Patent Application
20150225454
The present invention relates to tetrameric forms of peptides, with antimicrobial properties, particularly antifungal properties. The invention also relates to methods of making these tetrameric peptides. The invention also relates to the use of these multimers for inhibiting the growth of microorganisms, particularly fungus. The invention further relates to composition comprising these tetrameric peptides.
Antifungals are used to inhibit the growth of fungus and/or kill fungus (fungicide). For example, antifungal medications are drugs are used to treat infections caused by fungus and to prevent the development of fungal infections in patients, especially those with weakened immune systems. Pathogenic fungal infections are the seventh most common cause of infection-related deaths in the US.
In fact, recent surveys suggest that fungal diseases are posing a growing threat to the global biota.
In comparison to antibacterial drugs, the number of available antifungals is limited since common drug targets in fungi are homologues of similar molecular types in human which might also be inhibited. Examples of antifungals include polyenes, azoles, allylamines, fluocytosine and echniocandins (
Fungal infection of the eye, for example, fungal keratitis or keratomycosis, is common in tropical climates. In advanced countries, use of contact lens or lens care solution is the major risk factor for fungal keratitis. Ever since the first report on contact lens-associated fungal keratitis, several countries documented the incidence of ocular fungal pathogens infection (
The emergence of drug-resistance in fungus combined with an increased number of immune-compromised patients has limited the therapeutic options for clinicains and underscores the need for new classes of antifungals.
It is therefore desirable to develop antimicrobials particularly suited as antifungals, which are broad spectrum, suitable for many uses, especially for the treatment of ocular infections and preferably, resilient to the development of fungus resistance.
According to a first aspect, the present invention relates to an isolated peptide tetramer comprising the formula [(RGRKVVRR)2K]2KKi or an isolated peptide tetramer derivative of peptide tetramer [(RGRKVVRR)2K]2KKi with an initial peptide monomer (RGRKVVRR); comprising at least one amino acid substitution, at least one amino acid deletion, a rearrangement of at least one peptide monomer compared to the initial peptide monomer and/or at least one non-proteogenic amino acid modification in at least one peptide monomer compared to the initial peptide monomer or; wherein i=0 or 1
The invention also relates to uses thereof of the isolated peptide tetramer derivative or isolated peptide tetramer as described herein
The invention also includes a method of preparing the isolated peptide derivative or isolated peptide tetramer as described herein.
FIG. 1:Structure of monomer, linear retrodimer, peptide dimer B2088, peptide tetramer B4010, scrambled B4010 and natamycin.
Non-proteogenic amino acid refers to an amino acid which is not normally found in naturally occurring proteins which are typically made up of different numbers and combinations of 20 standard amino acids in their sequences.
Protected amino acid means an amino acid with a protecting group linked to either the carboxy group or the amine group protecting that group to allow the formation of a peptide bond with only the carboxy group or the amine group but not both. If the amine group is protected, only the carboxy group is available to form a peptide bond with another amine group. If the carboxy group is protected, only the amine group is available to form a peptide bond with another carboxy group. The terminal amine group or terminal carboxy group of a polypeptide may similarly be protected so that a peptide bond may be formed from the carboxy or amine end only.
The present invention relates to an isolated peptide tetramer comprising the formula [(RGRKVVRR)2K]2KKi or an isolated peptide tetramer derivative of peptide tetramer [(RGRKVVRR)2K]2KKi with an initial peptide monomer (RGRKVVRR); comprising at least one amino acid substitution, at least one amino acid deletion, a rearrangement of at least one peptide monomer compared to the initial peptide monomer and/or at least one non-proteogenic amino acid modification in at least one peptide monomer compared to the initial peptide monomer; wherein i=0 or 1. Further, any two, three or all of the peptide monomers may be modified with at least one amino acid amino acid substitution, at least one amino acid deletion, a rearrangement of the peptide monomer and/or at least one non-proteogenic amino acid modification. The sequence of the initial peptide monomer is RGRKVVRR (SEQ ID NO: 1).
If i=0, the isolated peptide tetramer is [(RGRKVVRR)2K]2K.
If i=1, the isolated peptide tetramer is [(RGRKVVRR)2K]2KK.
The isolated peptide tetramer may be derived from (RGRKVVRR)2K]2K or is [(RGRKVVRR)2K]2KK.
The isolated peptide tetramer derivative has the formula [(peptide)2K]2KK.
In particular, the isolated peptide tetramer derivative is branched with the structure:
The isolated peptide tetramer derivative is similarly branched with the structure:
Each of peptide 1, peptide 2, peptide 3 and peptide 4 is derived from the initial peptide monomer as described. Peptide 1, peptide 2, peptide 3 and peptide 4 may have the same sequence or may have different sequences
Peptide 1, peptide 2, peptide 3 and peptide 4 may independently comprise any one of SEQ ID NOs: 2-26
The amino acid substitution may be the substitution of one or more amino acid residues of the initial peptide monomer with any suitable amino acid. The substitution may be a substitution of one amino acid of the initial peptide monomer with any suitable amino acid. For example, the amino acid substitution may be at least one alanine substitution. The amino acid substitution may comprise successively substituting one amino acid of at least one initial peptide monomer (RGRKVVRR) with any suitable amino acid
In particular, the amino acid substitution comprises successively substituting one amino acid of at least one initial peptide monomer (RGRKVVRR) with alanine
Accordingly, the isolated peptide tetramer derivative may be selected from the group consisting of [(AGRKVVRR)2K]2KKi, [(RARKVVRR)2K]2KKi, [(RGAKVVRR)2K]2KKi, [(RGRAVVRR)2K]2KKi, [(RGRKAVRR)2K]2KKi, [(RGRKVARR)2K]2KKi, [(RGRKVVAR)2K]2KKi and [(RGRKVVRA)2K]2KKi; wherein i=0 or 1.
When i=0, the isolated peptide tetramer derivative is selected from the group consisting of [(AGRKVVRR)2K]2K, [(RARKVVRR)2K]2K, [(RGAKVVRR)2K]2K, [(RGRAVVRR)2K]2K, [(RGRKAVRR)2K]2K, [(RGRKVARR)2K]2K, [(RGRKVVAR)2K]2K and [(RGRKVVRA)2K]2
When i=1, the isolated peptide tetramer derivative is selected from the group consisting of [(AGRKVVRR)2K]2K, [(RARKVVRR)2K]2KK, [(RGAKVVRR)2K]2KK, [(RGRAVVRR)2K]2KK, [(RGRKAVRR)2K]2KK, [(RGRKVARR)2K]2KK, [(RGRKVVAR)2K]2KK and [(RGRKVVRA)2K]2KK
In a further embodiment, two or more of the amino acids of the initial peptide monomer may be substituted with alanine residues.
For example, the isolated peptide tetramer derivative may be selected from the group consisting of [(RGAAVVRR)2K]2KKi, [(RGRKVVAA)2K]2KKi, [(RGAKAVRR)2K]2KKi, [(RGRKAARR)2K]2KKi, [(RGAAAVRR)2K]2KKi, [(RGAKAARR)2K]2KKi, [(RGRAAARR)2K]2KKi, [(RGAAAARR)2K]2KKi, [(RGRKAAAA)2K]2KKi; wherein i=0 or 1.
When i=0, the isolated peptide tetramer derivative is selected from the group consisting of [(RGAAVVRR)2K]2K, [(RGRKVVAA)2K]2K, [(RGAKAVRR)2K]2KKi, [(RGRKAARR)2K]2K, [(RGAAAVRR)2K]2K, [(RGAKAARR)2K]2Ki, [(RGRAAARR)2K]2K, [(RGAAAARR)2K]2K, [(RGRKAAAA)2K]2K.
When i=1, the isolated peptide tetramer derivative is selected from the group consisting of [(RGAAVVRR)2K]2KK, [(RGRKVVAA)2K]2KK, [(RGAKAVRR)2K]2KK, [(RGRKAARR)2K]2KK, [(RGAAAVRR)2K]2KK, [(RGAKAARR)2K]2KK, [(RGRAAARR)2K]2KK, [(RGAAAARR)2K]2KK, [(RGRKAAAA)2K]2KK.
In a further embodiment, the isolated peptide tetramer derivative may comprise [VRGRVRKR)2K]2KKi; wherein i=0 or 1. In this embodiment, there is a rearrangement of the initial peptide monomer (RGRKVVRR); i.e. the sequence of the initial peptide monomer is scrambled. When i=0, the isolated peptide tetramer derivative is [VRGRVRKR)2K]2K. When i=1, the isolated peptide tetramer derivative is [VRGRVRKR)2K]2KK.
In another example, the amino acid deletion may comprise successively excluding an amino acid from the N-terminus of at least one initial peptide monomer. Accordingly, the isolated peptide tetramer derivative may be selected from the group consisting of [(GRKVVRR)2K]2KKi, [(RKVVRR)2K]2KKi, [(KVVRR)2K]2KKi, [(VVRR)2K]2KKi, [(VRR)2K]2KKi, [(RR)2K]2KKi and [(R)2K]2KKi; wherein i=0 or 1.
When i=0, the isolated peptide tetramer derivative is selected from the group consisting of [(GRKVVRR)2K]2K, [(RKVVRR)2K]2K, [(KVVRR)2K]2K, [(VVRR)2K]2K, [(VRR)2K]2K, [(RR)2K]2K and [(R)2K]2K.
When i=1, the isolated peptide derivative is selected from the group consisting of [(GRKVVRR)2K]2KK, [(RKVVRR)2K]2KK, [(KVVRR)2K]2KK, [(VVRR)2K]2KK, [(VRR)2K]2KK, [(RR)2K]2KK and [(R)2K]2KK.
The isolated peptide tetramer non-proteogenic amino acid modification may comprise (i) including at least one additional non-proteogenic amino acid at any position of and/or (ii) substituting at least one amino acid residue with a non-proteogenic amino acid in; at least one peptide monomer.
In particular, an additional non-proteogenic amino acid may be included at any position of at least one peptide monomer.
The isolated peptide tetramer or isolated peptide tetramer derivative according to any aspect of the invention may be for use as a medicament, pharmaceutical composition and/or antimicrobial composition and/or in therapy. The antimicrobial composition may be an antibacterial, antifungal or anti-protozoan composition. In particular, the antimicrobial composition comprises an antifungal composition.
The invention includes the use of an isolated peptide tetramer or isolated peptide tetramer derivative according to any aspect of the invention in the preparation of a medicament for preventing and/or treating at least one microbial infection. The microbial infection may be selected from the group consisting of bacterial infection, fungal infection and protozoan infection. In particular, the microbial infection comprises a fungal infection.
The invention also includes a method of inhibiting and/or reducing the growth of at least one microorganism comprising contacting the microorganism with at least one isolated peptide tetramer or isolated peptide tetramer derivative according to any aspect of the invention. This method may be an in vitro method. The microorganism may be selected from the group consisting of bacteria, fungi and protozoa. In particular, the microorganism comprises a fungi.
The invention also includes a method of preventing and/or treating at least one microbial infection comprising administering at least one isolated peptide tetramer or isolated peptide tetramer derivative according to any aspect of the invention.
The invention further includes a contact lens and/or eye drop solution, a pharmaceutical and/or antimicrobial composition, a composition for coating a device and/or a kit comprising the isolated peptide tetramer or isolated peptide tetramer derivative according to any aspect of the invention.
Methods of preparing the isolated peptide tetramer or isolated peptide tetramer derivative are included as part of the invention.
In one example, the method of preparing a peptide tetramer comprising four identical peptide monomers or isolated peptide tetramer derivative comprising four identical peptide monomers, comprises the steps of:
In another example, the method of preparing a peptide tetramer comprising four identical peptide monomers or isolated peptide tetramer derivative comprising four identical peptide monomers, comprises the steps of:
According to another aspect, the method of preparing a peptide tetramer comprising four identical peptide monomers or isolated peptide tetramer derivative comprising four identical peptide monomers comprises the steps of:
Further, the method of preparing a peptide tetramer comprising four identical peptide monomers or isolated peptide tetramer derivative comprising four identical peptide monomers comprises the steps of:
Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention.
Standard molecular biology techniques known in the art and not specifically described were generally followed as described in Green, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (2012).
The peptide monomer RGRKVVRRKK (SEQ ID NO: 27), linear retrodimer RGRKVVRRKKKRRVVKRGR (SEQ ID NO: 28), the peptide dimer B2088 (RGRKVVRR)2KK, the peptide tetramer B4010 [(RGRKVVRR)2K]2KK and scrambled B4010 [(VRGRVRKR)2K]2KK are illustrated in
In this procedure, every amino acid in the B4010 sequence (RGRKVVRR) is replaced with alanine residue and the antimicrobial and haemolytic activities will be evaluated by high throughput screening (HTS) methods. In the second method, every amino acid residue in B4010 sequence will be successively deleted and their activity-toxicity profile will be evaluated as before. After evaluating the MIC of all the peptides against at least three strains, the best peptides will be chosen and assessed for their activity in 50% rabbit tear fluid, 25% serum and in trypsin (enzyme: peptide ˜1:100).
Table 1 shows the modified peptides and their properties.
After identifying the peptides that display excellent activity in complex biological fluid, the following modifications by non-proteinogenic amino acid residues exemplified in Table 2 will be chosen.
Chemicals and Peptides: Sabouraud's Dextrose Agar was purchased from Acumedia (Michigan, MI, USA). Peptides were purchased from EZBiolabs Inc., (Carmel, Ind., USA). Lipids such as L-α-phophatidylcholine (PC), L-α-phostidylethanolamine (PE), L-α-phosphatidylserine (PS) and L-α-phosphatidylinositol (PI) were bought from Avanti Polar Lipids Inc. (AL, USA). Ergosterol, sodium azide (NaN3), Carbonyl cyanide m-chloro phenylhydrazone (CCCP), 4-aminopyridine (4-AP), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), Gadolinium III Chloride, tetra ethyl ammonium chloride (TEA) and 3′,3′-dipropylthiadicarbocyanine (diS-C3-5) dye were purchased from Sigma-Aldrich Corp. (MO, USA). ATP bioluminescence kit was obtained from Molecular Probes Inc (OR, USA). Amphotericin B and Natamycin were obtained in powder form from Sigma-Aldrich (S) Pte Ltd (Singapore).
Far UV-CD spectra of the peptides (0.6 mg/ml) were recorded on a JASCO J810 spectropolarimeter (JASCO, Tokyo, Japan) in 10 mM PBS (pH 7.0) using a 0.1 cm path length quartz cuvette at 25° C. Spectra were recorded from 260 nm to 190 nm in 0.1 nm steps at a scan rate of 50 nm/min. The final spectrum is the average of 4 scans. The CD data is expressed as mean residual ellipticity ((θ)mrw, deg cm2 dmol−1).
The yeast strains were cultivated and suspended in Sabouraud's Dextrose (SD) broth diluted at one sixth at a starting OD600=˜0.08 in a flat-bottomed microtitre plate. A serial dilution of peptide in the same broth was mixed with the inoculum to give a final peptide concentration of 0.4-22 μM. The antifungal activity was assessed by monitoring the OD600 in cycles of 30 minutes and an orbital shaking at 100 rpm using an Infinite M200 microplate reader (Tecan Group Ltd., Switzerland) for 24/48 h at 37° C. Cultures without peptides were used as positive controls and broth alone or with 22 μM peptide served as negative controls. The minimum concentration required for complete inhibition was assessed by both visible observations as well as by measuring the OD600 and taken as the MIC. Each experiment was repeated in triplicates.
Results:
The peptide monomer, RGRKVVRRKK (SEQ ID NO: 27) and the linear retrodimer RGRKVVRRKKKRRVVKRGR (SEQ ID NO: 28) displayed weak antifungal activity (Table 3) against both C. albicans and Fusarium strains. However, upon linking two copies of the sequence through a branched lysine (B2088,
The peptide B4010 displayed a broad spectrum antifungal activity against ATCC reference isolates as well as clinical isolates. (Table 3). The activity was 2-4 times superior to natamycin, the only US FDA approved ophthalmic drug. Table 4 shows the MIC and cytotoxicity of other designed peptides.
C. albicans
C. albicans
C. albicans
C. albicans
C. albicans
F. solani
F. solani
F. solani
C. albicans
C. albicans
C. albicans
C. albicans
C. albicans
The time-kill kinetics was measured for two strains of C. albicans (ATCC 10231 and DF2672R). The cultures were grown overnight in SD broth and the cell concentration was adjusted to 105-106 CFU/ml with phosphate buffer. The peptide or antifungals were added to the individual cultures. Appropriate final concentrations of B4010 (1.4-11 μM for ATCC and 0.37-3.7 μM for CA 2672R) or amphotericin B (0.55 μM-11 μM for CA 2672R strains) and natamycin (4.7 μM-75 μM for ATCC and 30 μM for CA 2672R strains) were adjusted for the inocula. The test solution was incubated at 37° C. with constant shaking. 100 μL of the suspension is withdrawn at predetermined time points, serially diluted (102 or 103 fold) and poured into the SDA plate. The plate was incubated for 48 h at 37° C. for colony counting. The data were expressed in terms of % cell viability relative to the positive control.
For studying the effects of metal ions, SD broth was adjusted with appropriate concentrations of salts to yield a final concentration of 100 and 135 mM for NaCl and KCl and 0.5-2 mM for CaCl2 and MgCl2. MIC determinations were carried out as described above. C. albicans ATCC10231 cells and salts in broth were taken as a positive control. Salts alone (NaCl, KCl, and CaCl2 and MgCl2) in broth or with 22 μM peptide served as a negative control.
To study the effects of metal ions on viability, the broth solution containing C. albicans (OD600=0.4-0.6) was adjusted to appropriate final concentrations of metal ions, incubated with 5.5 μM of B4010 for 6 h and the cell viability was determined as before after 48 h.
Results: The effects of physiological concentrations of metal ions (NaCl, CaCl2 and MgCl2) on the antifungal activity of B4010 were examined. The growth of C. albicans ATCC 10231 was monitored at various concentrations of the peptide and in the presence of physiologically relevant (close to the concentration present in tear fluid) concentrations of monovalent and divalent cations (
For all the experiments, unless otherwise stated, ATCC 10231 strain was used. B4010 (1 mg/ml) was incubated with trypsin (enzyme:peptide ratio 1:100) at 37° C. 20 μL aliquot of this mixture was withdrawn at various time intervals (0.5, 1, 2, 4 and 6 h) and mixed with 1 μL of trypsin inhibitor. The antifungal activity of the peptide was determined by adding this mixture to 180 μl of inoculum and monitoring the growth at OD600 for 24 h. Similar experiments performed in the presence of trypsin/trypsin inhibitor (without B4010) were used as a positive control. For assessing the antifungal activity in tear fluid (TF), the peptide was dissolved in freshly collected rabbit TF and incubated at 37° C. for 6 h. After incubation, an equal volume of an overnight culture of C. albicans (˜106 CFU/ml) was mixed with peptides in tear fluid and incubated at 37° C. for 24 h. The final concentrations of the peptide were at 4.4, 8.8 and 22 μM. A 100 μL aliquot of serial dilutions (102 or 103 times) of this mixture were inoculated on a SD agar plate and then incubated for 48 h at 37° C. Culture alone and culture with 50% tear fluid served as the positive control. The data was expressed in % killing with respect to culture without tear fluid. About 6-10% killing was observed in the presence of 50% tear fluid.
The effect of 5% human serum on the activities of B4010 against two C. albicans clinical isolates was determined. The human male serum was centrifuged at 13,000 rpm for 10 mins in order to remove the lipids and the supernatant was collected. The MIC values against clinical isolates of C. albicans 2672R and C. albicans 1976R were determined both in standard medium (SD broth) as well as in standard medium containing 5% serum supernatant as before.
Antifungal properties of the peptide were examined in three complex biological environments.
The peptide B4010 was incubated with trypsin (trypsin:peptide ratio=1:100) at 37° C. An aliquot of this mixture was withdrawn at various time intervals, added to the C. albicans inoculum and the growth was monitored (
The antifungal efficacy of B4010 was also monitored in the presence of complex biological fluids such as tear fluid and human serum.
The antifungal activity of B4010 was assessed in 50% tear fluid. In the absence of tear fluid, about 98±2% loss of viable cells were observed at a peptide concentration of 5.5 μM. However, the candidacidal activity was moderately suppressed by the presence of 50% tear fluid at lower (4.4 and 8.8 μM of B4010) concentration (
The effect of serum on antifungal efficacy of B4010 was assessed by measuring the change in the MIC of B4010 in the presence of 5% human serum against two clinical isolates of C. albicans. For both the strains the MIC values were shifted from 0.34 μM without serum to 5.5 μM in the presence of serum, suggesting 16-fold increase in the MIC in 5% sera.
Haemolytic activity of peptides and antifungal drugs were determined against rabbit red blood cells (Oren et al., 1997). Briefly, serial dilution of peptides/antifungals in PBS was mixed with rRBC (final concentration 4% v/v), incubated at 37° C. for 1 h and centrifuged at 3000 rpm for 10 minutes. The release of hemoglobin in the supernatant was monitored by measuring the hemoglobin absorbance at 576 nm. The readings from cell suspension in PBS (without any additives) or 1% Triton-X100 were used as 0% or 100% haemolysis.
Results of haemolytic assay: The ability of B4010 in disrupting the membrane integrity of mammalian cells was evaluated by haemolytic assay using rabbit red blood cells. The peptide had no significant haemolytic activity (<1% haemolysis) against rabbit erythrocytes even at 440 μM (
Table 5 summarises of the haemolysis assay of various peptide tetramers
IOBA-NHC cells were cultured under standard conditions (humidified atmosphere of 5% CO2 at 37° C.) in DMEM/F12 supplemented with 1 μg/ml bovine pancreas insulin, 2 ng/ml mouse epidermal growth factor, 0.1 μg/ml cholera toxin, 5 g/ml hydrocortisone, 10% fetal bovine serum (FBS), 50 UI/ml penicillin, and 50 UI/ml streptomycin. Cells from passages 50-80 were used in all experiments. Every day, normal culture development was observed by phase-contrast microscopy. Cells were removed by gentle trypsin incubation at confluence and counted. They were seeded into 96 well culture plates (Corning, Schiphol-Rijk, The Netherlands) for microtitration analysis of flow cytotoxicity assay (˜10,000 cells/well). Cultures were kept at 37° C. for 24 h. Subconfluent cells (culture surface covering nearly 70%) were then exposed to various peptide concentrations (0.22-225 μM). Cytotoxicity was measured periodically using MultiTox-Fluor Multiplex assay kit (Promega, Wis., USA) by measuring the AFC fluorescence (λex=485 and λem=520 nm). Cell viability was reported after 24 h incubation of the cells with the peptides since there was no detectable toxicity even after 8 h of incubation and the EC50 (effective concentration of peptide that reduces the viable cells by 50%) was determined.
Acute toxicity of B4010 was assessed with C57BL6 (6-8 weeks old) wild type mice. Two healthy wild type mice were chosen for each route of administration. B4010 was delivered through intra-peritoneal (200 mg/kg) or (100 mg/kg) intra-venous routes. Two animals were used for each administration and monitored through 24/48 h to determine mortality, or signs of toxicity.
Results of Cytotoxicity Assay:
Exposure of B4010 caused 50% loss of cell viability at 220 μM (
It was further examined whether topical application of B4010 would influence the corneal wound healing in vivo. All animal experiments were conducted in compliance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research, the guide for the Care and Use of laboratory animals (National Research Council) and under the supervision of Singhealth Experimental Medical Centre (SEMC). Four New Zealand white rabbits were divided into two groups, two each for control (saline) and two each for the study group treated with B4010. Rabbits were tranquilized by intra muscular injection of 1 ml of ketamine (100 mg/ml) and 0.5 ml of xylazil (20 mg/ml). Corneas were anesthetized by topical administration of 1% xylocaine. A 5-mm trephine was used to outline the wound margin and mechanical removal of epithelial cells was carried out by sterile mini blade (BD-Beaver) leaving the basal lamina intact as previously described (Crosson et al., 1986). The animals were treated by topical administration of 22 μM of B4010, 3 times per day. Cornea wound was visualized by staining with fluorescein sodium, which is used in the ophthalmology clinic as a non-toxic dye to disclose wounds of the cornea, and using a cobalt-blue filter with slit lamp biomicroscopy. Measurements of the residual wound area were performed during the reepithelialization process by Image-J 1.440.
Results: The epithelial wound healing rate was not altered by topical application of B4010 (22 μM) 3 times a day compared to the control groups (
Determination of MIC Against Wild Type (WT) and Mutant S. cerevisiae Strains
The antifungal activity of B4010 was also tested against S. cerevisiae ergΔ mutants carrying an altered sterols structure and composition
A few identical colonies from WT and mutant strains were immediately inoculated and grown overnight in SD broth containing 0.1 mg/ml ampicilin at 30° C. Cells were harvested by centrifugation, washed with sterile water and frozen. Cells were plated again on SD agar plates and incubated at 37° C. for 48 h. Two or three identical colonies of the mutant strains were collected and inoculated in SD broth. Peptides were dissolved in sterile water and mixed with WT and mutant strains and incubated at 37° C. for 48 or 72 h in a test tube. The lowest concentration of the peptide that inhibited the growth of the yeast strain was determined by visual inspection.
Results: The emergence of resistant pathogens poses a greater threat to the management of fungal infections. To determine if the sterol alterations have an affect on the antifungal activity of B4010, experiments were performed using S. cerevisiae strains that carry specific mutations in the ergosterol (ergΔ) pathways. Table 6 summarizes the MIC values of B4010 against wild type and various ergΔ mutants which carry altered sterol structure and compositions. Interestingly, all the ergΔ mutants displayed hyper susceptibility to B4010 i.e. 2-4 fold decrease in MIC values compared to the wild type strains.
ErgΔ mutants are hypersensitive to B4010. As a result of the mutation, the strains accumulate sterols with altered structure in B or C ring of ergosterol and the side chains. For simplicity, the major sterol is given in Table 5 for the strains. Overall, the mutant strains displayed 2-4 fold decrease in MIC values compared to the wild type strain. The mutant strains erg2Δ, erg3Δ, erg3Δerg6Δ and erg4Δerg5Δ displayed increased sensitivity to B4010 (4 folds decrease in MIC compared to the wild type). The mutant strains erg2Δerg6Δ and erg6Δ displayed 2 fold increased sensitivity to B4010.
In another study to probe the mechanism of action, the affinity of B4010 for insoluble polysaccharides such as chitin and β-D-glucan, the major component of fungal cell walls was examined. B4010 (5.5-22 μM) was incubated with chitin (from shrimp shells) or β-D-glucan for 2 h at 37° C. The mixture was centrifuged at 10,000 g and the supernatant was analyzed by SDS-PAGE (4-20%). A control experiment without chitin/β-D-glucan was also performed to quantify peptide binding to the carbohydrate polymers.
The peptide showed no affinity for cell wall polysaccharides as no coprecipitation was observed with either chitin or β-D-glucan (
Two complementary fluorometry assays were performed to determine if B4010 has a membrane targeting action.
To check if the peptide permeabilized the cytoplasmic membrane, the uptake of SG was assayed. This is a membrane impermeable dye which does not fluoresce when incubated with cells which have intact cell membrane. In membrane-compromised cells fluoresce strongly upon binding to intracellular nucleic acids.
For SG uptake assay, overnight cultures of Candida (ATCC 10231) were harvested by centrifugation, washed three times with HEPES buffer and resuspended in the same buffer. The cells were incubated with 1 μM SG in the dark prior to the addition of peptide. The increase in emission intensity at 520 nm was monitored after addition of various concentrations of peptide (λex=485 nm). 1% Triton-X100 was added at the end of the experiment to determine the % uptake
Results:
The ability of B4010 to permeabilise the cytoplasmic membrane was assessed by SYTOX Green uptake assay. The fluorogenic dye stains cells that have compromised plasma membrane and displayed strong fluorescence upon binding to intracellular nucleic acid. A marked and rapid increase in the fluorescence of C. albicans upon addition of B4010 was observed confirming the membranolytic action (
Addition of B4010 to C. albicans incubated with 1 μM SG resulted in rapid uptake of the dye with a concomitant increase in fluorescence intensity in a concentration-dependent manner (
diS-C3-5 Membrane Depolarisation
Change in membrane potential of C. albicans upon addition of B4010 was monitored by the release of potential sensitive probe diS-C3-5 dye. Briefly, 1 ml of overnight grown mid-log phase Candida cells in 5 mM HEPES buffer (pH 7.0) was mixed with 10 μM diS-C3-5 dye and incubated at 37° C. for 1-2 h in a thermo shaker. 800 μL of the dye-loaded cell suspension was transferred to a stirred quartz cuvette and the change in fluorescence intensity was monitored at an emission wavelength (λem) of 670 nm (excitation wavelength, 622 nm) using a Quanta Master spectrofluorimeter (Photon Technology International, NJ, USA). The excitation and emission bandwidths were set at 1 and 2 nm, respectively. Once a constant fluorescence level was achieved, 10 μL of concentrated peptide solution in HEPES buffer was added so that the final concentration of peptide was 0.22-22 μM. The change in fluorescence intensity was monitored continuously for 1 or 2 h. To study the effects of energy poisons and ion-channel inhibitors, the additives were added prior to the addition of 5.5 μM B4010. The change in fluorescence intensity was monitored as before.
Since yeasts maintain a negative resting membrane potential inside the cell, in order to determine if the candidacidal action of B4010 was due to dissipation of electrochemical gradients across the membrane, using a potential sensitive probe, diS-C3-5 was used.
To verify if the dissipation of membrane potential is linked to candidacidal activity, a cell viability assay was performed under identical conditions. A complete loss of viability was observed when the concentration of peptide exceeded 4×MIC. Together with the SG uptake assay, these results confirmed that B4010 caused rapid membrane potential dissipation and permeabilizes the cytoplasmic membrane of C. albicans in a concentration-dependent manner
The extracellular release of metal ions and ATP upon challenging C. albicans with B4010 was assessed to determine if perturbation of the membrane would affect its barrier function.
Overnight cultured late logarithmic phase C. albicans was harvested by centrifugation, washed 5 times with 10 mM HEPES (pH 7.0), resuspended in the same buffer and adjusted to an OD600=0.4. To 5 ml of this suspension, B4010 (final concentration 5.5 μM) was added and incubated at 37° C. for 2 h. The mixture was centrifuged at 3,000 g and the presence of K+, Ca2+ and Mg2+ in the supernatant was estimated by Perkin Elmer Dual-view Optima 5300 DV inductively coupled plasma-optical emission spectrometry (ICP-OCS Massachusetts, USA) available at CMMAC facilities (Department of Chemistry, National University of Singapore).
The extracellular ATP levels upon challenging C. albicans with B4010 were determined as reported before (Koshlukova et al., 1999). Cells (OD600≈0.6) were incubated for 1.5 h at 37° C. with or without various additives for 2 hours at 37° C. with orbital shaking. B4010 (5.5 μM) was added and incubated for another 1.5 h at 37° C. with shaking. Each tube was then centrifuged at 5000 g for 5 mins. Then 225 μL of boiling TE buffer (50 mM Tris, 2 mM EDTA, pH 7.8) was added to the 25 μl of the supernatant and mixed well. This mixture was boiled again for another 2 minutes and stored at 4° C. until further examination. 100 μL of a luciferin-luciferase ATP assay mixture was added to 100 μL of the supernatant and luminescence was monitored using the Infinite M200 microplate reader (Tecan Group Ltd., Switzerland). For the time-course experiment, cells (OD600=0.4) were treated with 5.5 μM peptide for various time intervals. For dose-dependent studies, the concentration of the peptide was varied from 0.4-44 μM. The extracellular ATP concentration was determined from calibration curve obtained by ATP assay kit (Molecular Probes, OR, USA) as per the manufacturer's instruction.
Results:
The background K+ and Ca2+ concentration in the supernatant were 43.4±2 μM and 2.5±0.5 μM, respectively. After incubation of C. albicans with B4010 (5.5 μM) for 2 h, more than two fold elevation of potassium (104±4.2 μM) and calcium (5.8±1.3 μM) concentration was observed, whereas no significant changes in the levels of Na+ and Mg2+ ions were observed. ATP bioluminescence assay indicated a rapid release of ATP from C. albicans upon addition of B4010 (
For the effects of energy poisons and ion channel inhibitors, yeast cells (105-106 CFU/ml) were incubated for 2 h with or without additives at 37° C. A final concentration of 5.5 μM B4010 peptide was added to the cell suspension and incubated further for another 1.5 h at 37° C. Each cell suspension was further diluted and 100 μl of the cells were plated and incubated at 37° C. SDA plates with additives (and no peptide) served as the positive control. Cell viability was obtained by counting the number of colonies formed in each plate after 48 h incubation at 37° C. The final concentrations of the additives were: CCCP (5 μM), NaN3 (5 mM), 4-AP (1 mM), NPPB (0.5 mM), Gadolinium II chloride (1 mM) and tetra ethyl ammonium chloride (15 mM). The reported values were averages of two independent duplicate experiments. Control experiments with all the additives were also performed to assess the toxicity of the additives to C. albicans.
To study the effect of metabolic activity of C. albicans on the susceptibility to B4010, the diS-C3-5 loaded cells were incubated with 5 μM CCCP (an uncoupler of proton gradients) or 5 mM NaN3 (which blocks both classical and alternative pathways of mitochondrial inhibition) and the changes in fluorescence intensity upon addition of B4010 were monitored. Addition of CCCP resulted in a strong reduction in fluorescence intensity indicating collapse of the membrane potential. Subsequent addition of B4010 had weak changes in the transmembrane potential (
The effect of CCCP/NaN3 on ATP release by B4010 was also investigated. Consistent with the cell viability assays, ATP bioluminescence assay indicated significant increase in the extracellular ATP levels in B4010-treated cells (
It was Observed that
B4010 caused rapid release of K+ as well as ATP and that the addition of external K+ decreased the extent of cell death with >16-fold increase in MIC at elevated ion concentrations prompted the question whether ion-channel inhibitors could protect C. albicans from B4010. The effects of non-specific organic cationic inhibitors (TEA and 4-AP) as well as yeast stretch-activated ion channel blocker Gd3+ on candidacidal activity of B4010 were tested. The peptide caused significant loss of viability in cells that were pretreated with TEA and 4-AP (
To determine if the loss of B4010 activity was associated with alterations in the membrane potential, the effect of additives for a change in intensity of diS-C3-5 dye loaded cells before and after the addition of B4010 was monitored. TEA or 4-AP did not alter the membrane potential whereas subsequent addition of B4010 resulted in complete loss of membrane potential and the magnitude of intensity changes was similar to the one observed in cells without prior addition to 4-AP or TEA (
Small unilamellar vesicles (SUVs) were prepared using PC/PE/PI or PS lipids (5:2.5:2.5) containing 15 wt % ergosterol or PC/Cholesterol (10:1) (Makovitzki et al., 2006). For the preparation of PC/PE/PI or PS/Erg SUV, the lipids were dissolved in chloroform/methanol (2:1, v/v) in a glass tube. Ergosterol was dissolved in same solvent and added to lipid mixture to make final ergosterol content of 15 wt %. The Lipid-ergosterol mixture was dried using nitrogen gas to form lipid layer. The film was hydrated in a buffer containing 20 mM PBS (pH 7), vortexed and sonicated. Each cycle of sonication (5 sec, 40° C.) is followed by freezing in liquid nitrogen and thawing on a water bath kept at 37° C. The procedure was repeated 5-6 times until an optically clear dispersion appeared. The SUV was divided into two parts. To the first part 50 mM calcein was added and incubated for 1 h. The excess calcein was removed by injecting 100 μL of the calcein-loaded SUV's into a Ultrahydrogel™ 250 (7.8 mm×300 mm) column equilibrated with 20 mM PBS (pH 7.0) and eluted isochratically at a flow rate of 1 ml/min for 1.5 column volumes. The calcein-loaded liposomes were mixed with calcein-free liposome (1:1) to adjust final liposome concentration. Peptide concentration was added to obtain 1:30 or 1:15 peptide:liposome ratio. Changes in the fluorescence signal after addition of peptide or Triton X-100 was measured on a PTI spectrofluorometer, using an excitation wavelength of 480 nm and emission wavelength of 512 nm. The Percentage calcein released at each time point was calculated using the equation:
Where A0 is the observed fluorescence intensity upon addition of peptides, Amin is the average intensity at the baseline (before peptide addition), Amax is the average intensity at the saturation phase after adding 1% Triton X-100. Similar protocol was followed for the preparation of PC/Cholesterol SUVs.
Results:
In tropical countries, mycoses have high incidence and are responsible for major health and economic problems. The expanding populations of immuno compromised patients, large-scale use of antifungals in food and increased use of medical devices and implants have further raised the incidence of fungal infections. Systemic Candida species account for 40% mortality rates and is the 4th leading cause of hospital acquired blood stream infections in the US. Examples of current antifungals and their properties are shown in Table 7. However, the problem of increase in antifungal resistance and limited choice of antifungal remains.
C. albicans, C. glabrata & C. krusei
This study reports that multimerisation of a weak antifungal peptide resulted in highly enhanced antifungal and membrane permeabilising activities compared to its monomeric counterpart. The special properties of the B-series of peptides are:
1. Kills many forms of fungus and yeast rapidly (less than 1 hour).
2. Able to kill mutant forms of yeast with changed sterol membrane composition.
3. Resistant to degradation.
4. Resistant to physiological concentrations of monovalent and divalent ions.
The most active peptide which carried 4 copies (B4010) displayed excellent antifungal activities against Candida and Fusarium strains. Scrambling the sequence resulted in a 2-4 fold decrease in antifungal activity, indicating the importance of amino acid sequence.
Kinetics of candidacidal activity for B4010 was compared with polyene antifungals. For both the ATCC and clinical isolates, B4010 caused 3 log reductions of viable cells in less than 1 h at 2× or 4×MIC values whereas the polyene antifungals require an elevated concentration and longer time to achieve similar end points. The antifungal properties of B4010 were assessed in the presence of monovalent and divalent cations as well as in complex biological fluids. The results showed that the antifungal activity of B4010 was not altered by physiologically relevant ionic strengths although at high concentration of K+ ions a reduced activity was observed. The strong depolarizing activity of K+ on yeast cells at elevated concentration may be responsible for the partial loss of antifungal activity.
When incubated with trypsin or tear fluid for 6 h, B4010 retained significant antifungal potency. These results suggest improved stability of B4010 in complex biological environments. In the presence of human serum, the MIC values of the peptide against two clinical isolates were elevated by about 16 fold. It is likely that the interaction of B4010 with albumin or other proteins may be responsible for the increased MIC values. Taken together these results highlight considerable antifungal efficacy of B4010 in complex biological fluids.
Several mechanisms have been documented in the evolution of resistance to azole and polyene antifungals by C. albicans. Qualitative or quantitative changes in the sterol structure and composition by altering the specific steps in the ergosterol biosynthetic pathways are amongst the most important mechanisms of azole and polyene resistance known. Analysis of the sterol composition in azole-resistant C. albicans strains from AIDS and leukemia patients indicated the accumulation of ergosta-7,22-dienol. It has been shown that mutations in ERG4, ERG6 and ERG3 of S. cerevisiae displayed enhanced resistance to polyene and azole antifungals. However, all the yeast mutants which carry altered sterol structure and composition are hyper sensitive to B4010. It is interesting to note that erg3Δ mutants that are intrinsically resistant to azoles and polyene antifungals are more susceptible to B4010 than are the erg2D and erg2D6D mutants. It is possible that the enhanced membrane fluidity resulting from sterol alterations may contribute to increased permeability of B4010, thus leading to the observed hyper susceptibility of the mutant strains.
The therapeutic potential of host defense peptides is also limited by increased cell toxicity and haemolytic activity. In comparison to natamycin and amphotericin B, the peptide was non-haemolytic to rabbit erythrocytes at higher concentration. However, the cytotoxicity of the peptide to HCE cells was comparable to natamycin and superior to that of amphotericin B. B4010 did not affect the corneal reepithelialization rate in rabbit nor displayed acute toxicity in mice indicating safety of the peptide in surgical settings.
Since many antifungal peptides require an intact cell wall to exert antifungal action, the affinity of B4010 for cell wall polysaccharides was examined. B4010 had no affinity for b-D-glucan or chitin as no coprecipitation was observed in the pull-down experiments. To probe the membrane targeting properties and to correlate the rapid candidacidal activity, the effect of concentration of B4010 on the kinetics of membrane permeabilization was monitored by SG uptake and diS-C3-5 release assays. At all concentrations, a rapid SG uptake was observed although the maximum uptake was achieved at 4× the MIC. These results support the time-kill kinetics assays that complete killing occurred at 4×MIC in 30 minutes. diS-C3-5 dye release studies suggested a weak dissipation of the membrane potential at lower concentrations and maximum dissipation was observed above 2×MIC. A loss of viability in yeast cells exposed to 4×MIC of B4010 under identical conditions suggests that dissipation of membrane potential is linked to candidacidal activity. The rapid dissipation of membrane potential and SG uptake may indicate direct interaction of the peptide with the cytoplasmic membrane.
Challenging C. albicans with B4010 caused 2 fold increase in K+- and Ca2+-ions indicating membrane damage. The extracellular release of ATP from yeast cells treated with B4010 is concentration-dependent with maximum efflux at 4×MIC. The kinetics of ATP efflux at 4×MIC reached a maximum within 30 minutes, in accordance with kinetics of candidacidal activity. SEM studies showed that C. albicans treated with B4010 displayed rough with disrupted morphologies and extensive blebbing, again pointing to the observations that membrane is the principal and presumably the critical target for the peptide.
This study showed that additives which alter the cytoplasmic membrane potential or energy metabolism confer substantial protection of C. albicans from B4010 induced lethality. At 5 μM, CCCP has been shown to cause depolarization of the cytoplasmic membrane whereas a high concentration (>50 μM) is required for mitochondrial depolarization. Cell viability assays confirmed that cells pretreated with 5 μM CCCP provided partial protection (45% viable cells) from B4010. diS-C3-5 assay indicated collapse of membrane potential in the presence of 5 μM CCCP. A similar effect was observed in the presence of anion channel inhibitor, DIDS. These results suggest that the alterations in transnegative electrochemical gradient may potentially be the underlying mechanism of protection of C. albicans from B4010. It has been shown that the membrane potential of S. cerevisiae is −76±5 mV whereas for C. albicans the value is −120 mV (55,56). The reduced membrane potential of S. cerevisiae may account for the observed higher MIC values S. cerevisiae (5.5 μM) compared to C. albicans (0.37-1.4 μM).
In support of this, a high concentration of extracellular K+ strongly depolarises the yeast cells without affecting cell viability and subsequent addition of B4010 caused little changes in the membrane potential and partial protection (40% viable cells) from B4010. Furthermore, ion-channel inhibitors, which did not alter the membrane potential, failed to protect yeast cells from B4010. However, the presence of NaN3 completely abrogated the candidacidal activity of B4010. It has been shown that NaN3 alters the fluidity of the plasma membrane of yeast without affecting the membrane potential. Consistent with this, addition of membrane fluidizer rescued the antifungal activity of B4010. Therefore, this suggests that the candidacidal action of B4010 is linked to plasma membrane potential and fluidity of the membrane.
The effect of B4010 on calcein-loaded SUVs containing purified lipids and ergosterol point unambiguously towards membrane-lytic action of the peptide. The results from these studies further indicated that tetrabranching and the aminoacid sequence are important for membrane disruption since a decreased calcein release was observed in the presence of B2088 and Sc_B4010 peptides. In cholesterol-containing zwitterionic SUVs, however, B4010 caused reduced calcein release suggesting weaker interactions with mammalian model membrane. A high % of calcein release and retention of extended conformation of the peptide in mixed liposome containing ergosterol supported the results obtained from the MD simulation results. In addition, the low MIC against yeasts and high EC50 values against HCE cells confirm that B4010 selectively damages the fungal membrane.
In conclusion, this study showed that assembling 4 copies of a weakly active peptide on a branched lysine core amplifies the properties and overcomes several limitations of the linear antimicrobial peptides. In addition, the peptide is hyper potent against several yeast strains with altered sterol structure and composition, thus suggesting their potential to combat resistance. The peptide is non-toxic when tested in vitro and in vivo. B4010 targets the cytoplasmic membrane and caused rapid dissipation of membrane potential and loss of intracellular components.
Additives that alter the membrane potential or metabolic activity have profound influence on the antifungal properties. Using SUVs, this study showed that the peptide was more selective to fungal model membrane than for the mammalian model membrane. The first arginine residue of the putative sequence played an important role in mediating the interactions with the negatively charged fungal model membrane. In support of this, replacement of the first arginine resulted in ˜2-4 fold decrease in antifungal activity. Although it is difficult to simulate the in vivo conditions, this study combines extensive experimental and computer simulation studies to advance understanding of the interaction of antifungal peptides with model lipids and provide guidance for the rational design of therapeutically important new antifungal drug with the promise of combating resistance.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201206715.3 | Sep 2012 | SG | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2013/000390 | 9/9/2013 | WO | 00 |
