title: The characterization of Lucilia cuprina acetylcholinesterase as a drug target, and the identification of novel inhibitors by high throughput screening.
abstract: 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.
authors.affiliation: Intervet Innovation GmbH, Zur Propstei, 55270 Schwabenheim, Germany. thomas.ilg@sp.intervet.com
authors.forename: Thomas
authors.initials: T
authors.lastname: Ilg
authors.forename: Jörg
authors.initials: J
authors.lastname: Cramer
authors.forename: Jürgen
authors.initials: J
authors.lastname: Lutz
authors.forename: Sandra
authors.initials: S
authors.lastname: Noack
authors.forename: Harald
authors.initials: H
authors.lastname: Schmitt
authors.forename: Heike
authors.initials: H
authors.lastname: Williams
authors.forename: Trevor
authors.initials: T
authors.lastname: Newton
mesh_terms: D000110:Acetylcholinesterase / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002219:Carbamates / Q000494:pharmacology; D002800:Cholinesterase Inhibitors / Q000494:pharmacology*; D003001:Cloning, Molecular; D004175:Diptera / Q000187:drug effects* / Q000502:physiology; D016503:Drug Delivery Systems / Q000379:methods*; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D020128:Inhibitory Concentration 50; D007300:Insect Control / Q000379:methods; D007306:Insecticides / Q000494:pharmacology*; D007700:Kinetics; D008969:Molecular Sequence Data; D010755:Organophosphates / Q000494:pharmacology; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013860:Thiocholine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000494:pharmacology; D014162:Transfection
chemical_list: D002219:Carbamates; D002800:Cholinesterase Inhibitors; D017261:Glycosylphosphatidylinositols; D007306:Insecticides; D010755:Organophosphates; D011994:Recombinant Proteins; D013860:Thiocholine; D000110:Acetylcholinesterase