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Trehalose‐6‐phosphate synthase from the cat flea Ctenocephalides felis and Drosophila melanogaster : gene identification, cloning, heterologous functional expression and identification of inhibitors by high throughput screening
Author(s) -
Kern C.,
Wolf C.,
Bender F.,
Berger M.,
Noack S.,
Schmalz S.,
Ilg T.
Publication year - 2012
Publication title -
insect molecular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.955
H-Index - 93
eISSN - 1365-2583
pISSN - 0962-1075
DOI - 10.1111/j.1365-2583.2012.01151.x
Subject(s) - biology , drosophila melanogaster , heterologous expression , cloning (programming) , heterologous , gene , ctenocephalides , felis , trehalase , genetics , microbiology and biotechnology , biochemistry , enzyme , recombinant dna , cats , computer science , programming language , embedded system
Trehalose phosphate synthase (EC 2.4.1.15; TPS) is the crucial enzyme for the biosynthesis of trehalose, the main haemolymph sugar of insects, and therefore a potential insecticidal molecular target. In this study, we report the functional heterologous expression of Drosophila melanogaster TPS, the gene identification, full length cDNA cloning and functional expression of cat flea ( Ctenocephalides felis ) TPS, and the Michaelis–Menten constants for their specific substrates glucose‐6‐phosphate and uridinediphosphate‐glucose. A novel high throughput screening‐compatible TPS assay and its use for the identification of the first potent insect TPS inhibitors from a large synthetic compound collection (>115 000 compounds) is described. One compound class that emerged in this screening, the 4‐substituted 2,6‐diamino‐3,5‐dicyano‐4H‐thiopyrans, was further investigated by analysing preliminary structure–activity relationships. Here, compounds were identified that show low µM to high nM half maximal inhibitory concentrations on insect TPS and that may serve as lead compounds for the development of insecticides with a novel mode of action.