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Short cationic antimicrobial peptides display superior antifungal activities towards candidiasis and onychomycosis in comparison with terbinafine and amorolfine Wenche Stensen, Rob Turner, Marc Brown, Nahid Kondori, John Sigurd Svendsen, and Johan Svenson Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00654 • Publication Date (Web): 31 Aug 2016 Downloaded from http://pubs.acs.org on September 1, 2016

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Molecular Pharmaceutics

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Short cationic antimicrobial peptides display superior antifungal activities towards candidiasis and onychomycosis in comparison with terbinafine and amorolfine

Wenche Stensena,b, Rob Turnerc, Marc Brownc,d, Nahid Kondorie, John Sigurd Svendsena,b*, Johan Svensonf,*

a

Department of Chemistry, UiT The Arctic University of Norway, Tromsø, Norway; bLytix

Biopharma AS, Tromsø, Norway; cMed Pharm Ltd, Guildford, U.K.; dTDDT, University of Hertfordshire, Hatfield, Herts, U.K.; eDepartment of Clinical Microbiology, Sahlgrenska University Hospital, Gothenburg, Sweden; fDepartment of Chemistry, Materials and Surfaces, SP Technical Research Institute of Sweden, Borås, Sweden.

Running title: Antifungal SAMPs

Keywords: onychomycosis, candidiasis, antimicrobial peptides, terbinafine, amorolfine, antifungal

ACS Paragon Plus Environment


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ABSTRACT

Novel antifungals are in high demand due to the challenges associated with resistant, persistent and systemic fungal infections. Synthetic mimics of antimicrobial peptides are emerging as a promising class of compounds for antifungal treatment. In the current study, five synthetic cationic antimicrobial tripeptides were evaluated as antifungal therapeutics against 24 pathogenic strains of fungi. Three of the peptides displayed strong general antifungal properties at low micromolar inhibitory concentrations. The most promising peptide, compound 5, was selected and evaluated as antifungal remedy for Candida albicans candidiasis in a human skin model and for the treatment of Trichophyton rubrum induced onychomycosis in an infected human nail model. Compound 5 was shown to display antifungal properties and a rapid mode of action superior to both the commercial comparators Loceryl® and Lamisil®. Compound 5 was also active against a clinical isolate of Candida albicans with acquired fluconazole resistance.

Table of content Graphic

Antifungal cationic SAMPs


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INTRODUCTION

Fungal infections are common and affect the majority of the general population at some stages in life. While the majority of the fungal infections are localized and respond well to non-systemical topical treatment, the prevalence of fungal resistance to antifungal drugs is rising alarmingly1. The rarer, severe forms of fungal infections, can cause meningitis and sepsis and can be deadly, particularly for immunocompromised individuals2. Chemo- or corticosteroid therapy, diabetes mellitus, long term catheterization, frequent hemodialysis and parenteral nutrition are all factors governing a suppressed immune system and subsequently an increased susceptibility for serious fungal complications3.

The most common fungal infection, predominantly localized to the skin or mucosal membranes, is candidiasis or “yeast” infection caused by Candida fungi, most frequently

Candida albicans4. C. albicans is generally part of a healthy microflora of the human body but can turn pathogenic under several medical conditions3. Another common type of fungal infection is onychomycosis which is an ungual disease. It represent a major problem which can be both painful and lead to permanent disfiguration5. Onychomycosis affects 10% of the general population and can take up to a year to cure depending on the choice of treatment which can be either systemic or topical6. The topical treatment of onychomycosis is hampered by the poorly permeable barrier formed by the nail plate and recurrence rate is significant and can be as high as 50% due to reinfection or insufficient treatment initially7.

Modified cationic antimicrobial peptides (AMPs) derived from the innate immune system are currently being investigated as novel agents in the struggle against bacteria resistant to conventional antibiotics8. The mode of action of AMPs is generally on the bacterial



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membrane via several permeabilizing routes which greatly limits resistance development9. Selectivity towards bacteria is attributed to the anionic net charge of the bacterial cell membrane compared to the zwitterionic mammalian cell membrane. A careful combination of hydrophobic and cationic structural elements is needed to create the optimal AMP which is often amphiphilic in nature allowing for efficient bacterial membrane association10. Introduction of unnatural hydrophobic and cationic amino acids into the peptides can boost both the antibacterial efficiency and the metabolic stability11-12. Systemic truncation and optimization of lactoferricin-derived peptides have led to a class of membrane active synthetic antimicrobial peptidomimetics (SAMPs) with high antibacterial potential and cellular selectivity8,

13

. The SAMPs are rapid bacterial killers and one such compound, LTX-109

(compound 5), is currently undergoing clinical trials against topical infections (Lytix Biopharma AS, Norway). In addition, compound 5 has recently been shown to effectively destroy Saccharomyces cerevisiae cells via plasma membrane disruption14.

The AMPs constitute an important part of the innate defense towards pathogens and have also been heralded as a new source of antifungal drugs15. Inspired by the central role of AMPs in the innate antifungal defense, the present study was launched to investigate whether the SAMP-type of AMPs display activity against human pathogenic fungi. This is not obvious as fungi produce an arsenal of proteases as virulence factors and these can be used to evade the natural innate defense16. In the current study, five potent antibacterial SAMPs were screened against a comprehensive library of 24 different fungal isolates. The included SAMPs are designed to be stable against proteolysis and can be prepared at a low cost. The most promising of these SAMPs was further formulated and evaluated in human infected skin and nail models and compared to commercial treatments.


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MATERIALS AND METHODS

Compound selection SAMPs with variation in C-terminal capping and central unnatural bulky hydrophobic amino acid were selected based on their ranging hydrophobicity which is linked to their antibacterial effect. To remain strongly amphiphilic, the cationic components of the compounds were kept constant in the current study. Compounds were used at >95 % purity and their individual purity and structural confirmation data can be found in ref 8 and 11.

Inhibition of fungal growth Minimum inhibitory concentration (MIC) was determined in MOPS (Sigma Aldrich Ltd) buffered RPMI 1640 (Invitrogen Ltd) medium using standard CLSI broth microdilution methodology17. The inocula used for yeast were in the range of 7.5 × 102 - 4.0 × 103 CFU/mL and 8.0 × 103 - 1.0 × 105 CFU/mL for the filamentous fungi. Viable counts were determined between 0 and 48 after inoculation.

Candidiasis skin model Human skin samples from cosmetic reduction surgery were used in MedPharm’s established candidiasis model. Briefly, epidermal sheets were inoculated with C. albicans (ATCC 10231) on the ventral side and mounted in MedPharm’s ChubTur® permeations cells with the Stratum

corneum side up. The cells were incubated at 35 ± 2 °C for a set period to allow the fungus to grow on the skin. Formulations were subsequently applied to the surface of the Stratum

corneum in three applications at hourly intervals. Compound 5 was formulated in a general 2% hydrogel matrix (compound 5: water: hydroxyethyl cellulose: benzyl alcohol (2:94:2:2% w/w)) and compared with commercial Lamisil® cream. The formulation was rinsed off with



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water before reapplying a new dose. After incubating the ChubTur® cells for 14 h with the third dosage, the cells were removed from incubation. The formulations were rinsed off the surface of the epidermal sheets and the ATP from viable organisms was assessed using MedPharm’s ATP viability assay. Hydrogel placebo and untreated samples were also included.

Onychomycosis nail model Full thickness human toenails (cadaver nails treated according to Traynor et al.

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) were

washed with 70% aqueous ethanol and vortexed for 1 min twice. This treatment was repeated with Ringer’s solution twice. After washing, the nails were placed in a sterile petri dish and allowed to air dry for 30 min. The dried nail were cut into 3 mm × 3 mm segments, measured for thickness, and infected on the underside using T. rubrum cell suspension. The nails were dried and mounted into a ChubTur® cell dorsal side up. The receptor chamber (4.4 ± 0.24 cm3 in volume) content was half filled with sterile Ringer’s solution. The cells were incubated at 25 ± 3 °C for a set period to allow full growth of the organism on the nail. After this time, the ChubTur® cells were treated with 10 µL of the different formulations applied on the upper surface of the nail. Loceryl® nail lacquer (amorolfine) was employed as commercial comparator, and deionized water as placebo. In addition, MedNail formulations and the established keratolytic enhancers salicylic acid, urea and papain, all compounds at a final concentration of 5% w/w were evaluated as pretreatments. The penetration enhancers were applied to the nail (100 µL/9 mm2) and incubated for 20 h at 25 °C before rinsing with deionized water. The nails were then dosed daily. Prior to re-applying each dose, the nails were carefully wiped with cotton swabs immersed in filtered sterilized deionized water. After incubation, the excess formulation was removed from the surface of the nails to avoid ATP


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quenching from the formulations before the nails were dismantled from the ChubTur® cells. Remaining viable fungi from the nails were assessed using the ATP viability assay.

ATP viability assay The viability of the fungal cells were determined using methods previously developed and described18. The quenching effect on the ATP recovery for the formulations was also investigated (Supporting information).

RESULTS AND DISCUSSION

Fungal and mammalian cell membranes are similar in overall polarity but the outer cell wall of the fungal cell is coated with negatively charged glycoproteins which have been shown to play a role for the interaction with cationic peptides19-20. The main difference between the two cell membranes lie in the presence of the neutral sterol ergosterol in the fungal membrane. As such Ergosterol is the target for several selective antifungal drugs21. Other targets, such as fungal specific sphingolipids, have been suggested to be involved in the selective action against Saccharomyces and filamentous fungi for the currently studied class of compounds and other defense peptides

14, 22-23

. Additional, more complex antifungal mechanisms for

naturally occurring antifungal peptides have been studied and were recently reported by van der Weerden et al.24-25

A small library of bioactive SAMPs was designed to represent variation in the central nongenetically encoded lipophilic amino acid as well as variation in the C-terminal modification (Figure 1).



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Figure 1. Chemical structures of the included SAMPs.

These structural features have previously been shown to influence the antibacterial activity of the SAMPs13. The studied SAMPs molecules are highly active against both S. aureus (MIC ≤5 µg/mL) and E. coli and display variation in hydrophobicity as indicated by the theoretical ClogP values (Table 1). A higher hydrophobicity is linked to both the antibacterial activity, particularly against E. coli and the hemolytic activities (Table 1).

Table 1. Physicochemical properties and antibacterial activity of compounds tested

Entry

Sequence1

1 2 3 4 5

R-Tbt-RNHCH(CH3)2 R-Npa-RNHCH2Bn R-Tbt-ROCH3 R-Tbt-RNH(CH2)5CH3 R-Tbt-RNHCH2Bn

1

Mw

CLog P2

MIC S. aureus (µg/mL)

MIC E. coli (µg/mL)

EC503 (µg/mL)

726.0 706.9 699.0 768.1 788.1

2.52 1.43 2.54 4.33 3.78

3 128 32 32 8 16 64 4 8 16 >128 32 32 64 32 64 64 16 32 16 16 16

4 128 4 128 2 8 4 2 4 2 4 16 128 8 32 16 8 32 32 16 32 8 32 16

16 >128 16 >128 32 32 8 8 64 8 8 32 128 32 32 32 32 64 64 32 64 64 16 16

1

Amphotericin B was employed as positive control.

2

Increased resistance to Amphotericin B

3

Increased resistance towards azoles

4

Resistant towards echinocandins

5

Generally increased resistance towards antimycotic drugs


1 32 4 16 4 8 32 4 8 4 2 2 64 8 2 16 8 16 16 4 16 4 4 4

5

Control1

4 64 8 64 8 16 4 4 16 4 4 4 64 16 4 16 16 32 32 8 16 8 8 8

0.5 1 0.25 4 1 1 0.5 1 0.25 0.25 0.25 0.5 2 0.25 0.5 0.5 0.5 2 1 0.5 0.5 0.25 1 0.12


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All included peptides displayed antifungal activities with low micromolar MIC-values against several strains. The concentrations needed to kill the fungal strains were generally somewhat higher than those needed to eradicate bacteria8. The most active peptides displayed MICvalues approximately one order of magnitude higher than amphotericin B which is a highly toxic antifungal agent crucial in treatment of severe systemic fungal infections. Sturdy fungi particularly resilient to amphotericin B treatment such as Aspergillus fumigatus, Aspergillus

terreus and Fusarium oxysporum were also the least sensitive to peptide treatment. Other strains with a natural general antimycotic resistance such as Mucor circinelloides, Rhizopus

oligosporus displayed intermediate sensitivity towards the peptides. C. glabrata and C. krusei, both with a naturally reduced sensitivity towards fluconazole and other azoles, were highly sensitive to the peptides. The antifungal activity of the peptides was linked to their physicochemical properties yielding antibacterial activity (Table 1.) suggesting a mechanism dictated by the same features. Bojsen et al. recently studied the kill kinetics of compound 5 towards S. cerevisiae and reported a similar rapid mode of action (at 5 × MIC) as that of amphotericin B14. The same study reported a membranolytic mode of action towards S.

cerevisiae14 for compound 5. The kill kinetics studies of compound 5 towards C. albicans also indicate a very rapid mode of action and a total eradication (limit of quantification 10 CFU/mL) of fungi after 2 h already at 2 × MIC (Supporting information) implying a similar mode of action towards Candida. Compounds 4 and 5, being significantly more hydrophobic than the other peptides due to their lipophilic C-terminal cappings, displayed a high overall antifungal activity. One interesting exception was the seemingly specific activity of compound 2, which lacks the central Tbt subunit, towards the Candida strains. Compound 2 is substantially less hydrophobic than the other four peptides and the results suggest that this higher hydrophilicity may increase the activity towards selected fungal species or that the


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shape and size of the central lipophilic residue results in greater membrane pertubation. Larsen et al. recently reported that very similar SAMPs, including compound 5, were inactive towards Candida strains22. In four independent laboratories the authors have been involved in the determination of the MIC value of compound 5 against C. albicans, with results not varying more than one titer from the value given in Table 2. In addition compound 5 was also tested against clinical isolate of C. albicans 877-1164 (isolated from oral cavity at Sahlgrenska University Hospital) with an acquired fluconazole resistance (MIC>256 µg/mL) and was shown to display a low 4 µg/mL MIC. Compounds 1 and 3 were active against only a few fungal strains which may be a reflection of their smaller and less hydrophobic C-terminal modifications. The cyclic arginine heptamer Novexatin® which is undergoing clinical trials toward onychomycosis has a net charge of +7 indicating that a net positive charge may also be beneficial for antifungal activity26. The related cationic peptidomimetics recently reported by Diamond et al. display comparable antifungal activities towards Candida species and support that C. tropicalis is more sensitive to these types of compounds and that C. krusei and

C. parapsilosis are less sensitive illustrating a similar mode of action27.

The high lipophilicity of compounds 4 and 5 provides them with a broad antifungal spectrum, but also an increased ability to inflict collateral damage to mammalian cells. The EC50 against human erythrocytes for compounds 4 and 5 are 32 and 175 µg/mL respectively. Thus compound 4 cannot be declared a lead compound due to the insufficient distinction between fungal and mammalian cells. Furthermore, compound 5 has been selected as a clinical development candidate for a topical antibacterial drug based on its broad antibacterial activity and superior pharmacological properties (Lytix Biopharma AS). Consequently it was decided that compound 5 with its broad antifungal activity should also be pursued in specific



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candidiasis and onychomycosis studies employing C. albicans and T. rubrum as fungal pathogens.

For the candidiasis study, compound 5 was formulated and compared with the squalene epoxidation inhibitor terbinafine (Lamisil® cream, standard prescription for treatment of topical fungal infections) in an assay employing human skin from cosmetic reduction surgery. Inoculated epidermal sheets were mounted in a ChubTur® cell and treated with formulated compound 5 and reference formulations at regular intervals. Recovery of ATP from the viable fungi was used as biomarker as presented in Figure 2.

Figure 2. ATP recovery from skin samples infected with C. albicans treated with terbinafine (n = 5), Compound 5 (n = 6), and various controls given as means ± SE.

The 2 % hydrogel formulation of compound 5 displayed a significant (p

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