Dopamine receptor antagonists as new mode-of-action insecticide leads for control of Aedes and Culex mosquito vectors

Abstract

Background: 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, Aedes aegypti, with toxicity to Ae. aegypti larvae as leads for novel insecticides. To extend DAR-based insecticide discovery, we evaluated the molecular and pharmacological characteristics of an orthologous DAR target, CqDOP2, from Culex quinquefasciatus, the vector of lymphatic filariasis and West Nile virus.

Methods/results: CqDOP2 has 94.7% amino acid identity to AaDOP2 and 28.3% identity to the human D1-like DAR, hD1. CqDOP2 and AaDOP2 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 CqDOP2 and AaDOP2 in comparison to hD1. Antagonists were toxic to both C. quinquefasciatus and Ae. aegypti larvae, with LC50 values ranging from 41 to 208 μM 72 h post-exposure. Orthologous DOP2 receptors identified from the African malaria mosquito, Anopheles gambiae, the sand fly, Phlebotomus papatasi and the tsetse fly, Glossina morsitans, had high sequence similarity to CqDOP2 and AaDOP2.

Conclusions: DAR antagonists represent a putative new insecticide class with activity against C. quinquefasciatus and Ae. aegypti, 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 in vivo 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.

Fig 1. Schematic depicting PIDP activities aimed at discovery of D₁-like DAR antagonists as new insecticides.

The workflow is based on the evolving “genome-to-lead” component of the PIDP first described in Meyer et al. [11]. 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 in vivo 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 CqDOP2 target, the AgDOP2, PpDOP2 and GmDOP2 targets identified from assembled genome sequences (S2 Fig), and the C. quinquefasciatus larval screen. Remaining components are the subject of works in review [15] and ongoing efforts. Abbreviations: Aa, Aedes aegypti; Ag, Anopheles gambiae; Am, Apis mellifera; Cq, Culex quinquefasciatus; Gm, Glossina morsitans; Is, Ixodes scapularis; Pp, Phlebotomus papatasi; NP, natural product.

Fig 2. Alignment of CqDOP2 and AaDOP2 amino acid sequences.

Highlighted areas indicate identical and conserved residues as designated by ClustalW [29]: black = identical residues; dark gray = strongly similar residues; light gray = weakly similar residues (for amino acid similarity groups, see: http://www.clustal.org/download/clustalx_help.html). Putative transmembrane (TM) domains I-VII are indicated as a line above the alignment.

Fig 3. Neighbor-joining sequence analysis of CqDOP2, AaDOP2 and representative biogenic amine receptors.

Abbreviations and NCBI accession numbers of species indicated are as follows: Aedes aegypti = Aa; AaDOP1 = D₁-like dopamine receptor 1 (JN043502); AaDOP2 = D₁-like dopamine receptor 2 (JN043503); AaDOP3 = D₂-like dopamine receptor (XM_001648573); Culex quinquefasciatus = Cq; CqDOP1 = D₁-like dopamine receptor 1 (XM_001842358); CqDOP2 = D₁-like dopamine receptor 2 (KM262648); CqDOP3 = D₂-like dopamine receptor (XM_001865540); Ixodes scapularis = Is; IsDOP1 = D₁-like dopamine receptor 1 (ISCW001496); IsDOP2 = D₁-like dopamine receptor 2 (ISCW008775); D. melanogaster = 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); Apis mellifera = 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); Bombyx mori = 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); Homo sapiens = 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). * Indicates receptors pharmacologically characterized in the current study.

Fig 4. Pharmacological characterization of AaDOP2 and CqDOP2 stably expressed in HEK293 cells.

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.

Fig 5. Characterization of select antagonists on receptor activity in HEK cells stably expressing CqDOP (○), AaDOP2 (●), or the human D₁ receptor (▪).

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.

Fig 6. Concentration response curves for C. quinquefasciatus (○) and Ae. aegypti (●) showing percent larval mortality at 72 h post exposure to DOP2 antagonists.

Each data point represents mean ± SEM (n ≥ 3 independent experiments).