREHFAYG (SEQ ID No. 9) or WSGHXGSSSKXS (SEQ ID No. 10), or a derivative thereof.

7. The isolated peptide according to any one of the claims 1 to 6, or according to SEQ ID No. 1 for use in the treatment or prevention of a microbial biofilm associated condition or infection in a human or animal subject.

8. The isolated peptide according to any one of the claims 1 to 6, or according to SEQ ID No. 1 for use in a treatment according to claim 4, wherein said microbial biofilm or biofilm associated condition or infection comprises Candida cells.

9. A composition for use in treatment of prevention of a fungal biofilm associated condition or infection in a human or animal subject, said composition comprising at least one peptide according to any one of the claims 1 to 6, or according to SEQ ID No. 1 and at least one antifungal agent.

10. The composition of claim 9, wherein said at least one antifungal agent is selected from the group of echinocandins or polyenes.

11. The composition of claim 9, wherein said at least one antifungal agent is caspofungin, anidulafungin, micafungin, or amphotericin B.

12. The composition of any one of the claims 9 to 11 , further comprising one or more pharmaceutically acceptable compounds, carries and/or adjuvants.

13. The composition of any one of the claims 9 to 12, wherein said subject has been implanted with a medical device, which is infected or at risk of being infected with a fungal biofilm.

14. The composition of claim 13 wherein said medical device is selected from the group consisting of catheters, stents, surgical plates, prostheses, valves or pins, artificial joints, pacemakers, contact lenses and bio-implants.

15. The composition of any one of the claims 9 to 14, wherein said fungal biofilm is a Candida biofilm.

16. A method for reducing, eradicating, inhibiting or preventing fungal biofilms or fungal biofilm formation, characterized in that a surface or medium outside the body of a human or animal subject carrying said fungal biofilm or susceptible to said fungal biofilm formation, is exposed to at least one peptide according to any one of the claims 1 to 6, or according to SEQ ID No. 1.

17. The method according to claim 16, wherein said surface or medium is exposed to at least one peptide according to any one of the claims 1 to 6, or according to SEQ ID No. 1 , and at least one antifungal agent.

18. The method of claim 17, wherein said at least one antifungal agent is selected from the group of echinocandins or polyenes.

19. The method of claim 17, wherein said at least one antifungal agent is caspofungin, anidulafungin, micafungin, or amphotericin B.

20. The method of any one of the claims 16 to 19 wherein said biofilm is exposed to said at least one peptide before, after or concurrent with exposing said biofilm to said antifungal agent.

21. The method of any one of the claims 16 to 20 wherein said fungal biofilm is a Candida biofilm.

22. A method for the treatment or prevention of a condition or infection associated with fungal biofilm development in a human or animal subject, said method comprising administering to said human or animal subject a composition comprising at least one peptide according to any one of the claims 1 to 6, or according to SEQ ID No. 1 , and at least one antifungal agent.

23. The method of claim 22, wherein said at least one antifungal agent is selected from the group consisting of echinocandins or polyenes.

24. The method of claim 22, wherein said at least one antifungal agent is caspofungin, anidulafungin, micafungin, or amphotericin B.

25. The method of any one of the claims 22 to 24, wherein said fungal biofilm is a Candida biofilm.