Identification of new compounds against fungal microbes

描述（由申请人提供）：该项目的目标是评估新型抗真菌药物的治疗潜力，该药物由我们的实验室通过筛选ChemBridge文库确定，目标是真菌糖基神经酰胺（glcer）的合成。最近，我们报道了真菌GlcCer是致病性真菌新隐球菌（Cn）引起致死性脑膜炎的必要条件1,2。特别是，我们发现缺乏合成葡萄糖神经酰胺的最终酶（葡萄糖神经酰胺合成酶1或Gcs1）的Cn突变株在吸入后被限制在肺肉芽肿中，因此没有到达血液，也没有扩散到大脑。后来，其他研究者证实并扩展了我们的发现，即参与GlcCer途径最后几个步骤的基因突变不仅影响感染人类的真菌，如Cn3、4、白色念珠菌5-7和烟曲霉8，还影响感染植物的真菌，如禾谷镰刀菌9。我们实验室的进一步研究表明，glcer促进Cn毒力的机制是通过允许真菌在中性/碱性环境中生长，例如在肺泡空间中2,10。在大多数二态真菌中，仅在肺部感染形式（酵母菌）中检测到GlcCer的产生，而在环境形式（霉菌）中检测不到，这也强调了GlcCer促进真菌在肺部生长的重要性

DESCRIPTION (provided by applicant): 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.