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. 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. 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.
Arthropod vectors transmit six of the 17 neglected tropical diseases (NTDs) currently recognized by the World Health Organization (WHO). Of these, the causative agents of dengue virus and lymphatic filariasis are transmitted by mosquitoes in the subfamily Culicinae (Phylum Arthropoda; Class Insecta; Family Culicidae) and exact an enormous burden on human health in tropical and subtropical regions of the globe. Aedes aegypti is the principal vector of dengue, chikungunya, and yellow fever viruses, and Culex quinquefasciatus is the vector of West Nile virus and the nematode Wuchereria bancrofti, the causative agent of lymphatic filariasis. An estimated 50–100 million dengue infections occur annually [1] and approximately 120 million people are infected with W. bancrofti [2] with additional billions at risk of contracting these and other mosquito-borne diseases. Chikungunya is an ongoing threat in Africa and Southern Asia, and a recent outbreak could potentially lead to its establishment in the Americas [3]. The WHO has established a roadmap to eradicate multiple NTDs by 2020, backed by the London Declaration on Neglected Tropical Diseases [4,5]. Achievement of this goal will require a multi-pronged, integrated approach involving new and existing vector control strategies, medicines, vaccines, and community outreach. Conventional insecticides will remain an important foundation of programs aimed at the control, elimination, and eradication of NTDs. Unfortunately the widespread development of insecticide resistant insect populations threatens continued control [6]. Vector control currently relies on a limited repertoire of active ingredients and the issue of insecticide cross-resistance is compounded by the fact that no new insecticides for insect vectors have become available for several decades [7]. In response, the Innovative Vector Control Consortium (IVCC) issued a call for three new insecticides with novel modes of action by 2023 to control malaria mosquitoes [8; http: //www. ivcc. com]. The search for chemistries with unique and pest-specific modes of action with limited environmental impact necessitates new, rational design approaches [9]. G protein-coupled receptors (GPCRs) are successful pharmaceutical targets with over one third of human drugs acting on these receptors or their downstream signaling processes [10]. Invertebrate GPCRs have long been suggested as targets for the development of new classes of insecticides [11,12]. The Purdue Insecticide Discovery Pipeline (PIDP) [13] is a GPCR-based platform established for discovery and development of novel mode-of-action insecticides for vector control [11,13,14,15]. Initially the PIDP is pursuing small molecule antagonists and agonists of invertebrate dopamine receptors (DARs) (Fig. 1) and has demonstrated proof of concept in the Ae. aegypti DAR system [11,13]. Vertebrate and invertebrate DARs are biogenic amine receptors in the Class A rhodopsin-like subfamily of GPCRs. DARs have been implicated in several neurological diseases of humans such as Parkinson' s disease and schizophrenia. Scientific investment in human DAR pharmacology and associated therapeutic interventions [16,17] provides a much needed foundation to drive equivalent discovery work in arthropod systems. Dopamine has a role in locomotion, learning, courtship, development, and several other complex behaviors in invertebrates [see 18,19, and 11 for an overview]. Several studies suggest that interference with dopaminergic processes may cause insect death or result in a variety of phenotypes such as incapacitation and disrupted development [11,13,15,20,21] that are highly attractive for insecticide development. The rational design of invertebrate DAR- and other GPCR-targeting chemistries could generate highly effective molecules for vector control. Invertebrates typically possess two D1-like receptors (Gαs coupled) and a single D2-like receptor (Gαi/o coupled) [19,22]. One of the D1-like DARs, hereafter referred to as DOP2, exhibits high amino acid sequence identity among the arthropods Ae. aegypti, Anopheles gambiae (African malaria mosquito), C. quinquefasciatus (northern house mosquito), Ixodes scapularis (Lyme disease tick), Drosophila melanogaster (fruit fly), and Apis mellifera (honey bee) across the transmembrane (TM) spanning domains (>70%) but limited sequence identity to the human D1-like DARs, hD1 and hD5 (<55%) [13]. Two D1-like DAR sequences, AaDOP1 and AaDOP2, were identified in the genome of the yellow fever mosquito, Ae. aegypti [23]. Assays using AaDOP1 or AaDOP2 expressing cells revealed elevated cAMP levels following exposure to dopamine in a concentration-dependent manner, providing support for the classification of these receptors [11]. Subsequently, the discovery of receptor antagonists with mosquitocidal properties was demonstrated by screening chemical libraries for AaDOP2 antagonists in cell-based assays [11,13]. Antagonists were evaluated in hit-to-lead studies that showed multiple compounds were selective for the AaDOP2 target versus hD1 and caused rapid and high mortality of Ae. aegypti larvae in vivo. Two D1-like DARs, CqDOP1 and CqDOP2 were identified from the assembled genome of C. quinquefasciatus [24] with CqDOP2 identified as the ortholog to AaDOP2 on the basis of amino acid similarity [13]. AaDOP2 and CqDOP2 provide an opportunity to address questions of relevance to insecticide discovery, namely (1) does sequence similarity between orthologous targets equate to conservation of pharmacological properties in vitro, (2) is sequence similarity predictive of the toxicity of target inhibitors in vivo, and (3) can differences in sequence between targets be exploited for development of taxon-selective chemistries? Here we present the first study to assess conservation in the molecular and pharmacological properties of orthologous dopamine receptors from species representing two of the most important mosquito genera affecting human health, Culex and Aedes. The human receptor, hD1, was evaluated in parallel to determine the relative potency and selectivity of DAR antagonists for mosquitoes versus humans. AaDOP2 antagonists were evaluated for toxicity to larvae of C. quinquefasciatus to explore the potential of extending the DAR antagonist-based insecticide discovery approach to this vector. Lastly, in silico analyses were conducted to evaluate DOP2 targets from three additional dipteran vectors of NTDs for inclusion in the PIDP; the An. gambiae mosquito, a vector of malaria, the Phlebotomus papatasi sand fly vector of leishmaniasis, and the Glossina morsitans tsetse fly vector of Human African Trypanosomiasis (sleeping sickness).
Mosquito larvae of the Liverpool strain IB12 of Ae. aegypti and the Johannesburg strain of C. quinquefasciatus were reared in an insectary on a 12 h day/night cycle at 75% RH at 28°C in 25 x 40 cm plastic pans (150 larvae per pan) on hamster pellets (Ae. aegypti) or ground flake fish food (C. quinquefasciatus). Adult Ae. aegypti and C. quinquefasciatus that eclosed under this rearing regimen had an average wing length of 3. 4 mm (measured as in [25]) and 3. 2 mm (measured as in [26]), respectively, suggesting diet was sufficient and larval crowding effects were minimal. The amino acid sequences of DARs from multiple arthropods were used to search the C. quinquefasciatus genome using the Basic Local Alignment Search Tool (tBLASTn, [27]). Gene models were confirmed by sequencing of amplified RT-PCR products following procedures described in Meyer et al [11]. Briefly, total RNA was extracted from adult C. quinquefasciatus females and treated with RNase-free DNase. RT-PCR amplification was performed using the SuperScript One-Step RT-PCR kit (Invitrogen, Carlsbad, CA) and the CqDOP2-specific primers CqDOP2_1F: 5' -ATGATGACTACGAATGCAACTGATTAC-3' and CqDOP2_1R: 5' -CTAAATGTACGTCTGCTCGCAC-3' . RT-PCR products separated by electrophoresis on a 1% agarose gel were excised and cloned using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA). Purified plasmids from the resulting clones were sequenced at the Purdue Genomics Core Facility (PGFC). DNA sequences were used to predict full-length coding regions and manual annotation was performed using Artemis software (version 9) [28]. The CqDOP2 conceptual protein sequence was aligned to that of AaDOP2 using ClustalW [29] and used to identify conserved amino acid residues and predict protein structural features [22,30,31]. To determine receptor expression in different life stages, total RNA was isolated from C. quinquefasciatus eggs, L4 larvae, pupae, and 5-day old adults (female and male) using the RNeasy Mini Kit (Qiagen) and following kit protocols, including DNase treatment. Generation of cDNA was performed using the iScript cDNA Synthesis Kit (Bio-Rad). Reverse transcription polymerase chain reaction (RT-PCR) was used with cDNA template, primers CqDOP2_1F and CqDOP2. 2R: 5' -CCAGCAGTGGAAGATAGAACG-3' , Taq polymerase (Phusion, New England Biolabs) and the following thermo-cycling conditions: 35 cycles at 94°C, 45 s; 55°C, 45 s; 72°C, 2 min. Subsequent products were separated by gel electrophoresis and photographed (EpiChemi II Darkroom, UVP Laboratory Products). Products of approximately 700 bp in length were excised, purified (MinElute Gel Extraction Kit, Qiagen), and sequenced at the PGCF. A neighbor-joining phylogenetic analysis was conducted using amino acid sequences of arthropod and mammalian GPCRs retrieved from GenBank. MEGA6 [32] was used to align and perform tree construction according to the procedure of Hall [33]. Diuretic hormone 44 receptor 1 (D. melanogaster) was used as an outgroup. Bootstrap analysis (1000 replicates) was performed as an estimate of branch reliability. To assess the potential of expanding the PIDP pipeline to orthologous DOP2 targets from a range of key dipteran vectors, additional tBLASTn searches of the assembled genomes of An. gambiae, P. papatasi (www. vectorbase. org) and G. morsitans [34] were performed using AaDOP2 and CqDOP2 sequences. The conceptual amino acid sequences for the resultant gene models, AgDOP2, PpDOP2, and GmDOP2, were aligned with AaDOP2 and CqDOP2 using ClustalW [29] and conserved structural features were predicted as described above. For functional characterization of the mosquito receptors, CqDOP2 and AaDOP2 were synthesized by Genscript (Piscataway, NJ) and cloned into the expression vector pcDNA3. 1+ (Invitrogen, Carlsbad, CA). Stable cell lines expressing the receptors in HEK293 cells were generated as described previously for AaDOP2 [11]. The AaDOP2 expressing cells used here were from the same clone previously utilized for AaDOP2 characterization [11]. Briefly, HEK293-CRELuc cells were plated in Dulbecco' s modified eagle' s medium (DMEM) supplemented with 5% bovine calf serum, 5% fetal clone I (Thermo Scientific, Waltham MA), 1% Antibiotic-Antimycotic (Life Technologies, Grand Island NY) and 2 μg/ml puromycin (Sigma-Aldrich, St. Louis, MO), transfected, and then subjected to selection with G418 (600 μg/ml). G418-resistant clones were selected and screened for receptor expression in the cAMP activated luciferase (CRELuc) reporter cell line construct [11]. For pharmacological characterization of receptor activity, cryopreserved cells stably expressing AaDOP2 (10,000 cells/well), CqDOP2 (5,000 cells/well), or human D1 (5,000 cells/well) were thawed, washed, and re-suspended in assay buffer (Hank’s balanced salt solution, Hyclone, Logan, UT) supplemented with 20 mM 2-[4- (2-hydroxyethyl) piperazin-1-yl]ethanesulfonic acid (HEPES, Hyclone, Logan, UT) and 0. 1% bovine serum albumin (MP Biomedicals, Santa Ana, CA), seeded in white 384 well plates (PerkinElmer, Waltham, MA), and incubated for 1 h at 37°C. Compounds were serially diluted in assay buffer containing 3-isobutyl-1-methylxanthine (IBMX, final concentration 0. 5 mM), added to the plates, and incubated for 1 h at 25°C to allow for cAMP accumulation. Reactions were stopped and cAMP was measured by a homologous time-resolved fluorescence (HTRF) assay according to the manufacturer’s recommendations (Cisbio, Bedford, MA). Fluorescence was read on a Synergy4 plate reader (BioTek, Winooski, VT). Dopamine hydrochloride, histamine dihydrochloride, 5-hydroxytryptamine hydrochloride (serotonin), (±) -octopamine hydrochloride, tyramine hydrochloride (Sigma-Aldrich, St. Louis, MO), (-) -epinephrine bitartrate, and L (-) -norepinephrine bitartrate (Research Biochemical International, Natick, MA) were used for initial receptor characterization studies. Antagonist profiles were generated by adding serially diluted antagonists followed by dopamine (3 μM for AaDOP2- and CqDOP2-, and 0. 5 μM for hD1-expressing cells). The antagonists amitriptyline hydrochloride, asenapine maleate, (±) butaclamol hydrochloride, chlorpromazine hydrochloride, doxepin hydrochloride, cis- (Z) -flupenthixol dihydrochloride, SCH23390 hydrochloride (Sigma-Aldrich, St. Louis, MO), and amperozide hydrochloride (Tocris bioscience, Ellisville, MO) were selected based on previous chemical screens against AaDOP2 and subsequent bioassays against Ae. aegypti larvae [11,13]. All serial dilutions were carried out using the Precision liquid handling station (BioTek, Winooski, VT). Data were collected from a minimum of three independent experiments conducted in duplicate. Statistical analysis of data was conducted with GraphPad Prism 6 software (GraphPad Software Inc. , San Diego, CA). A panel of nine AaDOP2 antagonists was selected based on toxicity determined from single-point dose, high-throughput screens against Ae. aegypti larvae [13,15]. Compounds were evaluated in parallel in concentration-response assays against third-instar larvae (L3) of C. quinquefasciatus and Ae. aegypti at room temperature (23–25°C). Larvae were transferred using a plastic pipette to the wells of a 24-well tissue culture plate (Corning Inc. , Corning NY) (five larvae per well) containing 1 ml de-ionized water and 400,200,100,50, or 25 μM test compound or water only as the control. Antagonists were diluted in water to the desired concentration immediately before transfer to tissue culture plates. Larval mortality was determined every 30 min for the first 3 h, then daily at 24,48, and 72 h post-exposure. Plates were gently shaken and larvae were lightly touched with a sterile probe (up to three times, as required) and stringent criteria were established for scoring such that larvae that failed to respond to both stimuli were recorded as dead. Four technical replicates were performed per dose, and each bioassay was performed a minimum of three times. Calculations of lethal concentration 50 (LC50) and lethal time 50 (LT50) were made using GraphPad Prism 6 software (GraphPad Software Inc. , San Diego, CA).
A 1,440 bp sequence encoding the predicted open reading frame of CqDOP2 was identified from cloned RT-PCR products (Genbank ID KM262648). Alignment of the conceptual CqDOP2 amino acid sequence with that of AaDOP2 showed high overall amino acid sequence identity (94. 7%) and 100% identity in the predicted TM-spanning domains (Fig. 2). The greatest divergence between the two sequences was observed in the third intracellular loop (ILIII) with nine amino acid differences and two additional amino acids in CqDOP2, and in the N-terminus with seven differences, and two additional amino acids in CqDOP2 and one additional residue in AaDOP2. The remaining amino acid differences were primarily conservative substitutions. CqDOP2 possesses key biochemical features considered essential for GPCR function and that were also identified in AaDOP2 (Table 1). Residues D140, S225, and S228 of CqDOP2 are predicted to interact directly with biogenic amines [19] while the" DRY" motif (residues 157–159), and aspartate in TMII (D105) are required for receptor activation. CqDOP2 also possesses several putative palmitoylation and phosphorylation sites. Sequences from ~700 bp RT-PCR products amplified from C. quinquefasciatus eggs, L4 larvae, pupae or adult male or female cDNA (S1 Fig) matched that of the expected region of CqDOP2, confirming the presence of CqDOP2 transcripts in the life stages examined. Neighbor-joining sequence analysis (Fig. 3) placed CqDOP2 in a clade with other invertebrate D1-like DOP2 receptors from Ae. aegypti (AaDOP2), Ap. mellifera (AmDOP2), B. mori (BmDOPR2), D. melanogaster (DmDAMB), and I. scapularis (IsDOP2). This group formed part of a larger clade comprising invertebrate octopamine receptors (DmOAMB, BmOAR1, and AmOA1), but not human DARs. The analysis also revealed a second cluster comprising other invertebrate D1-like receptors, including IsDOP1, AmDOP1, BmDOPR1, DmD-DOP1, and AaDOP1 and the human DARs, hD1 and hD5. The invertebrate D2-like receptor sequences from Ap. mellifera (AmDOP3), B. mori (BmDOPR3), and D. melanogaster (DmDD2R) were placed in a cluster with CqDOP3 and AaDOP3 and formed part of a larger clade with the human D2-like receptors hD2, hD3, and hD4. Sequences containing putative DOP2 coding regions were identified from An. gambiae (AgDOP2), P. papatasi (PpDOP2), and G. morsitans (GmDOP2) using tBLASTn searches. Percentage identity of amino acid sequences relative to AaDOP2 were as follows: AgDOP2 = 82. 6%; PpDOP2 = 81. 3%; GmDOP2 = 79. 0%. Alignment revealed preservation of aspartate and serine residues predicted to bind biogenic amines and key aspartate residues and the DRY motif required for receptor activation (S2 Fig). Putative palmitoylation and phosphorylation sites were also preserved with the exception of residue K426 in P. papatasi. Amino acid sequences were most divergent between species in the N-terminus and in ILIII. Although we have previously reported partial characterization of AaDOP2 using a luciferase-based system [11], we evaluated CqDOP2 and AaDOP2 here in parallel using a HTRF-based cAMP assay (Cisbio, Bedford, MA) to avoid assay-induced bias. Both CqDOP2 and AaDOP2 responded to dopamine, with EC50 values of 2. 3 and 1. 7 μM, respectively (Fig. 4). For both receptors, epinephrine and norepinephrine elicited an increase in cAMP, however, these biogenic amines were much less potent having EC50 values at least 10-fold higher than that of dopamine (Table 2). Treatment with histamine, octopamine, serotonin, or tyramine did not cause a measurable response in activity for either receptor. The dopamine-stimulated activity of CqDOP2 and AaDOP2 in response to a subset of AaDOP2 and hD1 antagonists mirrored each other, but differed from those observed for hD1 (Fig. 5, Table 3). Amitriptyline, asenapine, amperozide, chlorpromazine, and doxepin were markedly more potent at both mosquito receptors over the human receptor. In contrast, the D1 antagonists (±) butaclamol and SCH23390 were approximately 60-fold and 500-fold more selective for the human receptor over the mosquito receptors, respectively. To test the in vivo activity of select antagonists, concentration-response assays were conducted against L3 larvae of C. quinquefasciatus and Ae. aegypti. All DAR antagonists elicited ≥70% mortality of Ae. aegypti and C. quinquefasciatus larvae by 72 h at 400 μM, the highest dose tested (Fig. 6). The LC50 values at 72 h ranged from 41 to 208 μM depending on antagonist and mosquito species (Table 4). Chlorprothixene was the most toxic compound, eliciting the lowest LC50 values in both species (41±7 μM for C. quinquefasciatus and 62±9 μM for Ae. aegypti) and the lowest LT50 values (13. 9±2. 0 h for C. quinquefasciatus and 22. 2±3. 2 h for Ae. aegypti) (Table 4). Mortality to antagonists in both species was similar, although C. quinquefasciatus larvae were slightly more susceptible than Ae. aegypti larvae, having lower LT50 and LC50 values (Fig. 6, Table 4). Four compounds (chlorprothixene, chlorpromazine, methiothepin, and mianserin) caused >70% mortality in C. quinquefasciatus within the first 24 h (S3 Fig), and all but amitriptyline caused >70% mortality by 48 h (S4 Fig). In Ae. aegypti, >70% mortality was observed after 48 h for five compounds (chlorprothixene, chlorpromazine, methiothepin, mianserin, and asenapine) (S4 Fig).
This study provides evidence for CqDOP2 as a D1-like DAR and supports an orthologous relationship to AaDOP2. Within the mosquito subfamily Culicinae, tribes Aedini (including Ae. aegypti) and Culicini (including C. quinquefasciatus) are thought to have diverged in the early Cretaceous, an estimated 127 to 158 million years ago [41], yet the degree of sequence and pharmacological similarity, coupled with the detection of transcripts in all life stages, suggests that CqDOP2 and AaDOP2 play a conserved role in mosquito neurological processes and may explain the similar toxic effects of DAR antagonists to the larvae of C. quinquefasciatus and Ae. aegypti. These findings are an important step towards understanding the relationship between sequence similarity, DAR pharmacology in vitro and antagonist toxicity in vivo. Future mutagenesis and modeling work with these receptors may reveal key DAR structural features required for activity that may be used to predict the pharmacology of orthologous targets. This information could be used to assess the value of de-orphanization and development of additional orthologous targets, which represents a considerable time and cost investment. DAR antagonist toxicity to C. quinquefasciatus suggests these molecules may have activity against other culicine species and possibly other arthropod vectors. The three additional DOP2 targets, AgDOP2, PpDOP2 and GmDOP2, identified in this study (Figs. 1, S2) provide a powerful research tool to investigate DAR antagonist potency to other dipteran vectors and the pharmacological consequences of divergence in amino acid sequences in these species. Importantly, our finding that CqDOP2 and AaDOP2 are pharmacologically distinct from the human D1-like DAR, hD1, and exhibit a 35 to 227-fold range difference in response to select antagonists suggests that differences between mosquito and human DARs can be exploited for development of mosquito-selective compounds with low mammalian toxicity. The DAR antagonists tested in this study are tricyclic amines, with the exception of amperozide, a diphenylbutylpiperazine. Modeling of mosquito receptors may allow prediction of ligand binding sites and facilitate directed searches for small molecule inhibitors, potentially revealing new classes of DAR antagonists selective for mosquitoes. Agonists also have potential to disrupt AaDOP2 signaling [13] and should be examined against CqDOP2 and the DOP2 DAR targets of other dipteran vectors identified in this study. The rational, target-based insecticide discovery approach taken in this study provides further evidence that investment in small molecule antagonists of invertebrate GPCRs may deliver much needed novel mode-of-action products to the vector control market.
New mode-of-action insecticides are required to control arthropod vectors of neglected tropical diseases (NTDs). Rational drug design approaches offer attractive methods to identify new insecticidal chemistries that are potent and selective for molecular targets of arthropod vectors. Previously identified antagonists of a D1-like dopamine receptor (DAR) from the yellow fever mosquito, Aedes aegypti were toxic to the larvae of this species and are candidate novel insecticide leads. Building on this work, here we evaluated the molecular and pharmacological characteristics of an orthologous DAR from Culex quinquefasciatus, the vector of lymphatic filariasis and West Nile virus. We show that orthologous mosquito DARs have similar pharmacological profiles in vitro and that Ae. aegypti-active DAR antagonists are toxic to C. quinquefasciatus larvae in vivo. Sequence similarity between orthologous targets can be indicative of DAR target potential for discovery of potent, selective inhibitors. These findings justify expansion of insecticide discovery efforts to orthologous DARs from additional dipteran vectors of NTDs and provide support for DAR antagonists as a new class of chemistries for taxon-selective insecticides for vector control.
Dopamine Receptor Antagonists as New Mode-of-Action Insecticide Leads for Control of Aedes and Culex Mosquito Vectors