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

• Published in: Insect molecular biology 2012-08, Vol.21 (4), p.456-471
• Main Authors: Kern, C, Wolf, C, Bender, F, Berger, M, Noack, S, Schmalz, S, Ilg, T
• Format: Article
• Language: English
• Subjects: Animals; biosynthesis; Cloning, Molecular; complementary DNA; Ctenocephalides - genetics; Ctenocephalides - metabolism; Ctenocephalides felis; Drosophila melanogaster; Drosophila melanogaster - genetics; Drosophila melanogaster - metabolism; ectoparasite; Enzymes; Gene Expression; genes; Glucose-6-phosphate; Glucose-6-Phosphate - chemistry; Glucose-6-Phosphate - metabolism; Glucosyltransferases - antagonists & inhibitors; Glucosyltransferases - chemistry; Glucosyltransferases - genetics; Glucosyltransferases - metabolism; Hemolymph; high throughput screening; insecticidal properties; insecticide target; Insecticides; Insecticides - chemistry; Insecticides - metabolism; insects; Kinetics; mechanism of action; pesticide target; Pyrans - chemistry; screening; Small Molecule Libraries - analysis; Small Molecule Libraries - chemistry; Structure-Activity Relationship; Structure-activity relationships; Sugar; Trehalose; Trehalose - genetics; Trehalose - metabolism; trehalose phosphate synthase; trehalose-6-phosphate synthase
• ISSN: 0962-1075; 1365-2583
• DOI: 10.1111/j.1365-2583.2012.01151.x

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.