Resistance-gene-guided genome mining for novel antifungal compounds

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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.