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

Abstract

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.

TPS1 drug design for rice blast disease in magnaporthe oryzae

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Figure 4 Binding conformations of the best three compounds from the Molecular Libraries Small Molecule Repository (MLSMR), when docked onto Tps1. (a) 24789937, (b) 44825744, and (c) 16423676, and the binding conformation of Compound 1 (d) (Kern et al, 2012). (e) Binding affinity measurements.

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“…(a) 24789937, (b) 44825744, and (c) 16423676, and the binding conformation of Compound 1 (d) (Kern et al, 2012). (e) Binding affinity measurements.…”

“…As a comparison, we tested the binding affinity of a compound identified from a previous high-throughput screening for potent inhibitors of the Tps from the cat flea Ctenocephalides felis and Drosophila melanogaster (Kern et al, 2012) with M. oryzae Tps1 (Figure 4d and 4e). Surprisingly, the compound (referred to as ‘Compound 1’) did not bind well to Tps1, which may be due to structural variations between the different Tps enzymes; indeed, Compound 1 is a relatively smaller molecule as compared with the compounds identified by AutoDock Vina, with much potential for further modification.…”

A genetically encoded Förster resonance energy transfer sensor for monitoring in vivo trehalose-6-phosphate dynamics

“…Therefore, accurate measurements of free metabolites at cellular or subcellular resolution is possible only if unspecific effects can be excluded a priori.We expect the T6P–TRACK sensors to become a valuable tool for investigating the role of T6P in more challenging contexts such as plant development and growth [5]. Although trehalose is absent in mammals, it is essential for insects [33] and the sensors can be useful in the search for insecticide compounds targeting the Tre pathway. Because trehalose is similarly important for nematodes [34], our T6P sensors may serve to screen for drugs targeting the Tre pathway and acting against pathogenic nematodes.…”

Transciptomic Analysis of Larval Fat Body of Plutella xylostella under Low Temperature

“…Trehalose는 혈림프에서 발견되는 주요 당이며, 1차적인 에너지원과 chitin 합성의 시작물질로 알려져 있다 (Xiong et al, 2016;Thompson, 2003). 지방체는 척추동물의 간과 비 슷한 역할을 하며, 대사중심으로써 trehalose-6-phosphate synthase를 이용하여 trehalose를 생산한다 (Kern et al, 2012). 파밤나방에서 보고된 것처럼 (Park and Kim, 2013) * The expression level of each contig can be divided into upregulation (log 2 Fc>2), downregulation (log 2 Fc<-2), and not meaningful (-2<log 2 Fc<2), compared to the control.…”