5.5.5
FAMOXADONE: A NEW CHEMICAL CLASS OF FUNGICIDES AND INHIBITORS OF MITOCHONDRIAL BC1 FUNCTION

DB JORDAN1, RS LIVINGSTON1, JJ BISAHA1, MA PICOLLELLI1, RS SCHWARTZ1, JA STERNBERG2 and X-S TANG3

1DuPont Agricultural Products, Stine-Haskell Research Center, P.O. Box 30, Newark, DE 19714-0030, 2DuPont Agricultural Products, Experimental Station, P.O. Box 80402, Wilmington, DE 19880-0402 and 3DuPont Central Research and Development, Experimental Station, P.O. Box 80173, Wilmington, DE 19880-0173

Background and objectives
Famoxadone (DPX-JE874) is a preventative and curative fungicide under development for plant disease control [1]. We determined the mode of action of the molecule and its analogs as presented below.

Results and conclusions
Famoxadone and its oxazolidinone analogs (OAD's) are potent inhibitors of mitochondrial electron transport specifically inhibiting the function of the enzyme ubiquinol:cytochrome c oxidoreductase (cytochrome bc1). Visible absorbance spectral studies on the purified enzyme suggested that famoxadone bound close to the low potential heme of cytochrome b. Red shifts in the spectra of the enzyme's reduced cytochrome b were additive for famoxadone and antimycin A (a ligand known to bind near the high potential heme of cytochrome b) but they were not additive for famoxadone and myxothiazol (a ligand known to bind near the low potential heme of cytochrome b) indicating that the former pair of inhibitors had independent binding sites whereas the latter pair were mutually exclusive. This binding mode was confirmed in competitive binding experiments by studying the displacement of a radiolabelled OAD from submitochondria. Myxothiazol displaced radiolabelled OAD from submitochondria whereas antimycin A did not. EPR studies on the binding of famoxadone to submitochondria and purified cytochrome bc1 suggested its binding mode was more similar to that of myxothiazol than that of stigmatellin (another ligand known to bind near the low potential heme of cytochrome b). Zoospores of Phytopthora infestans, when given low concentrations of famoxadone and other OAD's, were observed to cease oxygen consumption and motility within seconds and after minutes the cells disintegrated releasing organelles and other cellular contents. Famoxadone was a potent inhibitor of the growth of Saccharomyces cerevisiae when the yeast was grown on nonfermentable carbon sources, such as glycerol and ethanol, and it was approximately a 1000-fold less potent inhibitor of growth when the yeast was grown on the fermentable carbon source, glucose. Such physiological observations are consistent with the loss of mitochondrial function imposed by famoxadone and OAD's. Single amino acid changes in the apocytochrome b of S. cervisiae's cytochrome bc1, located near the low potential heme, differentially altered the inhibition constants for inhibitors famoxadone, myxothiazol, azoxystrobin, and kresoxim-methyl. The results of these amino acid replacements support the assignment of famoxadone's binding site near the low potential heme of cytochrome b and suggest significantly different interactions of apocytochrome b's amino acid side chains with famoxadone in comparison to the other inhibitors.

References
1. Joshi MM, Sternberg JA, 1996. Proceedings Brighton Crop Protection Conference: Pests and Diseases, pp. 21-26.