High-throughput synergy screening identifies microbial metabolites as combination agents for the treatment of fungal infections

Abstract

The high mortality rate of immunocompromised patients with fungal infections and the limited availability of highly efficacious and safe agents demand the development of new antifungal therapeutics. To rapidly discover such agents, we developed a high-throughput synergy screening (HTSS) strategy for novel microbial natural products. Specifically, a microbial natural product library was screened for hits that synergize the effect of a low dosage of ketoconazole (KTC) that alone shows little detectable fungicidal activity. Through screening of approximately 20,000 microbial extracts, 12 hits were identified with broad-spectrum antifungal activity. Seven of them showed little cytotoxicity against human hepatoma cells. Fractionation of the active extracts revealed beauvericin (BEA) as the most potent component, because it dramatically synergized KTC activity against diverse fungal pathogens by a checkerboard assay. Significantly, in our immunocompromised mouse model, combinations of BEA (0.5 mg/kg) and KTC (0.5 mg/kg) prolonged survival of the host infected with Candida parapsilosis and reduced fungal colony counts in animal organs including kidneys, lungs, and brains. Such an effect was not achieved even with the high dose of 50 mg/kg KTC. These data support synergism between BEA and KTC and thereby a prospective strategy for antifungal therapy.

Fig. 1.

Synergistic effect of F101604 with a low dosage of KTC. (Top) The samples were treated as labeled in duplicate. The assay plates after incubation overnight at 35°C in a moistured chamber. Regrowth of samples (Top) in drug-free fresh RPMI media 1640 is shown (Middle). Fluorescence was measured at excitation wavelength (Ex) 544 nm and emission wavelength (Em) 590 nm and converted as percentage of growth inhibition (Bottom).

Fig. 2.

Nontoxicity to human HepG2 cells treated with low dosage of KTC and extract F101604. The same amount of HepG2 cells was seeded in each of the 96 wells. After 24-h incubation at 37°C CO₂ incubator with a humidified chamber, colors were developed based on the cell viability (see text).

Fig. 3.

Establishment of an immunocompromised mouse model for fungal infection by i.p. injection of CY at a dosage of 100 mg/kg (body weight) once daily for 3 consecutive days. (A) The number of WBCs counted at various days with CY and saline control treatment. (B) Percent survival of mice infected with high, medium, and low concentration of CP blastospores (see text).

Fig. 4.

Percent survival of systemic infected mice treated with test compounds. Blastospores (1 × 10⁴) of CP were injected into the immunocompromised mice at day 0, and the test compounds were administered orally by gavage 6 h postinfection and once daily thereafter for 5 days. For better observation, the number of dead mice was adjusted approximately ±3% in case of overlap when drawing the figure.