Identification of new compounds against fungal microbes

描述(由申请人提供)：本项目的目标是评估我们实验室通过筛选ChemBridge文库确定的新型抗真菌药物的治疗潜力，这些药物的目标是合成真菌葡萄糖神经酰胺(GlcCer)。最近，我们报道了致病真菌新生隐球菌(CN)引起致死性脑膜脑炎所必需的真菌GlcCer。特别是，我们发现一株CN突变株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)，吸入后被限制在肺肉芽肿内，因此不能到达血液，也不会传播到大脑。后来，其他研究人员证实并扩展了我们的发现，即GlcCer途径最后一步涉及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，而且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，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，如存在于肺泡腔2、10。GlcCer促进肺部真菌生长的重要性也因以下事实而得到强调：在大多数二相性真菌中，GlcCer仅在肺部感染形式(酵母)中检测到，而在环境形式(霉菌)11-13中检测不到，这表明 在这些真菌中，肺部感染可能需要GlcCer。在肺孢子虫肺炎(PCP)期间，GlcCer的合成似乎也很重要，因为在从暴发性肺部感染中分离出这种真菌时，已发现大量的葡萄糖神经酰胺合成酶转录本14。综上所述，这些研究表明，GlcCer很可能是肺部感染过程中所需的一种泛真菌毒力因子，以促进中性/碱性环境下肺泡腔的真菌生长，因此，它是一个有前景的新药物靶点。 因此，我们通过筛选ChemBridge文库来寻找GlcCer合成的抑制剂，以寻找在类似于肺的环境中抑制CN生长的化合物：中性/碱性pH、37℃和5%CO2。我们鉴定了两个化合物，它们显著减少了CN中GlcCer的合成，但在哺乳动物细胞中没有。重要的是，接受铅化合物BHBM治疗的小鼠中，90%的小鼠在致命的鼻腔注射CN后存活下来，它们的肺(和大脑)没有真菌细胞。此外，我们还发现，在体外，BHBM对肺部病原体肺孢子虫的活性是戊二胺的50倍以上。因此，我们推测，靶向真菌GlcCer途径将是阻碍空气传播真菌疾病发展的一种有效的新的治疗策略。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(S)；2)研究鉴定的化合物在体内真菌感染模型中的作用。

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.