Rapamycin and Less Immunosuppressive Analogs Are Toxic to Candida albicans and Cryptococcus neoformans via FKBP12-Dependent Inhibition of TOR

Abstract

Candida albicans and Cryptococcus neoformans cause both superficial and disseminated infections in humans.Current antifungal therapies for deep-seated infections are limited to amphotericin B, flucytosine, and azoles. A limitation is that commonly used azoles are fungistatic in vitro and in vivo. Our studies address the mechanisms of antifungal activity of the immunosuppressive drug rapamycin (sirolimus) and its analogs with decreased immunosuppressive activity. C. albicans rbp1/rbp1 mutant strains lacking a homolog of the FK506-rapamycin target protein FKBP12 were found to be viable and resistant to rapamycin and its analogs. Rapamycin and analogs promoted FKBP12 binding to the wild-type Tor1 kinase but not to a rapamycin-resistant Tor1 mutant kinase (S1972R). FKBP12 and TOR mutations conferred resistance to rapamycin and its analogs in C. albicans, C. neoformans, and Saccharomyces cerevisiae. Our findings demonstrate the antifungal activity of rapamycin and rapamycin analogs is mediated via conserved complexes with FKBP12 and Tor kinase homologs in divergent yeasts. Taken together with our observations that rapamycin and its analogs are fungicidal and that spontaneous drug resistance occurs at a low rate, these mechanistic findings support continued investigation of rapamycin analogs as novel antifungal agents.Cryptococcus neoformans and Candida albicans are two common opportunistic fungal pathogens. Current antifungal agents in clinical use include amphotericin B, fluconazole, and flucytosine (29), which have side effects, lack fungicidal activity, or lack activity against emerging resistant mutants (66). Thus, additional antifungal agents are needed.Several new antifungal agents are in development. The candins are 1,3-␤-glucan synthase inhibitors and are potently active against Candida species and Aspergillus fumigatus (23,40,51,52). The pneumocandin caspofungin acetate-MK-0991 is in phase III clinical trials for candida infections and has been approved for refractory aspergillosis. Broad-spectrum triazoles, including voriconazole, posaconazole, and ravuconazole, are being studied in human trials (50). Combination therapy with different antifungal agents may also improve therapy (20,28,62).Rapamycin (sirolimus) is a natural product of the bacterium Streptomyces hygroscopicus originally discovered in a screen for antimicrobial activity against C. albicans and later found to have potent immunosuppressive activity (4, 63). Rapamycin diffuses into the cell and associates with the peptidylprolyl isomerase FKBP12. Rapamycin inhibits FKBP12 enzymatic activity; however, this inhibition is not related to immunosuppressive or antifungal activity. The targets of the FKBP12-rapamycin complex are the TOR kinases. Two TOR proteins, Tor1 and Tor2, have been characterized in the yeasts S. cerevisiae and Schizosaccharomyces pombe, and a single TOR homolog has been identified in C. albicans, C. neoformans, Drosophila melanogaster, and humans (10,18,33,39,49,56,64). The Tor kinase has been functionally conserved from yeast to ...

Protein Folding Sculpting Evolutionary Change

“…When it is missing cells would normally die. They survive only because the reservoir of Hsp90 is available to direct massive maturation of calcineurin (an Hsp90 client) [11][12][13][14], which drives many protective changes.…”

Direct effects of non-antifungal agents used in cancer chemotherapy and organ transplantation on the development and virulence ofCandidaandAspergillusspecies

“…67 Prior studies established that rapamycin and its analogs (particularly analogs 23 and 2) were fungicidal against C. albicans because of FKB12-dependent inhibition of TOR; the rapamycin MICs in these studies ranged from less than 0.02 to 0.20 μg/ml with an MFC of 0.39 μg/ml. 66,68 At sublethal concentrations, rapamycin blocked C. albicans filamentation via expression of genes associated with nitrogen use, such as mep2 and gln3. 10,65,69 Mutants defective in these genes were avirulent in mice with disseminated candidiasis.…”

Antifungal activity of medicinal plants from Northeastern Brazil

“…These yeasts often cause superficial infections such as vaginitis; however, if the immune defenses of the host become compromised by anticancer therapy, human immunodeficiency virus infection, organ transplantation, or therapy with broad-spectrum antibiotics, they can cause severe systemic infections (Cruz et al, 2002;Morschauser, 2002).…”