We propose to discover the mode of action of anti-fungal drugs that require group III hybrid histidine kinases (HHKs). HHKs regulate the HOG (high-osmolarity glycerol) pathway and are conserved in fungi and absent in mammals, making them an ideal drug target. Fungicides that require the HOG pathway to kill pathogens are used in agriculture, but are too toxic for use in human patients. The drug target of these agricultural fungicides is unclear and represents a gap in our knowledge with significant implications. An understanding of the actual drug target will unveil a new path to antifungal drug development for patients. We propose a new, unrecognized mode of action among fungicides. We will test the hypothesis that antifungal agents in the phenylpyrrole and dicarboximide classes, such as fludioxonil, do not act directly on the putative target ? HHKs ? to trigger the HOG pathway, but rather act upstream. In preliminary work, we find that fungicides inhibit nitrosoglutathione reductase (GSNOR); the downstream HHK responds indirectly to elevated nitrosoglutathione (GSNO) levels and converts from a kinase into a phosphatase upon nitrosylation of cysteine 1062 near the HHK receiver domain. Excess nitrosative stress promotes mammalian cell toxicity, an unwanted aspect of exposure to fungicides in the food chain. However, understanding this mode of action will catalyze an unprecedented strategy for developing new antifungal drugs for patients that directly target HHKs, based on the novel pharmacophore at Cys1062 to be studied here. We propose 2 aims: 1) Investigate fludioxonil's action on GSNOR and the action of structurally related fungicides on this upstream target; and 2) Analyze the impact of GSNO on HHK function as a phosphatase, and the ability to harness this action by screening, identifying and chemically modifying nitrosylation mimetics that induce the conversion of HHKs from a kinase into phosphatase. While the action of fungicides on GSNOR reveals risk, their potency offers new drug development potential in targeting the HOG pathway and specifically HHK. Our work will thus reveal the unrecognized target of fungicides, clarify a new pharmacophore directly on fungal HHKs, and pave the way to new anti-fungal drugs based on fresh knowledge. The work is significant because anti-fungal drugs with new modes of action are badly needed. Systemic fungal infections are rapidly increasing and only one new class of drug for patients has come to market over the last 15 years. Our work will impact this field by validating a drug target that is unique to fungi for potential development of a new class of compounds that directly target HHKs by converting them from exhibiting kinase activity into exerting phosphatase activity.