Rand S. Schwartz

Mode of action of famoxadone

Famoxadone is a preventative and curative fungicide recently developed for plant disease control. The molecule and its oxazolidinone analogs (OADs) are potent inhibitors of mitochondrial electron transport, specifically inhibiting the function of the enzyme ubiquinol:cytochrome c oxidoreductase (cytochrome bc 1 ). Visible absorbance spectral studies on the purified enzyme suggested that famoxadone bound close to the low potential heme of cytochrome b. This binding mode was confirmed in competitive binding experiments by studying the displacement of a radiolabelled OAD from submitochondria. EPR studies on the binding of famoxadone to submitochondria and purified bc 1 suggested its binding mode was more like that of myxothiazol than that of stigmatellin (ligands known to bind near the low potential heme). Zoospores of Phytophthora infestans, when given low concentrations of famoxadone and other OADs, were observed to cease oxygen consumption and motility within seconds and later the cells disintegrated, releasing the cellular contents. Famoxadone was a potent inhibitor of the growth of Saccharomyces cerevisiae when grown on non-fermentable carbon sources and it was an approximately 50-fold less potent inhibitor of growth when the yeast was grown on a fermentable carbon source, glucose. Such physiological observations are consistent with the loss of mitochondrial function imposed by famoxadone and OADs. Single amino acid changes in the apocytochrome b of bakers yeast cytochrome b located near the low potential heme altered the inhibition constants for the inhibitors famoxadone, myxothiazol, azoxystrobin and kresoxim-methyl differentially, thus strongly suggesting different binding interactions of the protein with the inhibitors.

Cyclobutane carboxamide inhibitors of fungal melanin: biosynthesis and their evaluation as fungicides.

A new fungicide lead has been identified by enzyme screening of a focused combinatorial library. The lead compound 4, a potent inhibitor of scytalone dehydratase (SD), exhibits fungicidal activity upon foliar application but does not show systemic activity. The X-ray crystal structure of the enzyme-inhibitor complex and an appreciation for the relationship between physical properties and systemic activity enabled us to rapidly improve upon this initial lead. The geminal halogen-methyl group combination was found to be optimal for interaction with the bounding serine and asparagine side-chain residues. Replacement of CF3 with methyl was a key discovery, giving inhibitors with slightly diminished enzyme inhibition potency while significantly increasing systemic activity. Amides prepared from amines with 2,4-dichloro substitution on the phenyl ring gave the most potent enzyme inhibitors. Two compounds from this series showed systemic activity comparable to the commercial standard and were selected for outdoor testing in flooded plots which simulate rice paddies.

Design of scytalone dehydratase inhibitors as rice blast fungicides: derivatives of norephedrine.

Five X-ray crystal structures of scytalone dehydratase complexed with different inhibitors have delineated conformationally flexible regions of the binding pocket. This information was used for the design and synthesis of a norephedrine-derived cyanoacetamide class of inhibitors leading to potent fungicides.

Design of scytalone dehydratase inhibitors as rice blast fungicides: (N- phenoxypropyl)-carboxamides

Insights gained from a crystal structure of scytalone dehydratase led to the design of carboxamide inhibitors with a phenoxypropyl group substituted on the nitrogen atom Potent enzyme inhibitors were synthesized around this motif, the best of which provided excellent control of rice blast disease in greenhouse assays and outdoor field trials.

A new potent inhibitor of fungal melanin biosynthesis identified through combinatorial chemistry

A new fungicide lead has been identified by in vitro screening of a focused combinatorial library. Amides (768) were synthesized in pools of four and assayed as inhibitors of scytalone dehydratase. Deconvolution of one of the most active pools led to the discovery of a potent inhibitor of the enzyme 3b (K(i) = 26 pM), which has fungicidal properties.

Oxazolidinones: a new chemical class of fungicides and inhibitors of mitochondrial cytochrome bc1 function

Famoxadone is a preventative and curative fungicide recently commercialized for plant-disease control. The molecule and its oxazolidinone analogs are potent inhibitors of mitochondrial ubiquinol : cytochrome c oxidoreductase (cytochrome bc1) and they bind in the Q0 site of the enzyme near the low potential heme of cytochrome b. Inhibitor binding constants for five mutant cytochrome bc1 enzymes from Saccharomyces cerevisiae having single amino acid changes in their apocytochrome b located near the low potential heme were compared with their two parental wild-type enzymes. The five individual amino acid changes altered the inhibition constants for the inhibitors famoxadone, myxothiazol, azoxystrobin, and kresoxim-methyl in dissimilar fashion. The log scale differences in binding constants relative to those of their parentals provide fingerprints for the effects of the amino acid changes on binding of the individual inhibitors, thus reflecting the structural diversity of the inhibitors. ©1999 Society of Chemical Industry

Catalytic mechanism of scytalone dehydratase from Magnaporthe grisea

The catalytic mechanism of scytalone dehydratase was examined by studying alternative substrates and site-directed mutations of active-site residues. Searches for an enol intermediate by looking for a half-reaction with authentic scytalone and 3,4-dihydro-6,8-dihydroxy-1-(2H)-2-[ 13 C]naphthalenone were negative. An alternative substrate, 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one (DDBO), was nearly equal to scytalone as substrate for the enzyme, and DDBOs anomeric effect in stabilizing a partial carbocation center at C3 does not substantially contribute to the mechanism. Kinetic analysis of site-directed mutations of active-site amino acid side chains within the enzymes active site provided an account for the role of these residues in the enzyme-catalyzed dehydration reactions. A concerted E2 elimination for the catalytic mechanism is proposed.