A Rapid in Vivo Colorimetric Library Screen for Inhibitors of Microbial Respiration

• Published in: ACS chemical biology Vol. 7; no. 10; pp. 1659 - 1665
• Main Authors: Vallières, Cindy, Fisher, Nicholas, Lemoine, Marc, Pamlard, Olivier, Beaupierre, Sandra, Guillou, Catherine, Meunier, Brigitte
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 19.10.2012
• Subjects: Anti-Infective Agents - chemistry; Anti-Infective Agents - pharmacology; Chemical Sciences; Colorimetry; DNA Mutational Analysis; Electron Transport Complex III - drug effects; Fermentation; High-Throughput Screening Assays; Models, Molecular; NADH, NADPH Oxidoreductases - antagonists & inhibitors; Organic chemistry; Oxygen Consumption - drug effects; Saccharomyces cerevisiae - drug effects; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism
• ISSN: 1554-8929; 1554-8937
• DOI: 10.1021/cb3002717

A number of fungicides that target the respiratory chain enzymes complexes II and III are used in agriculture. They are active against a large range of phytopathogens. Unfortunately, the evolution of fungicide resistance has quickly become a major issue. Resistance is often caused by mutations in the inhibitor binding domains of the complexes, and new molecules are required that are able to bypass such resistance mutations. We report here on a rapid in vivo high-throughput method, using yeast and the redox dye TTC to screen chemical libraries and identify inhibitors of respiratory function. We applied that screening process, followed by a series of tests, to a diverse library of 4,640 molecules and identified a weak inhibitor of complex III without toxic effect on the cell. Interestingly, that drug (D12) is fully active against the mutant enzyme harboring the G143A mutation that confers a high level of resistance toward most of the fungicides targeting complex III but is not active against bovine complex III. Using a collection of yeast strains harboring mutations in the inhibitor binding sites (Qo and Qi sites), we showed that D12 targeted the Qo site and that its inhibitory activity was weakened by the mutation L275F. A phenylalanine is naturally present at position 275 in mammalian complex III, which could explain the differential sensitivity toward D12. The molecule is not structurally related to commercial inhibitors of complex III and could potentially be used as a lead compound for the development of antimicrobial agents.