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Selective inhibition of acetylcholinesterase 1 from disease-transmitting mosquitoes: design and development of new insecticides for vector control
Umeå University, Faculty of Science and Technology, Department of Chemistry.
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Acetylcholinesterase (AChE) is an essential enzyme with an evolutionary conserved function: to terminate nerve signaling by rapid hydrolysis of the neurotransmitter acetylcholine. AChE is an important target for insecticides. Vector control by the use of insecticide-based interventions is today the main strategy for controlling mosquito-borne diseases that affect millions of people each year. However, the efficiency of many insecticides is challenged by resistant mosquito populations, lack of selectivity and off-target toxicity of currently used compounds. New selective and resistance-breaking insecticides are needed for an efficient vector control also in the future. In the work presented in this thesis, we have combined structural biology, biochemistry and medicinal chemistry to characterize mosquito AChEs and to develop selective and resistance-breaking inhibitors of this essential enzyme from two disease-transmitting mosquitoes.We have identified small but important structural and functional differences between AChE from mosquitoes and AChE from vertebrates. The significance of these differences was emphasized by a high throughput screening campaign, which made it evident that the evolutionary distant AChEs display significant differences in their molecular recognition. These findings were exploited in the design of new inhibitors. Rationally designed and developed thiourea- and phenoxyacetamide-based non-covalent inhibitors displayed high potency on both wild type and insecticide insensitive AChE from mosquitoes. The best inhibitors showed over 100-fold stronger inhibition of mosquito than human AChE, and proved insecticide potential as they killed both adult and larvae mosquitoes.We show that mosquito and human AChE have different molecular recognition and that non-covalent selective inhibition of AChE from mosquitoes is possible. We also demonstrate that inhibitors can combine selectivity with sub-micromolar potency for insecticide resistant AChE.

Place, publisher, year, edition, pages
Umeå: Umeå University , 2017. , 67 p.
Keyword [en]
acetylcholinesterase, non-covalent inhibitor, vector control, insecticide, mosquito, vector-borne disease, high throughput screening, rational design
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry; läkemedelskemi
Identifiers
URN: urn:nbn:se:umu:diva-134625ISBN: 978-91-7601-723-4 (print)OAI: oai:DiVA.org:umu-134625DiVA: diva2:1094489
Public defence
2017-06-02, KB.E3.03 (stora hörsalen), KBC-huset, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2017-05-12 Created: 2017-05-10 Last updated: 2017-05-12Bibliographically approved
List of papers
1. Acetylcholinesterases from the Disease Vectors Aedes aegypti and Anopheles gambiae: Functional Characterization and Comparisons with Vertebrate Orthologues
Open this publication in new window or tab >>Acetylcholinesterases from the Disease Vectors Aedes aegypti and Anopheles gambiae: Functional Characterization and Comparisons with Vertebrate Orthologues
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2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 10, e0138598Article in journal (Refereed) Published
Abstract [en]

Mosquitoes of the Anopheles (An.) and Aedes (Ae.) genus are principal vectors of human diseases including malaria, dengue and yellow fever. Insecticide-based vector control is an established and important way of preventing transmission of such infections. Currently used insecticides can efficiently control mosquito populations, but there are growing concerns about emerging resistance, off-target toxicity and their ability to alter ecosystems. A potential target for the development of insecticides with reduced off-target toxicity is the cholinergic enzyme acetylcholinesterase (AChE). Herein, we report cloning, baculoviral expression and functional characterization of the wild-type AChE genes (ace-1) from An. gambiae and Ae. aegypti, including a naturally occurring insecticide-resistant (G119S) mutant of An. gambiae. Using enzymatic digestion and liquid chromatography-tandem mass spectrometry we found that the secreted proteins were post-translationally modified. The Michaelis-Menten constants and turnover numbers of the mosquito enzymes were lower than those of the orthologous AChEs from Mus musculus and Homo sapiens. We also found that the G119S substitution reduced the turnover rate of substrates and the potency of selected covalent inhibitors. Furthermore, non-covalent inhibitors were less sensitive to the G119S substitution and differentiate the mosquito enzymes from corresponding vertebrate enzymes. Our findings indicate that it may be possible to develop selective non-covalent inhibitors that effectively target both the wild-type and insecticide resistant mutants of mosquito AChE.

National Category
Other Chemistry Topics
Identifiers
urn:nbn:se:umu:diva-110996 (URN)10.1371/journal.pone.0138598 (DOI)000362511000010 ()26447952 (PubMedID)
Funder
Swedish Research Council
Available from: 2015-11-17 Created: 2015-11-02 Last updated: 2017-05-10Bibliographically approved
2. Discovery of selective inhibitors targeting acetylcholinesterase 1 from disease-transmitting mosquitoes
Open this publication in new window or tab >>Discovery of selective inhibitors targeting acetylcholinesterase 1 from disease-transmitting mosquitoes
2016 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 59, no 20, 9409-9421 p.Article in journal (Other academic) Published
Abstract [en]

Vector control of disease-transmitting mosquitoes is increasingly important due to the re-emergence and spread of infections such as malaria and dengue. We have conducted a high throughput screen (HTS) of 17,500 compounds for inhibition of the essential AChE1 enzymes from the mosquitoes Anopheles gambiae and Aedes aegypti. In a differential HTS analysis including the human AChE, several structurally diverse, potent, and selective noncovalent AChE1 inhibitors were discovered. For example, a phenoxyacetamide-based inhibitor was identified with a 100-fold selectivity for the mosquito over the human enzyme. The compound also inhibited a resistance conferring mutant of AChE1. Structure-selectivity relationships could be proposed based on the enzymes' 3D structures; the hits' selectivity profiles appear to be linked to differences in two loops that affect the structure of the entire active site. Noncovalent inhibitors of AChE1, such as the ones presented here, provide valuable starting points toward insecticides and are complementary to existing and new covalent inhibitors.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2016
National Category
Organic Chemistry
Identifiers
urn:nbn:se:umu:diva-119925 (URN)10.1021/acs.jmedchem.6b00967 (DOI)000386641300010 ()
Note

First published in thesis in manuscript form.

Available from: 2016-05-03 Created: 2016-05-02 Last updated: 2017-05-10Bibliographically approved
3. N-Aryl-N'-ethyleneaminothioureas effectively inhibit acetylcholinesterase 1 from disease-transmitting mosquitoes
Open this publication in new window or tab >>N-Aryl-N'-ethyleneaminothioureas effectively inhibit acetylcholinesterase 1 from disease-transmitting mosquitoes
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2017 (English)In: European Journal of Medicinal Chemistry, ISSN 0223-5234, E-ISSN 1768-3254, Vol. 134, 415-427 p.Article in journal (Refereed) Published
Abstract [en]

Vector control of disease-transmitting mosquitoes by insecticides has a central role in reducing the number of parasitic- and viral infection cases. The currently used insecticides are efficient, but safety concerns and the development of insecticide-resistant mosquito strains warrant the search for alternative compound classes for vector control. Here, we have designed and synthesized thiourea-based compounds as non-covalent inhibitors of acetylcholinesterase 1 (AChE1) from the mosquitoes Anopheles gambiae (An. gambiae) and Aedes aegypti (Ae. aegypti), as well as a naturally occurring resistant-conferring mutant. The N-aryl-N'-ethyleneaminothioureas proved to be inhibitors of AChE1; the most efficient one showed submicromolar potency. Importantly, the inhibitors exhibited selectivity over the human AChE (hAChE), which is desirable for new insecticides. The structure-activity relationship (SAR) analysis of the thioureas revealed that small changes in the chemical structure had a large effect on inhibition capacity. The thioureas showed to have different SAR when inhibiting AChE1 and hAChE, respectively, enabling an investigation of structure-selectivity relationships. Furthermore, insecticidal activity was demonstrated using adult and larvae An. gambiae and Ae. aegypti mosquitoes.

Keyword
Acetylcholinesterase 1, Aedes aegypti, Anopheles gambiae, Insecticides, Thiourea, Vector control
National Category
Organic Chemistry
Identifiers
urn:nbn:se:umu:diva-134612 (URN)10.1016/j.ejmech.2017.03.050 (DOI)000401677500035 ()28433681 (PubMedID)
Available from: 2017-05-09 Created: 2017-05-09 Last updated: 2017-06-29Bibliographically approved
4. Binding Mode of Reversible Inhibitors in Mosquito Acetylcholinesterase 1 Governs their Selective and Resistance-Breaking Potency
Open this publication in new window or tab >>Binding Mode of Reversible Inhibitors in Mosquito Acetylcholinesterase 1 Governs their Selective and Resistance-Breaking Potency
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(English)Manuscript (preprint) (Other academic)
National Category
Organic Chemistry
Research subject
läkemedelskemi
Identifiers
urn:nbn:se:umu:diva-134619 (URN)
Available from: 2017-05-10 Created: 2017-05-10 Last updated: 2017-05-10

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