The goal of this project is to assess the therapeutic potential of novel antifungal agents, identified by our laboratory via screening the ChemBridge library, that target the synthesis of fungal glucosylceramide (GlcCer). Recently, we reported that the fungal GlcCer is required for the pathogenic fungus Cryptococcus neoformans (Cn) to cause a lethal meningo-encephalitis1, 2. In particular, we showed that a Cn mutant strain lacking the final enzyme for the synthesis of glucosylceramide (glucosylceramide synthase 1 or Gcs1), after inhalation, was confined in the lung granuloma and, thus, did not reach the bloodstream and did not disseminate to the brain. Later, other investigators corroborated and extended our findings that mutation of genes involved in the last steps of the GlcCer pathway affect fungal pathogenesis not only of fungi infecting humans, such as Cn3, 4, Candida albicans5-7, and Aspergillus fumigatus8, but also of fungi infecting plants, such as Fusarium graminearum9. Further studies in our lab showed that the mechanism by which GlcCer promotes virulence of Cn is by allowing fungal growth in a neutral/alkaline environment, such as that present in the alveolar spaces2, 10. The importance of GlcCer to promote fungal growth in the lung is also underscored by the fact that in most dimorphic fungi, production of GlcCer is detected only in the lung infective form (yeast) and not in the environmental form (mold)11-13, suggesting that also in these fungi GlcCer may be required for lung infection. The synthesis of GlcCer seems to be important also during Pneumocystis pneumonia (PCP) as glucosylceramide synthase transcripts have been found to be abundant at the time of isolation of the fungus from a fulminate lung infection14. Taken together, these studies suggest that GlcCer is most likely a pan-fungal virulence factor required during lung infection to promote fungal growth at the neutral/alkaline environment of alveolar spaces, and as such, it is a promising novel drug target. Therefore, we looked for inhibitors of GlcCer synthesis by screening a ChemBridge library for compounds that inhibit Cn growth in an environment similar to the lung: neutral/alkaline pH, 37C and 5% CO2. We identified 2 compounds that significantly decreased the synthesis of GlcCer in Cn but not in mammalian cells. Importantly, 90% of mice treated with the lead compound, BHBM, survived a lethal intranasal injection of Cn and their lungs (and brains) were free from fungal cells. Moreover, we found that BHBM is over 50-fold more active than pentamidine against the lung pathogen Pneumocystis in vitro. Therefore, we hypothesize that targeting the fungal GlcCer pathway will be an effective novel therapeutic strategy for impeding the development of airborne fungal diseases. To test this hypothesis, we propose the following aims: 1) identify and validate the target(s) of the active compounds;and 2) study the effect of the identified compounds in in vivo models of fungal infections.
. Cryptococcosis and PCP are significant public health problems not only in the US, but also globally. The Centers for Disease Control estimates more than 1 million new cases/year of cryptococcosis worldwide in patients with AIDS, and reported that Cryptococcus-related deaths exceed 650,000/year, making HIV-associated deaths caused by cryptococcosis more frequent than those caused by tuberculosis. Pneumocystis infection remains the most common serious opportunistic illness in HIV-infected persons15 with a mortality rate of 20-50%16, 17. PUBLIC HEALTH RELEVANCE: Fungal infections have dramatically increased during the last decade and new treatment strategies are needed. This proposal focuses on the characterization and validation of new compounds with potent antifungal activity, providing new insights into the development of better therapeutic strategies.
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