The Plant Defensin Ppdef1 Is a Novel Topical Treatment for Onychomycosis

Abstract

Onychomycosis, or fungal nail infection, causes not only pain and discomfort but can also have psychological and social consequences for the patient. Treatment of onychomycosis is complicated by the location of the infection under the nail plate, meaning that antifungal molecules must either penetrate the nail or be applied systemically. Currently, available treatments are limited by their poor nail penetration for topical products or their potential toxicity for systemic products. Plant defensins with potent antifungal activity have the potential to be safe and effective treatments for fungal infections in humans. The cystine-stabilized structure of plant defensins makes them stable to the extremes of pH and temperature as well as digestion by proteases. Here, we describe a novel plant defensin, Ppdef1, as a peptide for the treatment of fungal nail infections. Ppdef1 has potent, fungicidal activity against a range of human fungal pathogens, including Candida spp., Cryptococcus spp., dermatophytes, and non-dermatophytic moulds. In particular, Ppdef1 has excellent activity against dermatophytes that infect skin and nails, including the major etiological agent of onychomycosis Trichophyton rubrum. Ppdef1 also penetrates human nails rapidly and efficiently, making it an excellent candidate for a novel topical treatment of onychomycosis.

Keywords: NMR; Ppdef1; Trichophyton rubrum; antifungal; onychomycosis; plant defensin.

Figure 1

Sequence alignment of Ppdef1 with several members of the plant defensin family. MUSCLE alignment of known plant defensins CDL12_12515 (from Handroanthus impetiginosus PIN14865.1), DmAMP1 (from Dahlia merckii P0C8Y4.1), RsAFP2 (from Raphanus sativus AAA69540.1), NaD1 (from Nicotiana alata Q8GTM0.1), and MtDef4 (from Medicago truncatula XP_003628977.1) with Ppdef1. Amino acid identity and similarity are indicated by coloured shading. Conserved residues are coloured red. Gaps have been inserted to maximize alignment. Loops are defined as regions between cysteine residues (L1–L7).

Figure 2

Quality assessment of recombinant Ppdef1. Ppdef1 was expressed in P. pastoris and purified using cation exchange chromatography followed by size exclusion chromatography. (A) The final purified material yielded a single peak when analysed with RP-HPLC. (B) MALDI-TOF analysis confirmed that the purified material contained a single species with a mass that is consistent with the expected mass of the folded defensin (5478.5 Da).

Figure 3

NMR solution structure of Ppdef1. (A) Cartoon representation of Ppdef1 (green) overlaid with NaD1 (grey, PDB ID: 1MR4), showing the extended loop 5 of Ppdef1. (B) Solvent accessible surface of Ppdef1 coloured according to its electrostatic potential at pH 7.0 (scale from −5 kT/e (red) to +5 kT/e (blue)). Charged residues are numbered with sidechains represented as sticks.

Figure 4

Ppdef1 permeabilizes C. albicans cells within 30 min. C. albicans cells were treated for 30 min with Ppdef1 and then assessed for membrane permeabilization using PI. (A) Flow cytometry analysis of cells treated with 0 (grey filled), 12.5 (black line), 25 (red line), 50 (blue line), or 100 µg/mL (green line) Ppdef1. A concentration-dependent increase in the proportion of dead cells (right peak) was observed. (B) Percent survival (corresponding to the left peak in (A)) of C. albicans cells treated with either 50 or 100 µg/mL of various antifungal drugs.

Figure 5

Ppdef1 retains activity in the presence of keratin. Growth of T. rubrum hyphae was monitored after treatment with increasing concentrations of Ppdef1 in either ½ PDB (red), ½ PDB with 2 mg/mL hydrolysed keratin (black), or ½ PDB with 2 mg/mL ground human fingernail (blue). Ppdef1 decreased the growth of T. rubrum in a concentration-dependent manner in all the growth conditions assessed. Relative growth was calculated relative to the untreated fungi in each medium. Error bars represent the standard error for the mean of the two biological replicates.

Figure 6

Ppdef1 kills T. rubrum growing on keratin as a sole nutrient source. Cell viability of hyphae grown on nail fragments for 72 h was monitored using PrestoBlue^®. T. rubrum was treated with two concentrations of Ppdef1 (blue), efinaconazole (orange), or water (grey) for 24 h before cell viability was measured. Uninfected nails were included as a control (dark grey). Ppdef1 treatment at 200 µg/mL caused a significant decrease in the number of viable fungi compared to the untreated control (* p < 0.05) and 200 µg/mL efinaconazole (** p < 0.01). Efinaconazole did not cause a significant decrease in viable fungi at either concentration. Error bars indicate the standard error of the mean.

Figure 7

Ppdef1 penetrates human nails. Ppdef1 (1%), terbinafine (1%), and efinaconazole (10%) were applied to human fingernails daily for 10 days, and the amount of drug that had passed through the nail was monitored. The percent of the applied dose detected each day is indicated by the bars corresponding to each day. Greater than 20% of the Ppdef1 penetrated the nail on Days 7–10. In contrast, less than 5% of both terbinafine and efinaconazole penetrated the nails on all days, except for terbinafine on Days 7 and 9, where penetration was still less than 10%. Data are the average of six replicates for Ppdef1, and four replicates for terbinafine and efinaconazole. Error bars indicate the standard error of the mean.

Figure 8

Ppdef1 is active in an infected nail model. T. rubrum-infected nails were treated with Ppdef1 reduced drug product, Jublia^®, Penlac^®, and Ppdef1 reduced base formulation (i.e., vehicle) for 7 days. At the end of the study, the amount of viable fungus remaining was assessed by measuring the amount of ATP recovered (presented as percentage luminescence units relative to the infected control). All three of the antifungal drugs as well as the Ppdef1 vehicle caused a significant decrease in viable fungi in the infected nail model with Ppdef1 and Jublia^® both resulting in less than 4 percent of the untreated infected control. Ppdef1 and Jublia^® caused a significantly greater fungal kill than ciclopirox (**** p < 0.0001) and vehicle (** p < 0.01). Error bars represent the standard error of the mean (n = 6, with the exception of ciclopirox, n = 3).