A “Genome-to-Lead” Approach for Insecticide Discovery: Pharmacological Characterization and Screening of <em>Aedes aegypti</em> D₁-like Dopamine Receptors

<div><h3>Background</h3><p>Many neglected tropical infectious diseases affecting humans are transmitted by arthropods such as mosquitoes and ticks. New mode-of-action chemistries are urgently sought to enhance vector management practices in countries where arthropod-borne diseases are endemic, especially where vector populations have acquired widespread resistance to insecticides.</p> <h3>Methodology/Principal Findings</h3><p>We describe a “genome-to-lead” approach for insecticide discovery that incorporates the first reported chemical screen of a G protein-coupled receptor (GPCR) mined from a mosquito genome. A combination of molecular and pharmacological studies was used to functionally characterize two dopamine receptors (<em>Aa</em>DOP1 and <em>Aa</em>DOP2) from the yellow fever mosquito, <em>Aedes aegypti</em>. Sequence analyses indicated that these receptors are orthologous to arthropod D₁-like (Gα_s-coupled) receptors, but share less than 55% amino acid identity in conserved domains with mammalian dopamine receptors. Heterologous expression of <em>Aa</em>DOP1 and <em>Aa</em>DOP2 in HEK293 cells revealed dose-dependent responses to dopamine (EC₅₀: <em>Aa</em>DOP1 = 3.1±1.1 nM; <em>Aa</em>DOP2 = 240±16 nM). Interestingly, only <em>Aa</em>DOP1 exhibited sensitivity to epinephrine (EC₅₀ = 5.8±1.5 nM) and norepinephrine (EC₅₀ = 760±180 nM), while neither receptor was activated by other biogenic amines tested. Differential responses were observed between these receptors regarding their sensitivity to dopamine agonists and antagonists, level of maximal stimulation, and constitutive activity. Subsequently, a chemical library screen was implemented to discover lead chemistries active at <em>Aa</em>DOP2. Fifty-one compounds were identified as “hits,” and follow-up validation assays confirmed the antagonistic effect of selected compounds at <em>Aa</em>DOP2. <em>In vitro</em> comparison studies between <em>Aa</em>DOP2 and the human D₁ dopamine receptor (hD₁) revealed markedly different pharmacological profiles and identified amitriptyline and doxepin as <em>Aa</em>DOP2-selective compounds. In subsequent <em>Ae. aegypti</em> larval bioassays, significant mortality was observed for amitriptyline (93%) and doxepin (72%), confirming these chemistries as “leads” for insecticide discovery.</p> <h3>Conclusions/Significance</h3><p>This research provides a “proof-of-concept” for a novel approach toward insecticide discovery, in which genome sequence data are utilized for functional characterization and chemical compound screening of GPCRs. We provide a pipeline useful for future prioritization, pharmacological characterization, and expanded chemical screening of additional GPCRs in disease-vector arthropods. The differential molecular and pharmacological properties of the mosquito dopamine receptors highlight the potential for the identification of target-specific chemistries for vector-borne disease management, and we report the first study to identify dopamine receptor antagonists with <em>in vivo</em> toxicity toward mosquitoes.</p> </div

Neighbor-joining sequence analysis of <i>Aedes aegypti Aa</i>DOP1 and AaDOP2 and representative biogenic amine receptors.

<p>The deduced amino acid sequences for the mosquito dopamine receptors <i>Aa</i>DOP1 and AaDOP2 and additional receptors for dopamine, muscarinic acetylcholine, octopamine, serotonin, and tyramine from <i>Drosophila melanogaster</i> and <i>Apis mellifera</i>, as well as the human D₁-like and D₂-like dopamine receptors were aligned for use in the analysis. Bootstrap values (100 replicates) are indicated with numbers at supported branches. The outgroup is a <i>D. melanogaster</i> diuretic hormone receptor, a Class B GPCR. Abbreviations: <i>Aa</i> = <i>Ae. aegypti</i>; <i>Is</i> = <i>I. scapularis</i>; <i>Dm</i> = <i>D. melanogaster</i>; <i>Am</i> = <i>A. mellifera</i>; <i>Hs</i> = <i>H. sapiens</i>. Sequences: <i>Is</i>dop1, D₁-like dopamine receptor (ISCW001496); <i>Is</i>dop2, D₁-like dopamine receptor (ISCW008775); <i>Dm</i>D-Dop1, D₁-like dopamine receptor (P41596); <i>Dm</i>DAMB, D₁-like dopamine receptor (DopR99B/DAMB: AAC47161), <i>Dm</i>DD2R, D₂-like dopamine receptor (DD2R-606: AAN15955); <i>Dm</i>Dih, diuretic hormone 44 receptor 1 (NP_610960.1); <i>Dm</i>mAChR, muscarinic acetylcholine receptor (AAA28676); <i>Dm</i>OAMB, octopamine receptor in mushroom bodies, isoform A (NP_732541); DM5HT1A, serotonin receptor 1A, isoform A (NP_476802); <i>Dm</i>Tyr, tyramine receptor (CG7431: NP_650652); <i>Am</i>DOP1, D₁-like dopamine receptor (dopamine receptor, D1, NP_001011595); <i>Am</i>DOP2, D₁-like dopamine receptor (dopamine receptor 2, NP_001011567), <i>Am</i>DOP3, D₂-like dopamine receptor (<i>Am</i>DOP3, NP_001014983); <i>Am</i>mAChR, muscarinic acetylcholine receptor (XP_395760); <i>Am</i>OA1, octopamine receptor (oar, NP_001011565); <i>Am</i>5HT1A, serotonin receptor (5ht-1, NP_001164579); <i>Am</i>Tyr, tyramine receptor (XP_394231); <i>Hs</i>D1, D₁-like dopamine receptor (D(1A), NP_000785); <i>Hs</i>D2,D₂-like dopamine receptor (D(2), NP_000786); <i>Hs</i>D3, D₂-like dopamine receptor (D(3), NP_000787); <i>Hs</i>D4, D₂-like dopamine receptor (D(4), NP_000788); <i>Hs</i>D5, D₁-like dopamine receptor (D(1B)/D5, NP_000789).</p

Drug discovery and development pipeline for new insecticidal chemistries.

<p><b>A:</b> The illustration shows critical steps involved with the “genome-to-lead” (described in this manuscript) and “lead-to-product” phases. Abbreviations: (EPA) Environmental Protection Agency; (FDA) Food and Drug Administration; (SAR) structure-activity relationship study. The intended administration route of a particular chemistry dictates the federal agency that will receive the registration package; <b>B:</b> Expanded details of the “hit-to-lead” phase including those pursued in this study.</p

Confirmation and secondary assays for “hit” antagonists of <i>Aa</i>DOP2 and human D₁ receptor.

<p>Select chemistries and the assay control (SCH23390) were tested in dose-response cAMP assays in the presence of 3 µM dopamine in <i>Aa</i>DOP2- or 100 nM dopamine in hD₁-expressing cells (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001478#pntd-0001478-g005" target="_blank">Figure 5</a>). Compounds with IC₅₀ values ≥10 µM are considered to lack activity at <i>Aa</i>DOP2 and were not tested at hD₁. N.D. = not determined; hD₁ = Human D₁ dopamine receptor.</p

Alignment of the complete <i>Aedes aegypti Aa</i>DOP1 and <i>Aa</i>DOP2 amino acid sequences.

<p>Highlighted areas designate residues with shared biochemical characteristics, as designated by the ClustalW <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001478#pntd.0001478-Chenna1" target="_blank">[33]</a> output, where black shading = identical residues; dark shading = strongly similar residues; light shading = weakly similar residues. Also noted are the residues composing the N- and C-termini and the transmembrane (TM) domains I–VII.</p

Responses of <i>Aa</i>DOP1 and <i>Aa</i>DOP2 to biogenic amines and synthetic dopamine receptor agonists.

<p>HEK293 cells stably expressing both a CRELuc reporter construct and either of the receptors were stimulated with potential agonists. Dose-response curves were plotted and the EC₅₀ values were calculated. Compounds with EC₅₀ values ≥10 µM are considered to lack intrinsic activity at <i>Aa</i>DOP2.</p

Pharmacological characterization of the <i>Aedes aegypti Aa</i>DOP1 and <i>Aa</i>DOP2 receptors.

<p>The mosquito receptors were stably expressed in HEK 293-CRELuc cells for dose-response assays and determination of EC₅₀ values (shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001478#pntd-0001478-t001" target="_blank">Table 1</a>). <b>A, C</b>: <i>Aa</i>DOP1, <b>B, D</b>: <i>Aa</i>DOP2. Representative curves for <b>A, B</b>: biogenic amines; <b>C, D</b>: synthetic dopamine receptor agonists; <b>E</b>: Inhibitory effect of 10 µM SCH23390 in the presence of 1 µM dopamine (n = 4) shown for both mosquito dopamine receptors. ** <i>p</i><0.01; *** <i>p</i><0.001; <b>F</b>: Dose-response curve of dopamine for <i>Aa</i>DOP2 in the absence or presence of 10 µM SCH23390 used to identify an appropriate “signal window” for chemical library screening. The concentration of dopamine selected for screening (300 nM) is indicated with a box. CPS = counts per second; M = molarity.</p

Dose-response curves for selected screen “hit” compounds that exhibited antagonistic effects on <i>Aa</i>DOP2.

<p>Direct cAMP accumulation assays were used for dose-response assays and determination of IC₅₀ values for SCH23390 (antagonist control) and seven <i>Aa</i>DOP2 antagonists (shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001478#pntd-0001478-t003" target="_blank">Table 3</a>) identified in the chemical library screen.</p

Toxicity of antagonist screen hits in <i>Ae. aegypti</i> larval bioassays.

<p><b>A: </b><i>Ae. aegypti</i> larval bioassay showing toxicity of amitriptyline and doxepin at a single dose point (400 µM) compared to the water control; Ami = amitriptyline, Dox = Doxepin; * indicates <i>p</i><0.05; <b>B: </b><i>Ae. aegypti</i> larval bioassay involving amitriptyline in a dose-response format (25 µM–400 µM).</p

Standardized Metadata for Human Pathogen/Vector Genomic Sequences

<div><p>High throughput sequencing has accelerated the determination of genome sequences for thousands of human infectious disease pathogens and dozens of their vectors. The scale and scope of these data are enabling genotype-phenotype association studies to identify genetic determinants of pathogen virulence and drug/insecticide resistance, and phylogenetic studies to track the origin and spread of disease outbreaks. To maximize the utility of genomic sequences for these purposes, it is essential that metadata about the pathogen/vector isolate characteristics be collected and made available in organized, clear, and consistent formats. Here we report the development of the GSCID/BRC Project and Sample Application Standard, developed by representatives of the Genome Sequencing Centers for Infectious Diseases (GSCIDs), the Bioinformatics Resource Centers (BRCs) for Infectious Diseases, and the U.S. National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health (NIH), informed by interactions with numerous collaborating scientists. It includes mapping to terms from other data standards initiatives, including the Genomic Standards Consortium’s minimal information (MIxS) and NCBI’s BioSample/BioProjects checklists and the Ontology for Biomedical Investigations (OBI). The standard includes data fields about characteristics of the organism or environmental source of the specimen, spatial-temporal information about the specimen isolation event, phenotypic characteristics of the pathogen/vector isolated, and project leadership and support. By modeling metadata fields into an ontology-based semantic framework and reusing existing ontologies and minimum information checklists, the application standard can be extended to support additional project-specific data fields and integrated with other data represented with comparable standards. The use of this metadata standard by all ongoing and future GSCID sequencing projects will provide a consistent representation of these data in the BRC resources and other repositories that leverage these data, allowing investigators to identify relevant genomic sequences and perform comparative genomics analyses that are both statistically meaningful and biologically relevant.</p></div