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

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 ≈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.

[1]  A. Bolmström,et al.  Quality control guidelines for National Committee for Clinical Laboratory Standards--recommended broth macrodilution testing of ketoconazole and itraconazole , 1996, Journal of clinical microbiology.

[2]  P. Verweij,et al.  Antifungal Activity of Nonantifungal Drugs , 2003, European Journal of Clinical Microbiology and Infectious Diseases.

[3]  L. Mele,et al.  Retrospective study of candidemia in patients with hematological malignancies. Clinical features, risk factors and outcome of 76 episodes , 1999, European journal of haematology.

[4]  D. Kontoyiannis,et al.  Rationale for Combination Antifungal Therapy , 2001, Pharmacotherapy.

[5]  J. Lehár,et al.  Systematic discovery of multicomponent therapeutics , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[6]  M. Hamann,et al.  Marine natural products and their potential applications as anti-infective agents , 2003, The Lancet Infectious Diseases.

[7]  J. Mekalanos,et al.  Small-Molecule Inhibitor of Vibrio cholerae Virulence and Intestinal Colonization , 2005, Science.

[8]  T. Potter,et al.  Host-specific Variation in Infection by Toxigenic Fungi and Contamination by Mycotoxins in Pearl Millet and Corn , 2005, Mycopathologia.

[9]  L. Saiman,et al.  Risk factors for candidemia in Neonatal Intensive Care Unit patients , 2000, The Pediatric infectious disease journal.

[10]  J. Perfect,et al.  Combination Antifungal Therapy , 2004, Antimicrobial Agents and Chemotherapy.

[11]  E. Bruck,et al.  National Committee for Clinical Laboratory Standards. , 1980, Pediatrics.

[12]  F. Barchiesi,et al.  In vitro activities of terbinafine in combination with fluconazole and itraconazole against isolates of Candida albicans with reduced susceptibility to azoles , 1997, Antimicrobial agents and chemotherapy.

[13]  J. Heeres,et al.  Ketoconazole — a new broad spectrum orally active antimycotic , 1979, Experientia.

[14]  J. Galgiani,et al.  Pharmacology and toxicity of high-dose ketoconazole , 1987, Antimicrobial Agents and Chemotherapy.

[15]  D. Hughes,et al.  Diversifying microbial natural products for drug discovery , 2003, Applied Microbiology and Biotechnology.

[16]  D. Kazierad,et al.  Comparison of midazolam and simvastatin as cytochrome P450 3A probes , 2006, Clinical pharmacology and therapeutics.

[17]  K. Ying,et al.  cDNA microarray analysis of differential gene expression and regulation in clinically drug-resistant isolates of Candida albicans from bone marrow transplanted patients. , 2006, International journal of medical microbiology : IJMM.

[18]  S. Ōmura,et al.  New beauvericins, potentiators of antifungal miconazole activity, Produced by Beauveria sp. FKI-1366. I. Taxonomy, fermentation, isolation and biological properties. , 2004, The Journal of antibiotics.

[19]  S. Solomon,et al.  Candida parapsilosis fungemia associated with parenteral nutrition and contaminated blood pressure transducers , 1987, Journal of clinical microbiology.

[20]  S. Ōmura,et al.  Inhibition of acyl-CoA: cholesterol acyltransferase activity by cyclodepsipeptide antibiotics. , 1992, The Journal of antibiotics.

[21]  M. Larocco,et al.  Infection in the bone marrow transplant recipient and role of the microbiology laboratory in clinical transplantation , 1997, Clinical microbiology reviews.

[22]  M. Gbéassor,et al.  Inhibition of human pathogenic fungi by ethnobotanically selected plant extracts , 2003, Mycoses.

[23]  M. Klepser,et al.  Comparison of Etest, chequerboard dilution and time-kill studies for the detection of synergy or antagonism between antifungal agents tested against Candida species. , 2002, The Journal of antimicrobial chemotherapy.

[24]  T. Eckmanns,et al.  The influence of high-efficiency particulate air filtration on mortality and fungal infection among highly immunosuppressed patients: a systematic review. , 2006, The Journal of infectious diseases.

[25]  J. Graybill,et al.  Effect of fluconazole on fungicidal activity of flucytosine in murine cryptococcal meningitis , 1996, Antimicrobial agents and chemotherapy.

[26]  J. Heitman.  A Fungal Achilles' Heel , 2005, Science.

[27]  M. Ghannoum,et al.  In vitro determination of optimal antifungal combinations against Cryptococcus neoformans and Candida albicans , 1995, Antimicrobial agents and chemotherapy.

[28]  S. Ōmura,et al.  Tensidols, New Potentiators of Antifungal Miconazole Activity, Produced by Aspergillus niger FKI-2342 , 2006, The Journal of Antibiotics.

[29]  J. Sobel,et al.  Nosocomial acquisition of Candida parapsilosis: an epidemiologic study. , 1993, The American journal of medicine.

[30]  S. Lindquist,et al.  Hsp90 Potentiates the Rapid Evolution of New Traits: Drug Resistance in Diverse Fungi , 2005, Science.

[31]  J. L. Goodman,et al.  A controlled trial of fluconazole to prevent fungal infections in patients undergoing bone marrow transplantation. , 1992, The New England journal of medicine.

[32]  J. Rex,et al.  Resistance of Candida species to fluconazole , 1995, Antimicrobial agents and chemotherapy.

[33]  Victoria J. Fraser,et al.  Delaying the Empiric Treatment of Candida Bloodstream Infection until Positive Blood Culture Results Are Obtained: a Potential Risk Factor for Hospital Mortality , 2005, Antimicrobial Agents and Chemotherapy.

[34]  J. Sobel,et al.  A randomized and blinded multicenter trial of high-dose fluconazole plus placebo versus fluconazole plus amphotericin B as therapy for candidemia and its consequences in nonneutropenic subjects. , 2003, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[35]  J. Perfect,et al.  Adverse Drug Reactions to Systemic Antifungals , 1992, Drug safety.

[36]  M. Ghannoum,et al.  Antifungal Agents: Mode of Action, Mechanisms of Resistance, and Correlation of These Mechanisms with Bacterial Resistance , 1999, Clinical Microbiology Reviews.