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 Ϸ20,000 microbial extracts, 12 hits were identified with broadspectrum 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.antifungal ͉ beauvericin ͉ ketoconazole

In vitro antifungal activities of amphotericin B in combination with acteoside, a phenylethanoid glycoside from Colebrookea oppositifolia

“…In this regard, natural products are attractive prototypes in the search for new entities of plant origin that either have broad-spectrum antifungal activities or are modulators of the currently available antifungal agents; thus, these entities are focused mainly on a reduction in toxicity, enhancement of bioavailability, improvement of the antifungal spectrum and counteraction of resistance of existing drugs (Newman & Cragg, 2007;Zhang et al, 2007;Hemaiswarya et al, 2008). There are several reports in the literature where natural products have demonstrated enhancement of antimicrobial activity (Khan et al, 2006;Stavri et al, 2007;Werle, 2008;Kumar et al, 2008;Lechner et al, 2008;Sharma et al, 2010).…”

Mutation of G234 amino acid residue inCandida albicansdrug-resistance-related protein Rta2p is associated with fluconazole resistance and dihydrosphingosine transport

“…All mice were inoculated by injection of 0.2 ml C. albicans suspension into the lateral tail vein (1 £ 10 6 c.f.u/mouse) to establish the systemic candidiasis model. 18 The mice were initially treated with FLC (1.0 mg/kg) or sterile saline 2 h after infection by C. albicans, and then once a day intraperitoneally for 7 days. Mice were monitored daily for survival until Day 30.…”

Synergistic antifungal indolecarbazoles from Streptomyces sp. CNS-42 associated with traditional Chinese medicine Alisma orientale

“…Given the high mortality rate resulting from fungal infections in immunocompromised patients and the limited number of highly effective, yet safe treatment agents, the development of new antifungal therapeutics is critical. 1,2,5 A strategy limiting the pressure on drug targets would increase the lifespan of antifungal agents and reduce the frequency of treatment failures. 3,6 For instance, using synergistic drugs aimed at more than one target and slowing down the emergence of drug-resistant pathogens will be one of the key approaches to antifungal therapy.…”

“…3,6 For instance, using synergistic drugs aimed at more than one target and slowing down the emergence of drug-resistant pathogens will be one of the key approaches to antifungal therapy. Our research group demonstrated that beauvericin (BEA) showed strong synergism with ketoconazole against diverse fungal pathogens both in vitro and in vivo, 5 and that this synergetic effect was not caused by their pharmacokinetic interaction. 7 Looking for natural products with potentially new Mode of Action from untapped sources provides an essential strategy.…”

“…5,8,9 Combinations of BEA (0.5 mg kg − 1 ) and KTC (0.5 mg kg − 1 ) prolonged survival of the host infected with Candida parapsilosis and reduced fungal colony counts in animal organs including kidneys, lungs and brains in an immunocompromised mouse model. 5 Further screening on a sub-library of microbial natural products has been done, contained 900 crude extracts from the fermentation broth of endophytic bacteria 300 strains on three different media (AM2, NM2 and MPG). HTSS on these extracts gave a 1.2% hit rate and generated 90% of the maximum activity in combination with 1/4 MIC of KTC.…”