Intervet Innovation GmbH, Zur Propstei, 55270 Schwabenheim, Germany.
Insect Biochem Mol Biol. 2011 Jul;41(7):470-83. doi: 10.1016/j.ibmb.2011.04.003. Epub 2011 Apr 23.
Acetylcholinesterase (AChE, EC3.1.1.7.) is the molecular target for the carbamate and organophosphate pesticides that are used to combat parasitic arthropods. In this paper we report the functional heterologous expression of AChE from Lucilia cuprina (the sheep blowfly) in HEK293 cells. We show that the expressed enzyme is cell-surface-exposed and possesses a glycosyl-phosphatidylinositol membrane anchor. The substrates acetyl-, propionyl- and butyrylthiocholine (AcTC, PropTC, ButTC), and also 11 further thiocholine and homo-thiocholine derivatives were chemically synthesized to evaluate and compare their substrate properties in L. cuprina AChE and recombinant human AChE. The Michaelis-Menten constants K(M) for AcTC, PropTC and ButTC were found to be 3-7-fold lower for the L. cuprina AChE than for the human AChE. Additionally, 2-methoxyacetyl-thiocholine and isobutyryl-thiocholine were better substrates for the insect enzyme than for the human AChE. The AcTC, PropTC and ButTC specificities and the Michaelis-Menten constants for recombinant L. cuprina AChE were similar to those determined for AChE extracted from L. cuprina heads, which are a particularly rich source of this enzyme. The median inhibition concentrations (IC(50) values) were determined for 21 organophosphates, 23 carbamates and also 9 known non-covalent AChE inhibitors. Interestingly, 11 compounds were 100- to >4000-fold more active on the insect enzyme than on the human enzyme. The substrate and inhibitor selectivity data collectively indicate that there are structural differences between L. cuprina and human AChE in or near the active sites, suggesting that it may be possible to identify novel, specific L. cuprina AChE inhibitors. To this end, a high throughput screen with 107,893 compounds was performed on the L. cuprina head AChE. This led to the identification of 195 non-carbamate, non-organophosphate inhibitors with IC(50) values below 10μM. Analysis of the most potent hit compounds identified 19 previously unknown inhibitors with IC(50) values below 200nM, which were up to 335-fold more potent on the L. cuprina enzyme than on the human AChE. Some of these compounds may serve as leads for lead optimization programs to generate fly-specific pesticides.
乙酰胆碱酯酶（AChE，EC3.1.1.7.）是用于防治寄生性节肢动物的氨基甲酸酯类和有机磷类杀虫剂的分子靶标。在本文中，我们报告了来自 Lucilia cuprina（绵羊蝇）的 AChE 在 HEK293 细胞中的功能异源表达。我们表明，表达的酶是细胞表面暴露的，并且具有糖基磷脂酰肌醇膜锚。乙酰基、丙酰基和丁酰基硫代胆碱（AcTC、PropTC、ButTC）以及另外 11 种进一步的硫代胆碱和同型硫代胆碱衍生物被化学合成，以评估和比较它们在 L. cuprina AChE 和重组人 AChE 中的底物特性。与重组人 AChE 相比，L. cuprina AChE 对 AcTC、PropTC 和 ButTC 的米氏常数 K（M）低 3-7 倍。此外，2-甲氧基乙酰基硫代胆碱和异丁酰基硫代胆碱是昆虫酶的更好底物，而不是人 AChE。重组 L. cuprina AChE 的 AcTC、PropTC 和 ButTC 特异性和米氏常数与从 L. cuprina 头部提取的 AChE 测定的值相似，L. cuprina 头部是该酶的特别丰富来源。对 21 种有机磷化合物、23 种氨基甲酸酯化合物和 9 种已知的非共价 AChE 抑制剂进行了半数抑制浓度（IC（50）值）的测定。有趣的是，有 11 种化合物对昆虫酶的活性比人酶高 100 至>4000 倍。底物和抑制剂的选择性数据表明，L. cuprina 和人 AChE 的活性部位或附近存在结构差异，这表明可能有可能识别新型的、特异性的 L. cuprina AChE 抑制剂。为此，对 107893 种化合物进行了 L. cuprina 头 AChE 的高通量筛选。这导致了 195 种非氨基甲酸酯、非有机磷抑制剂的鉴定，其 IC（50）值低于 10μM。对最有效的命中化合物的分析确定了 19 种以前未知的抑制剂，其 IC（50）值低于 200nM，对 L. cuprina 酶的活性比人 AChE 高 335 倍。其中一些化合物可能作为先导化合物优化方案的起点，以产生针对蝇类的杀虫剂。
