Calcofluor white combination antifungal treatments for Trichophyton rubrum and Candida albicans

Abstract

Superficial mycoses caused by dermatophyte fungi are among the most common infections worldwide, yet treatment is restricted by limited effective drugs available, drug toxicity, and emergence of drug resistance. The stilbene fluorescent brightener calcofluor white (CFW) inhibits fungi by binding chitin in the cell wall, disrupting cell wall integrity, and thus entails a different mechanism of inhibition than currently available antifungal drugs. To identify novel therapeutic options for the treatment of skin infections, we compared the sensitivity of representative strains of the dermatophyte Trichophyton rubrum and Candida albicans to CFW and a panel of fluorescent brighteners and phytoalexin compounds. We identified the structurally related stilbene fluorescent brighteners 71, 85, 113 and 134 as fungicidal to both T. rubrum and C. albicans to a similar degree as CFW, and the stilbene phytoalexins pinosylvan monomethyl ether and pterostilbene inhibited to a lesser degree, allowing us to develop a structure-activity relationship for fungal inhibition. Given the abilities of CFW to absorb UV(365 nm) and bind specifically to fungal cell walls, we tested whether CFW combined with UV(365 nm) irradiation would be synergistic to fungi and provide a novel photodynamic treatment option. However, while both treatments individually were cytocidal, UV(365 nm) irradiation reduced sensitivity to CFW, which we attribute to CFW photoinactivation. We also tested combination treatments of CFW with other fungal inhibitors and identified synergistic interactions between CFW and some ergosterol biosynthesis inhibitors in C. albicans. Therefore, our studies identify novel fungal inhibitors and drug interactions, offering promise for combination topical treatment regimes for superficial mycoses.

Figure 1. Structures of representative stilbene and non-stilbene fluorescent brighteners and stilbene phytoalexin compounds.

Residues (R) at which related products differ from the representative compound are displayed. A. CFW. B. Fluorescent brightener 351. C. Brilliant yellow. D. Fluorescent brightener ER-III. E. 4,4′-diamino-2–2′-stilbenedisulfonic acid. F. Fluorescent brightener 135. G. Pinosylvan monomethyl ether.

Figure 2. Disc diffusion assays showing enhanced C. albicans inhibition when treated with CFW and ergosterol biosynthesis inhibitors.

Starting at the bottom right disc and moving clockwise, the amounts of each drug added per disc included 14.11, 3.52, 1.41 and 0 ng of itraconazole, 8.32, 2.08, 0.83, and 0 ng of miconazole, and 6.07, 1.52, 0.61, and 0 ng of fenpropimorph. Drugs were diluted in DMSO, and DMSO comprised the no-drug control.