Discovery of Selective Inhibitors Targeting Acetylcholinesterase 1 from Disease-Transmitting Mosquitoes

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 <i>Anopheles gambiae</i> and <i>Aedes aegypti</i>. 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

Enzyme kinetics.

<p>(A) Substrate preference for <i>Aa</i>AChE1 illustrated by Michaelis-Menten curves. The same trend was observed for <i>Ag</i>AChE1 <i>and Ag</i>AChE1-G119S. (B) Substrate inhibition of <i>Aa</i>AChE1, <i>Ag</i>AChE1 and <i>Ag</i>AChE1-G119S using ATChI as substrate. (C) Typical graph for <i>k</i>_<i>i</i>-calculations of <i>Ag</i>AChE1 with eserine as inhibitor. (D) Typical dose-response curve of <i>Aa</i>AChE1 with inhibitor, used for determination of <i>IC</i>_<i>50</i>-values for various compounds.</p

Characterization of inhibitors.

<p>*[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138598#pone.0138598.ref041" target="_blank">41</a>]</p><p>**[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138598#pone.0138598.ref042" target="_blank">42</a>]</p><p>Characterization of inhibitors.</p

Pt-transfer from Atox1 to WD4.

<p>Analytical gelfiltration, Black: 280 nm, Grey: 254 nm. <b>A</b>. Apo-Atox1 mixed with 0.5 eq. apo-WD4. <b>B</b>. Control, WD4. No Atox1-CisPt added, otherwise experiment conducted as D. <b>C</b>. Control, Atox1-CisPt. No WD4 added, otherwise experiment conducted as D. <b>D</b>. Transfer experiment. Atox1-CisPt, filtrated and concentrated, mixed with 0.5 eq. WD4 and incubated for 4 h prior to SEC analysis. See also <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070473#pone.0070473.s009" target="_blank">Figure S9</a></b>.</p

CisPt-triggered unfolding of Atox1 variants and WD4.

<p>Black: Apo-protein. Grey: +1 eq. Cu. Blue: +1 eq. Cu and +5 eq. CisPt. Red: +5 eq. CisPt. <b>A</b>. WT Atox1. <b>B</b>. Cys41Ala Atox1. <b>C</b>. 3Cys3Ala Atox1. <b>D</b>. Cys15Ala Atox1. <b>E</b>. Met10Ala Atox1. <b>F</b>. WT WD4.</p

t_1/2 of CisPt induced unfolding of apo- and holo-Atox1 variants and WD4 WT.

<p>5 eq. CisPt was added to either apo- or holo- (+1 eq. Cu) protein and loss of secondary structure was measured by far-UV CD in RT over two weeks. CD data at 220 nm plotted vs. time and fitted to single exponential decays.</p

Near-UV CD for Atox1 variants and WD4.

<p>Selected traces from Cu/CisPt titrations. Black: Apo-protein. Grey: +1 eq. Cu. Blue: +1 eq. Cu and +2 eq. CisPt. Red: +1 eq. CisPt +2 eq. Cu. <b>A</b>. WT Atox1. <b>B</b>. Cys41Ala Atox1. <b>C</b>. 3Cys3Ala Atox1. <b>D</b>. Cys15Ala Atox1. <b>E</b>. Met10Ala Atox1. <b>F</b>. WT WD4. See also <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070473#pone.0070473.s001" target="_blank">Figure S1</a></b>.</p

Analytical gelfiltration of Atox1/WD4.

<p>Protein +1 eq. Cu and 5 eq. CisPt. Black: 280 nm. Grey: 254 nm. <b>A</b>. WT Atox1. <b>B</b>. Cys41Ala Atox1. <b>C</b>. 3Cys3Ala Atox1. <b>D</b>. Cys15Ala Atox1. <b>E</b>. Met10Ala Atox1. <b>F</b>. WT WD4. Sample incubation time is 10 min. See also <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070473#pone.0070473.s003" target="_blank">Figure S3</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070473#pone.0070473.s008" target="_blank">S8</a></b>.</p

AChE1 glycopeptides detected by LC-MS.

<p>n.d. = not determined</p><p>AChE1 glycopeptides detected by LC-MS.</p

No loss of Cu upon CisPt binding to Atox1 WT.

<p>Samples treated with 1∶1 of CisPt and incubated for various times. Samples were centrifuged and Cu concentration was measured in flow trough (cut off in filter 3000 Da). Dark grey: CisPt treated holo Atox1. Light gray: Holo-Atox1. The last two columns are positive controls where Atox1 was omitted.</p