Identification of new compounds against fungal microbes

描述（由申请人提供）：本项目的目标是评估新型抗真菌药物的治疗潜力，这些药物是由我们实验室通过筛选ChemBridge文库鉴定的，靶向真菌葡萄糖神经酰胺（GlcCer）的合成。 最近，我们报道了真菌GlcCer是致病真菌新型隐球菌（Cn）引起致命的脑膜脑炎所需的1，2。特别是，我们发现，一个Cn突变株缺乏最终的葡萄糖神经酰胺合成酶（葡萄糖神经酰胺合成酶1或GCS 1），吸入后，被限制在肺肉芽肿，因此，没有达到血流，并没有传播到大脑。后来，其他研究人员证实并扩展了我们的发现，即参与GlcCer途径最后步骤的基因突变不仅影响感染人类的真菌（如Cn 3，4，白色念珠菌5 -7和烟曲霉8）的真菌发病机制，而且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，GlcCer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，例如存在于肺泡空间中的环境2，10。GlcCer促进肺中真菌生长的重要性也通过以下事实强调：在大多数二型真菌中，仅在肺感染形式（酵母）中检测到GlcCer的产生，而在环境形式（霉菌）中未检测到GlcCer的产生11-13，这表明GlcCer也是肺中真菌生长的重要因素。 在这些真菌中，肺部感染可能需要GlcCer。GlcCer的合成似乎在肺孢子虫肺炎（PCP）期间也很重要，因为在从暴发性肺部感染中分离真菌时发现葡萄糖神经酰胺合酶转录物丰富14。 综上所述，这些研究表明，GlcCer最有可能是肺感染期间促进真菌在肺泡间隙的中性/碱性环境中生长所需的泛真菌毒力因子，因此，它是一种有前途的新型药物靶标。 因此，我们通过在ChemBridge文库中筛选在类似于肺的环境中抑制Cn生长的化合物来寻找GlcCer合成的抑制剂：中性/碱性pH，37 ℃和5%CO2。我们鉴定了2种化合物，其显著降低Cn中GlcCer的合成，但在哺乳动物细胞中不降低。重要的是，用先导化合物BHBM治疗的小鼠中有90%在致死的鼻内注射Cn后存活，并且它们的肺（和大脑）没有真菌细胞。此外，我们发现BHBM在体外对肺病原体肺孢子虫的活性是喷他脒的50倍以上。 因此，我们假设，针对真菌GlcCer途径将是一种有效的新的治疗策略，以阻止空气传播的真菌疾病的发展。为了验证这一假设，我们提出了以下目标：1）鉴定和验证活性化合物的靶标;和2）研究所鉴定的化合物在真菌感染的体内模型中的作用。

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.