THE DEPENDENCE OF THE FATTENING ABILITY OF CHICKENS UPON SEX

<div><p>Background</p><p>New mode-of-action insecticides are sought to provide continued control of pesticide resistant arthropod vectors of neglected tropical diseases (NTDs). We previously identified antagonists of the AaDOP2 D1-like dopamine receptor (DAR) from the yellow fever mosquito, <i>Aedes aegypti</i>, with toxicity to <i>Ae</i>. <i>aegypti</i> larvae as leads for novel insecticides. To extend DAR-based insecticide discovery, we evaluated the molecular and pharmacological characteristics of an orthologous DAR target, <i>Cq</i>DOP2, from <i>Culex quinquefasciatus</i>, the vector of lymphatic filariasis and West Nile virus.</p><p>Methods/Results</p><p><i>Cq</i>DOP2 has 94.7% amino acid identity to <i>Aa</i>DOP2 and 28.3% identity to the human D1-like DAR, hD1. <i>Cq</i>DOP2 and <i>Aa</i>DOP2 exhibited similar pharmacological responses to biogenic amines and DAR antagonists in cell-based assays. The antagonists amitriptyline, amperozide, asenapine, chlorpromazine and doxepin were between 35 to 227-fold more selective at inhibiting the response of <i>Cq</i>DOP2 and <i>Aa</i>DOP2 in comparison to hD1. Antagonists were toxic to both <i>C</i>. <i>quinquefasciatus</i> and <i>Ae</i>. <i>aegypti</i> larvae, with LC50 values ranging from 41 to 208 μM 72 h post-exposure. Orthologous DOP2 receptors identified from the African malaria mosquito, <i>Anopheles gambiae</i>, the sand fly, <i>Phlebotomus papatasi</i> and the tsetse fly, <i>Glossina morsitans</i>, had high sequence similarity to <i>Cq</i>DOP2 and <i>Aa</i>DOP2.</p><p>Conclusions</p><p>DAR antagonists represent a putative new insecticide class with activity against <i>C</i>. <i>quinquefasciatus</i> and <i>Ae</i>. <i>aegypti</i>, the two most important mosquito vectors of NTDs. There has been limited change in the sequence and pharmacological properties of the DOP2 DARs of these species since divergence of the tribes Culicini and Aedini. We identified antagonists selective for mosquito versus human DARs and observed a correlation between DAR pharmacology and the <i>in vivo</i> larval toxicity of antagonists. These data demonstrate that sequence similarity can be predictive of target potential. On this basis, we propose expanded insecticide discovery around orthologous DOP2 targets from additional dipteran vectors.</p></div

IC₅₀ values (nM±SEM) for inhibition of dopamine-stimulated cAMP response in HEK293 cell lines by DAR antagonists.

<p>Values were determined from concentration response curves measuring the cAMP response. Fold selectivity was determined by dividing human D₁ IC₅₀ values by each respective mosquito DOP2 IC₅₀ value for each antagonist.</p><p>IC₅₀ values (nM±SEM) for inhibition of dopamine-stimulated cAMP response in HEK293 cell lines by DAR antagonists.</p

Schematic depicting PIDP activities aimed at discovery of D₁-like DAR antagonists as new insecticides.

<p>The workflow is based on the evolving “genome-to-lead” component of the PIDP first described in Meyer et al. [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003515#pntd.0003515.ref011" target="_blank">11</a>]. High-throughput (HTP), cell-based screens expressing arthropod D₁-like DARs (Target Panel) are employed to identify chemistries active against one or more arthropod targets. Vector-selective chemistries are identified using counter screens expressing the human hD_1–5 and the honeybee DAR (Non-target Panel). Subsequently, the <i>in vivo</i> toxicity of chemistries is confirmed in single-point dose and concentration response screens against mosquito larvae. Top hits are evaluated for activity against the adult stage of one or more vector species and taxon-level selectivity for the dipteran suborders Nematocera and Brachycera, and the subclass Acari. Information from structure activity relationship studies is used to direct iterative chemical screens. Chemical leads may enter the “Lead-to-Product” phase of the pipeline. New components of the pipeline described in the present study include the pharmacologically characterized <i>Cq</i>DOP2 target, the <i>Ag</i>DOP2, <i>Pp</i>DOP2 and <i>Gm</i>DOP2 targets identified from assembled genome sequences (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003515#pntd.0003515.s002" target="_blank">S2 Fig</a>), and the <i>C</i>. <i>quinquefasciatus</i> larval screen. Remaining components are the subject of works in review [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003515#pntd.0003515.ref015" target="_blank">15</a>] and ongoing efforts. Abbreviations: <i>Aa</i>, <i>Aedes aegypti; Ag</i>, <i>Anopheles gambiae; Am</i>, <i>Apis mellifera</i>; <i>Cq</i>, <i>Culex quinquefasciatus; Gm</i>, <i>Glossina morsitans; Is</i>, <i>Ixodes scapularis; Pp</i>, <i>Phlebotomus papatasi;</i> NP, natural product.</p

EC₅₀ values (μM±SEM) for dopamine and other biogenic amines estimated from concentration response curves measuring the cAMP response from HEK293 cells expressing <i>Aa</i>DOP2 or <i>Cq</i>DOP2 (n ≥ 8 conducted in duplicate).

<p>No response was observed for histamine, octopamine, serotonin, or tyramine for concentrations up to 100 μM.</p><p>EC₅₀ values (μM±SEM) for dopamine and other biogenic amines estimated from concentration response curves measuring the cAMP response from HEK293 cells expressing <i>Aa</i>DOP2 or <i>Cq</i>DOP2 (n ≥ 8 conducted in duplicate).</p

Pharmacological characterization of <i>Aa</i>DOP2 and <i>Cq</i>DOP2 stably expressed in HEK293 cells.

<p>Normalized cAMP response (mean ± SEM) seen as a function of concentration of dopamine, norepinephrine, and epinephrine for each receptor. The graphs are based on the compiled data (n ≥ 8 independent experiments, conducted in duplicate) and normalized using GraphPad Prism software to the maximal dopamine response for each experiment.</p

Alignment of <i>Cq</i>DOP2 and <i>Aa</i>DOP2 amino acid sequences.

<p>Highlighted areas indicate identical and conserved residues as designated by ClustalW [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003515#pntd.0003515.ref029" target="_blank">29</a>]: black = identical residues; dark gray = strongly similar residues; light gray = weakly similar residues (for amino acid similarity groups, see: <a href="http://www.clustal.org/download/clustalx_help.html" target="_blank">http://www.clustal.org/download/clustalx_help.html</a>). Putative transmembrane (TM) domains I-VII are indicated as a line above the alignment.</p

Neighbor-joining sequence analysis of <i>Cq</i>DOP2, <i>Aa</i>DOP2 and representative biogenic amine receptors.

<p>Abbreviations and NCBI accession numbers of species indicated are as follows: <i>Aedes aegypti</i> = Aa; AaDOP1 = D₁-like dopamine receptor 1 (JN043502); AaDOP2 = D₁-like dopamine receptor 2 (JN043503); AaDOP3 = D₂-like dopamine receptor (XM_001648573); <i>Culex quinquefasciatus</i> = Cq; CqDOP1 = D₁-like dopamine receptor 1 (XM_001842358); CqDOP2 = D₁-like dopamine receptor 2 (KM262648); CqDOP3 = D₂-like dopamine receptor (XM_001865540); <i>Ixodes scapularis</i> = Is; IsDOP1 = D₁-like dopamine receptor 1 (ISCW001496); IsDOP2 = D₁-like dopamine receptor 2 (ISCW008775); <i>D</i>. <i>melanogaster</i> = Dm; DmD-Dop1 = D₁-like dopamine receptor (P41596); DmDAMB = D₁-like dopamine receptor (DopR99B/DAMB: AAC47161); DmDD2R = D₂-like dopamine receptor (DD2R-606: AAN15955); DmDih = diuretic hormone 44 receptor 1 (NP_610960.1); DmmAChR = muscarinic acetylcholine receptor (AAA28676); DmOAMB = octopamine receptor in mushroom bodies, isoform A (NP_732541); Dm5HT1A = serotonin receptor 1A, isoform A (AAM68432); DmTyr = tyramine receptor (CG7431: NP_650652); <i>Apis mellifera</i> = Am; AmDOP1 = D₁-like dopamine receptor (NP_001011595); AmDOP2 = D₁-like dopamine receptor (NP_001011567); AmDOP3 = D₂-like dopamine receptor (NP_001014983); AmmAChR = muscarinic acetylcholine receptor (XP_395760); AmOA1 = octopamine receptor (oar, NP_001011565); Am5HT1A = serotonin receptor (NP_001164579); AmTyr = tyramine receptor (NP_001032395.1); <i>Bombyx mori</i> = Bm; BmDOPR1 = D₁-like dopamine receptor (AB162715); BmDOPR2 = D₁-like dopamine receptor (AB162716); BmDOP3 = D₂-like dopamine receptor (XM_004925908); BmmAChR = muscarinic acetylcholine receptor (XM_004922849); BmOAR1 = octopamine receptor (NM_001098278); Bm5HTR = serotonin receptor (X95604); BmTAR2 = tyramine receptor (NM_001171178); <i>Homo sapiens</i> = h; hD1, D₁-like dopamine receptor (D(1A), NP_000785); hD2 = D₂-like dopamine receptor (D(2), NP_000786); hD3 = D₂-like dopamine receptor (D(3), NP_000787); hD4 = D₂-like dopamine receptor (D(4), NP_000788); hD5 = D₁-like dopamine receptor (D(1B)/D5,NP_000789). <b>*</b> Indicates receptors pharmacologically characterized in the current study.</p

Comparison of protein features for <i>Cq</i>DOP2 and <i>Aa</i>DOP2.

<p>The number of amino acids composing the N- and C-termini and the intracellular and extracellular loops are relative to the transmembrane domain (TM) sequences shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003515#pntd.0003515.g002" target="_blank">Fig. 2</a>.</p><p>^a<i>Aa</i>DOP2 features from [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003515#pntd.0003515.ref011" target="_blank">11</a>]</p><p>^bValues refer to the number of amino acids composing these features</p><p>^cPresumed to form a disulfide bond for protein stabilization</p><p>^dPredicted as important for binding the amine moieties of catecholamines</p><p>^eImplicated in G-protein coupling</p><p>^fPredicted to form hydrogen bonds with catechol hydroxyl groups</p><p>^gAromatic residues implicated in ligand interaction</p><p>Comparison of protein features for <i>Cq</i>DOP2 and <i>Aa</i>DOP2.</p

Characterization of select antagonists on receptor activity in HEK cells stably expressing <i>Cq</i>DOP (○), <i>Aa</i>DOP2 (●), or the human D₁ receptor (▪).

<p>Graphs are based on compiled cAMP measurements (n ≥ 3 independent experiments) normalized using GraphPad Prism software to the dopamine response for each experiment and shown as mean ± SEM.</p

Concentration response curves for <i>C</i>. <i>quinquefasciatus</i> (○) and <i>Ae</i>. <i>aegypti</i> (●) showing percent larval mortality at 72 h post exposure to DOP2 antagonists.

<p>Each data point represents mean ± SEM (n ≥ 3 independent experiments).</p