Cryptococcus neoformans is a pathogenic fungus that is found world-wide and causes meningioencephalitis, particularly in immunocompromised individuals. It is invariably fatal unless treated, and the current antifungals are inadequate to effectively cure this disease, due to inherent toxicities or the inability to kill the fungus and prevent relapse. Recent studies have indicated that there are over 1,000,000 new cases of cryptococcosis in the world each year, which results in over 600,000 deaths. New agents to treat Cryptococcus are needed. We have shown that chitosan, the deacetylated form of chitin, is a critical component of the Cryptococcal cell wall and is absolutely required for virulence using amouse model of cryptococcosis. Our studies have further identified the key enzymes required for chitosan production. This assay development proposal is based on our findings that deletion strains that eliminate chitosan formation share a common set of phenotypes, termed 'chitosan deficiency', which we propose to exploit to identify novel antifungal drugs. The key phenotypes of chitosan deficiency can be combined for the development of robust, miniaturized high throughput screening (HTS) assays. These are reduced growth at elevated temperatures and changes in staining of the cell wall. The proposed assay development strategy is supported by the many genetic deletion strains which we have derived that will enable chitosan deficiency hit selection and profiling. We also show preliminary capabilities to characterize mechanism of action of selected hits through direct enzyme inhibition. The chitin that is deacetylated is primarily generated through the action of a single chitin synthase and a specific chitin synthase regulator, even though Cryptococcus encodes seven other synthases and two other synthase regulators. The deacetylation is also dependent upon a family of three chitin deacetylases that we hypothesize to act in concert with the synthase/regulator. This application is a proposal to develop primary assays that will be suitable for a high-throughput screen (HTS) to identify candidate compounds that will inhibit the biosynthesis of chitosan in cryptococcus. We will also develop secondary screens to rule out non-specific cytotoxic compounds and we present preliminary enzyme assay data that supports our ability to develop the necessary tertiary assays to characterize and validate the screening hits. Our lab has the needed equipment and space to develop the reagents, cell lines and assays outlined in this proposal. We also have considerable industrial HTS expertise in the lab that will ensure that the assays and strategies developed through this proposal will successfully meet the standards of any HTS facility that would implement them. This proposal represents a novel approach to antifungal inhibitor discovery and targets proteins that have no homologies in humans or other mammals.
Fungal infections have become more prevalent in recent years due to the increase in the immunocompromised patient populations from AIDS, organ transplants and chemotherapies. Cryptococcus neoformans is a pathogenic fungus which kills over 600,000 people per year, mostly in underdeveloped countries and current therapies are inadequate because they are not effective enough or have significant toxicity. Biosynthesis of the fungal cell wall is an attractive target for antifungal therapies because the cell wall is an essential organelle that is not present in the human host. Chitosan is a polysaccharide that is essential for growth in the mammalian host, and this project will develop assays for a high throughput screen for inhibitors of chitosan biosynthesis.