Resistance-gene-guided genome mining for novel antifungal compounds

项目摘要 全世界每年约有150万人死于系统性真菌感染。高死亡人数 强调了目前治疗的局限性，此外，对 现有的抗真菌药物。因此，迫切需要新的和更好的抗真菌剂。令人惊讶的是，真菌 是抗真菌药的良好来源棘白菌素就是一个很好的例子真菌产生的抗真菌剂 是次级代谢物（SM）--对生存能力不是必需的，但具有选择性优势的化合物 对生产有机体，通常通过抑制其竞争对手的生长。事实上，他们往往抑制重要的 生物活性使它们成为有医学价值的化合物的主要来源。真菌SM生物合成 通路由协调调节的基因簇编码。基因组计划显示， 比已知的SM多得多的SM基因簇，因此，更大的真菌次级代谢组 (all由所有1，000，000+种真菌制成的SM）比以前认为的要大得多。开发 为了有效地研究真菌SM的多样性，我们需要能够识别产生具有所需结构的化合物的簇。 活动一种很有前途的方法，称为抗性基因导向的基因组挖掘， 事实上，一些SM生物合成基因簇（BGC）含有一个在SM生物合成中没有作用的基因， 而是编码一种被簇产生的化合物抑制的蛋白质的抗性形式。 该基因的表达使生产生物体对其产生的化合物具有抗性。我们有 开发了一种方法，和计算机应用程序，使我们能够识别SM BGC在联合 基因组研究所数据库，我们预测将产生化合物，抑制两种蛋白质是必不可少的 供真菌生长。它们是FKS 1，真菌细胞壁生物合成所需的酶的催化亚基， 和YEF 3，一种仅在真菌中发现的翻译延伸因子。这些蛋白质不存在于哺乳动物中， 抑制它们的活性不应该对人类有毒。因为在它们的原生生物体中表达SM， 通常非常困难，我们建议表达预测产生FKS 1和YEF 3抑制剂的代表性BGC 在模型真菌构巢曲霉中，我们已经将其工程化用于真菌SM的异源表达。 我们提出了几种方法，建立在我们在这一领域的成功，我们将评估其有效性。 将纯化异源表达的化合物并确定其结构。他们将接受考验 对致病真菌白色念珠菌和新型隐球菌以及野生型A. nidulans，其是曲霉属致病性物种的有用替代物。

PROJECT SUMMARY Approximately 1.5 million people die each year, worldwide, from systemic fungal infections. The high death toll highlights the limitations of current treatments and, in addition, there is a growing problem with resistance to existing antifungals. There is, thus, a compelling need for new and better antifungal agents. Surprisingly, fungi are a good source of antifungals, the echinocandins being a prime example. The antifungals produced by fungi are secondary metabolites (SMs)--compounds that are not essential for viability but confer a selective advantage to the producing organism, often by inhibiting growth of its competitors. The fact that they often inhibit important biological activities has made them a prime source of medically valuable compounds. Fungal SM biosynthetic pathways are encoded by clusters of coordinately regulated genes. Genome projects have revealed that there are vastly more SM gene clusters than known SMs, and, thus, that the greater fungal secondary metabolome (all SMs made by all 1,000,000+ species of fungi) is much larger than was previously thought. To exploit the diversity of fungal SMs efficiently, we need to be able to identify clusters that produce compounds with desired activities. A promising approach to this end, called resistance-gene-directed genome mining, takes advantage of the fact that some SM biosynthetic gene clusters (BGCs) contain a gene that has no role in SM biosynthesis but, rather, encodes a resistant form of the protein inhibited by the compound produced by the cluster. Expression of this gene makes the producing organism resistant to the compound it produces. We have developed an approach, and computer application, that has allowed us to identify SM BGCs in the Joint Genomes Institute database that we predict will produce compounds that inhibit two proteins that are essential for fungal growth. They are FKS1, the catalytic subunit of an enzyme required for fungal cell wall biosynthesis, and YEF3, a translation elongation factor found only in fungi. These proteins are not present in mammals and inhibiting their activities should not be toxic to humans. Because expressing SMs in their native organisms is usually very difficult, we propose to express representative BGCs predicted to produce FKS1 and YEF3 inhibitors in the model fungus Aspergillus nidulans, which we have engineered for heterologous expression of fungal SMs. We propose several approaches, building on our successes in this area, that we will evaluate for efficacy. Heterologously expressed compounds will be purified and their structures will be determined. They will be tested for activity against the pathogenic fungi Candida albicans and Cryptococcus neoformans as well as wild-type A. nidulans which is a useful surrogate for pathogenic species of Aspergillus.