ABSTRACT: Project 3 – Trehalose Pathway for Antifungal Targets and Inhibitors This project is centered on a specific target pathway that encompasses the “holy grail” of antifungal target development which includes the important fungal specificity of the target compared to mammalian system and its fungicidal properties. In this proposal, we will extend our studies in Cryptococcus neoformans and Candida albicans which have been previously used to carefully validate the trehalose pathway in the pathobiology of these basidiomycetes and ascomycetes. There is also substantial data for trehalose pathway in other major pathogenic fungi including Aspergillus and Mucor and thus trehalose inhibitors could also be studied in these moulds. This proposal is primarily split into two major foci to develop understanding of the targets Tps 1 (Trehalose-6-phosphate synthase) and Tps 2 (Trehalose-6- phosphate phosphatase) and to identify inhibitors of these critical enzymes to further develop them into drugs. In the first aim, there will be a detailed structure analysis of the two major proteins to allow an informed approach to molecules which could potentially inhibit and/or interact with these enzymes. A structural/functional analysis will allow for development of screens to identify interacting molecules from chemical libraries. A second part of this proposal is to begin an understanding of resistance mechanism(s) and identification of interacting or client partners (proteins or pathways). It is our belief that these strategies allow an optimization of how these trehalose targets can be used for antifungal development. The second aim will be a comprehensive search and validation of trehalose inhibitors with strategies to evaluate their antifungal activity in animals. It is necessary to focus on fungicidal activity, ability to identify and use synergistic targets to improve fungicidal activity and reduce resistance potential. In our opinion the trehalose pathway represents one of the foremost target areas in antimicrobial research and it remains poorly developed. It is an extremely fungicidal target for fungi in vivo; it has no mammalian counterpart; and it has wide spectrum significance in the pathogenic fungal kingdom and can extend to other microbes impacting human disease.