title: Identification of new inhibitors of E. coli cyclopropane fatty acid synthase using a colorimetric assay.
abstract: Bacterial cyclopropane synthases catalyze the cyclopropanation of unsaturated fatty acids by transferring a methylene group from S-adenosyl-L-methionine (AdoMet) to the double bond of the lipids. Mycobacterium tuberculosis cyclopropane synthases have been shown to be implicated in pathogenicity, and therefore constitute attractive targets for the development of new drugs against tuberculosis. However, no in vitro assay for these cyclopropane synthases has yet been described. The homologous E. coli enzyme, cyclopropane fatty acid synthase, is thus a valuable model for inhibitor screening. Here, we report the adaptation to the E. coli CFAS of a previously reported enzyme-coupled colorimetric assay based on the quantification, using Ellman's reagent, of homocysteine produced from S-adenosyl-L-homocysteine, a product of the reaction, in the presence of AdoHcy nucleosidase and S-ribosylhomocysteinase. Using this assay we measured the kinetic parameters for CFAS: Km (AdoMet)=80 microM, kcat=4 min(-1). We adapted this assay to microtiter plates and tested 15 potential inhibitors of CFAS. Among them, two new inhibitors, a lipid analog and a thioether analog of AdoHcy, showed IC50 of 4 microM and 11 microM, respectively. This new assay will thus be useful for high-throughput screening of compound libraries for discovering novel antituberculous drug candidates.
authors.affiliation: Synthèse Structure et Fonction de Molécules Bioactives, UMR7613 CNRS-UPMC, Université Pierre et Marie Curie, boîte 182, 4, place Jussieu, F-75252 Paris cedex 05, France.
authors.forename: Dominique
authors.initials: D
authors.lastname: Guianvarc'h
authors.forename: Thierry
authors.initials: T
authors.lastname: Drujon
authors.forename: Thearina Ear
authors.initials: TE
authors.lastname: Leang
authors.forename: Fabienne
authors.initials: F
authors.lastname: Courtois
authors.forename: Olivier
authors.initials: O
authors.lastname: Ploux
mesh_terms: D003124:Colorimetry / Q000379:methods*; D015195:Drug Design; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D048168:Escherichia coli K12 / Q000201:enzymology*; D007700:Kinetics; D008780:Methyltransferases / Q000037:antagonists & inhibitors*; D015394:Molecular Structure; D013053:Spectrophotometry
chemical_list: D004791:Enzyme Inhibitors; D008780:Methyltransferases; C019964:cyclopropane synthetase