Potential of GPCR-Targeting Insecticides for Control of Arthropod

Chapter 4

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Potential of GPCR-Targeting Insecticides for Control of Arthropod Vectors Shruti Sharan1 and Catherine A. Hill*,1,2 1Department

of Entomology, Purdue University, 901 W. State Street, West Lafayette, Indiana 47907-2089, United States 2Purdue Institute for Inflammation, Immunology and Infectious Diseases, Purdue University, West Lafayette, Indiana 47907-2089, United States *E-mail: [email protected]. Telephone: +1 765 496-6157.

Resistance to conventional, small molecule insecticides threatens global efforts to control arthropod-borne infectious diseases. New insecticidal chemistries with novel modes of action and activity against resistant pest populations are needed as replacement products. Small molecule insecticides that disrupt G protein-coupled receptor (GPCR) targets in the arthropod have been proposed for development of next generation insecticides. The potential of such products is considered high for several reasons. Firstly, GPCRs are the targets of multiple human pharmaceuticals. Approximately 40% of human drugs act at human GPCRs, highlighting the therapeutic value of this target class. Secondly, formamidine insecticides, reported to operate via agonism of the invertebrate octopamine receptor, serve as proof of concept for GPCR insecticides. Yet despite the perceived promise, progress towards development of GPCR insecticides has been limited. Only two insecticides acting at invertebrate GPCRs have been registered and selective, synthetic ligands for lead development are not available. Arthropod biogenic-amine binding receptors are the subject of renewed attention in insecticide discovery research, with an emphasis on octopamine, dopamine and serotonin receptors. Here we review recent progress towards development of GPCR-targeting insecticides, with a focus on new, small molecule products for control of arthropod vectors, © 2017 American Chemical Society Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

explore reasons as to the lack of products in this class, and consider factors that must be overcome to realize the goal of GPCR insecticides.


The Call for New Mode of Action Insecticides for Vector Control More than half the world’s population is at risk of infectious diseases transmitted by arthropods and one sixth of the illness and disability suffered worldwide is due to vector-borne infectious diseases such as malaria, dengue, yellow fever, Zika, leishmaniasis and filariasis (1). Vector-borne diseases account for more than 17% of all infectious diseases and cause more than one million deaths annually (2). Malaria causes more than 400,000 deaths per year, with highest mortality recorded in children less than five years of age (1). Malaria deaths have been declining since 2000, but the disease is on the rise again in some regions (3, 4). More than 2.5 billion people in over 100 countries is at risk of contracting dengue, a disease that is re-emerging in many parts of the world (1). The incidence of vector-borne diseases is increasing globally due to human population growth, habitat destruction and climate change. At the same time, resistance to traditional chemical insecticides, the primary tool used to control vector-borne diseases, is widespread in many vector populations (5–7). The ability to suppress and eradicate some diseases and to control epidemics is under threat. The need for new vector control products with activity against resistant pest populations and favorable environmental profiles is pressing. Insecticide-based vector control has been the cornerstone of every multi-national campaign against vector borne diseases to date. This approach is also essential to the proposed elimination of diseases such as malaria and filariasis, and often the only option to control epidemics and diseases for which there are no medicines and vaccines. Vector control relies on several classes of pesticides as recognized by the Insecticide Resistance Action Committee (IRAC) (8, 9), an international association of crop protection companies established in 1984 focused on ensuring the long term efficacy of products to control insects, mites and ticks. In total, IRAC recognizes 55 different classes of insecticides among 30 groups (29 known and one unknown group). Vector control programs rely primarily on four classes of chemical insecticides, namely organochlorines, organophosphates (OPs), carbamates and pyrethroids to protect public health (10). Currently pyrethroid insecticides dominate global vector control efforts, representing more than 60% of the Indoor Residual Spray (IRS) and 100% of Long-Lasting Insecticidal Net (LLINs) products, respectively (3). The use of organochlorines and OPs is on the decline due to resistance and toxicity issues although DDT, arguably the best-known organochlorine pesticide and a widely recognized environmental toxicant, is still in use to control malaria in some countries (3). More than 30 years have elapsed since the introduction of synthetic pyrethroids (SPs) and this class remains the mainstay of public health pest control. Resistance to pyrethroids in the major Anopheles species vectors of malaria is widespread (11, 12) and poses the greatest single threat to malaria control (3). Similarly, there are very real concerns that resistance to SPs in Aedes species 56 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


mosquitoes, also a widespread phenomenon (13), threatens control of arboviral diseases. The loss of the SP class leaves the global human population vulnerable to existing and new vector-borne diseases. The mode of action (MoA) classification scheme developed and endorsed by IRAC is based on the best available evidence of the MoA of an insecticide (9) i.e., knowledge of the molecular target(s) disrupted by the chemistry in the insect. Notable classes of insecticides recognized by IRAC and widely employed in pest control include SPs, OPs and carbamates, organochlorines and fiproles, neonicatinoids, spinosyns, avermectins and insect growth regulators (IGRs). Chemistries in these classes disrupt a variety of molecular targets that include the sodium channel, acetylcholinesterase enzyme, GABA-gated chloride channel, nicotinic acetylcholine receptor and the glutamate-gated chloride channel, respectively in the insect, or in the case of IGRs, act as mimics of juvenile hormone (9). In response to the resistance crisis, scientists are calling for chemistries that operate via new MoAs (i.e., at alternative orthosteric sites on known molecular targets) (3, 4). Even more radical, is the proposal to develop chemistries that act at novel molecular targets in the arthropod (14). Although traditional insecticides are expected to dominate pest control programs for the next several decades, the single insecticide approach is considered an unlikely option for sustained pest control due to resistance development. To counteract this issue, resistance management will require the employment of multiple active ingredients, each with different MoAs and demonstrated potency to the insect vector. The not for profit Innovative Vector Control Consortium (IVCC) (3) plans the release of three classes of insecticides with novel MoAs by 2019 (4). Via partnerships with agrochemical companies, IVCC has made two new anti-malarial formulations available, namely Syngenta’s non-pyrethroid Actellic CS and Bayer’s long-lasting Deltamethrin formulation K-Othrine Polyzone (3). However, as far as the authors are aware, the field of insecticide discovery is yet to realize the goal of new chemical entities that operate at new molecular targets. The formamidine class of insecticides includes the presumed octopamine receptor agonists, amitraz and chlordimeform, and is the only IRAC class thought to act via arthropod G protein-coupled receptors (GPCRs) (9). GPCRs constitute one of the largest families of proteins in vertebrate and invertebrate genomes. These transmembrane spanning signaling molecules transfer extracellular signals to the cytosol or nucleus and are activated by a wide variety of stimuli including neurotransmitters, hormones and growth factors (15). GPCRs regulate many processes such as cell proliferation, differentiation and development, and in humans, are central to many pathophysiological conditions. Based on the IRAC MoA classification, invertebrate GPCRs are an under utilized target for pesticides, but the reasons for the modest size of this product class are unclear. Here we explore progress in target-driven approaches for development of GPCR insecticides. Framing this chapter are questions regarding the key factors that have affected development of GPCR insecticides and that must be addressed to ensure the success of this product class. Given the urgent need for new vector control products, these questions are considered in the context of small molecule modifiers of biogenic amine-binding GPCRs, with a focus on new products to control arthropod vectors of infectious disease. 57 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


G Protein-Coupled Receptors (GPCRs) GPCRs are the largest family of cell surface receptors (16), and are widely distributed in life forms from bacteria to fungi and higher animals. Members of the GPCR family share the same basic architecture of seven transmembrane (7TM) α helices, an extracellular N terminus and an intracellular C terminus, connected by three intracellular (IL1, IL2, and IL3) and three extracellular loops (EL1, EL2 and EL3) (15). GPCRs interact with a diverse range of ligands that include hormones, neurotransmitters, ions, odorants and photons of light (16). Activation of the receptor by binding of a ligand triggers intracellular signaling, mediated by membrane-bound heterotrimeric G proteins, which regulate a broad range of biological processes (17–19), including regulation of andenylate cyclases, phosphoplipases and the mitogen-activated protein kinase (MAPK) pathway (15). The G proteins consist of a Gβγ monomer and a guanine diphosphate-bound Gα subunit in their in-active state. In mammals, several isoforms of each subunit are expressed, many of which have splice variants, which engenders many G protein combinations (15). Four known families of G proteins are recognized based on the sequence identity and signaling activity of the α subunit: Gαs, Gαi, Gαq/11 and Gα12/13 (Figure 1). The Gαs and Gαi/o subfamilies were named for their ability to stimulate and inhibit adenylyl cyclase (AC) isoforms, respectively. The Gαq/11 subfamily is linked to the stimulation of phospholipase Cb (PLCb) while the Gα12/13 subfamily activates the small G protein Rho pathways. Following ligand activation, GPCRs catalyze the exchange of GDP for GTP on the Gα subunit, leading to a decreased affinity of Gα for Gβγ. The resulting dissociation of the heterotrimer allows the GTP-bound Gα and free Gβγ to interact with several downstream effectors, including adenylyl cyclases, phosphodiesterases, phospholipases, tyrosine kinases and ion channels (20). The repertoire of GPCRs in many animals has been catalogued by in silico approaches. The human genome encodes more than 800 GPCRs, while approximately 1000 GPCRs were identified in the roundworm, Caenorhabditis elegans, and the cognate ligands for most of these receptors are unknown. In contrast, only two GPCR signaling pathways have been identified in the budding yeast, Saccharomyces cerevisiae (15). Arthropods typically possess hundreds of GPCRs, providing numerous candidates for target evaluation. Two hundred GPCRs were identified in the genome of the fruit fly, Drosophila melanogaster (21, 22), while some 84 and 111 GPCRs have been annotated in the genomes of the silkworm, Bombyx mori and the red flour better, Tribolium castaneum, respectively. Community annotation efforts identified 276 GPCRs in the malaria mosquito, Anopheles gambiae (23), approximately 135 receptors in the yellow fever mosquito, Aedes aegypti (24), 115 in the northern house mosquito, Culex quinquefasciatus (25), and approximately 199 receptors in the Lyme disease tick, Ixodes scapularis (26).

58 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 1. Schematic diagram showing four G protein-coupling mechanisms (A: GαS, B, Gαi: C: Gαq/11 and D, Gα12/13) recognized in mammals and corresponding cAMP, MAP/ERK, PLC/IP3 and Rho-dependent signaling pathways. Molecular components comprising these pathways are shown: G protein-coupled receptors (green), G proteins (aqua), effector molecules (orange) and enzymes (white). Coupling mechanisms identified for the the Aedes aegypti (AaDOP2), Anopheles gambiae (AgDOP2), Culex quinquefasciatus (CqDOP2) and human hD1 dopamine receptors via heterologous expression in human embryonic kidney (HEK) cell lines and functional assay, are shown. AC, andeylate cyclase, ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; DAG, diacylglycerol; ERK, extracellular signal-regulated kinase; IP1, inositol phosphate; IP3, inositol trisphosphate; MEK, mitogen activated protein kinase kinase; PKA, protein kinase A; PKC, protein kinase C; PIP2, Phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; Raf, RAF kinase; Rho, Rho GTPase, RhoGEF, Rho guanine nucleotide exchange factor.

The classification of arthropod GPCRs follows that developed for mammalian GPCRs and is loosely derived on the classification first adopted for D. melanogaster GPCRs by Brody and Cravchik (22). Human GPCRs are subdivided into five main classes on the basis of sequence homology and the GRAFS system (rhodopsin, secretin, glutamate, adhesion and frizzled/taste) (15), also referred to as classes A, B, C, D and F (27). Orthologs of the human Class A (rhodopsin-like), B (secretin-like) and C (metabo-tropic-glutamate like) 59 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


GPCRs, all of which include targets for development of therapeutic products, have been identified in arthropods. The invertebrate odorant receptors (ORs) are not discussed in this review. In early studies, these receptors were considered GPCRs based on homology to mammalian ORs. However, while mammalian ORs are bona fide GPCRs and couple to Gα proteins, subsequent studies revealed that the insect ORs are “atypical” in that they likely oligomerize with the OR “Orco” that appears to function as a ligand-gated channel (28–30).

The Argument for GPCR-Targeting Insecticides The picture that is emerging for human GPCRs is one of remarkable biochemical plasticity. In addition to their diverse ligand-binding capabilities, human GPCRs couple to a wide range of intracellular signaling molecules and effector systems, and play crucial roles in virtually every organ system (16). GPCRs mediate biological activities that include vision, smell, taste, behavioral and mood regulation, immune defense and processes controlled by the nervous and endocrine systems. GPCRs have been implicated in a multitude of human disorders and numerous diseases have been linked to mutations and polymorphisms in these receptors (16). There is growing awareness of the involvement of GPCRs in multiple disease states such as neurological and cardiac disease, cancer, and immunological disorders (31–33). It is estimated that nearly half of all modern drugs regulate GPCR activity in some way; small molecules targeting ~50 GPCRs constitute >50% of currently marketed drugs (16). Cellular location, physiological function, and abundance in the cellular membrane, are among additional factors that make human GPCRs “druggable” targets, and these receptors are under wide investigation for development of novel therapeutics. Arthropod GPCRs regulate many biological processes, including reproduction, osmoregulation, growth, development and behavior (34). These receptors are presumed to exhibit a similar level of complexity to vertebrate GPCRs in terms of ligand binding and signaling modes, yet relatively less is known regarding their pharmacology. Arthropod GPCRs have been proposed as candidates for the development of next generation pesticides since the modification of receptor function by blocking or over stimulating its actions may either result in the death of a pest or disrupt its normal fitness and/or reproductive capacity, leading to a reduction in population number and disease transmission. Theoretically, arthropod GPCRs could be targeted by synthetic small molecule and natural product formulations that act either as agonists or antagonists. Alternatives include RNA interference (RNAi)- or Crispr/Cas9-based technologies that reduce or eliminate receptors that are essential to life. Target-driven insecticide discovery is enjoying renewed interest. The availability of sequenced and annotated invertebrate genomes has facilitated the identification of thousands of gene models, and in conjunction with techniques such as proteomics, functional genomics, and knockdown technologies, has provided information on essential physiological processes that could be exploited for pest control (34). Genome assemblies for multiple species of vectors, 60 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


including mosquito vectors of malaria, arboviruses and filariasis, the tsetse fly vector of African trypanosomiasis, sand fly vectors of leishmaniasis, kissing bug vector of Chagas, and an ixodid (hard) tick vector of Lyme disease, anaplasmosis and babesiosis are available via the National Institutes of Health (NIH) funded Bioinformatics Resource Center, VectorBase (3, 35). In 2011, the i5k initiative was launched with the goal to sequence the genomes of 5,000 insects and other arthropods considered of global importance for agriculture, food security, medicine and energy productions (36). These and other “omics” initiatives are expected to provide an unparalleled resource for target identification and comparative studies of orthologous targets, including GPCRs, across a wide range of phyletic groups. Numerous studies have proposed GPCRs as targets for insecticide development (19, 20, 23, 34, 37–40). Investigations have emphasized Class A receptors that bind small molecule biogenic amines, neuropeptides and peptide hormones. Formamidines are the only insecticide class recognized by IRAC that operate via disruption of GPCR function (9). The formamidine, amitraz and its metabolite dimethylphenylmethyformamidine (DPMF) are reported to agonize the presumably arthropod specific α- and β-adrenergic-like octopamine receptors (40, 41). The search for novel control compounds that target octopamine and dopamine receptors has received significant interest, and the former target class is the subject of a recent review (42). Genome-wide RNA interference (RNAi)-based transcript knockdown studies provided target validation of dopamine and serotonin receptors in T. castaneum. Short interfering-RNA (siRNA) screens identified six GPCRs required for either larval development or ecdysis (43). Work by Regna et al. (44) using transgenic D. melanogaster provided validation of the D1-like dopamine receptor, DOP2. RNA interference (RNAi) knockdown of DOP2 transcripts resulted in significant fly lethality and results suggested a link to immune function and regulation of ecdysis.

Advances in Understanding of the Pharmacology of Arthropod GPCRs Efforts to characterize the pharmacology of arthropod receptors have largely focused on members of the biogenic amine-binding Class A receptors (17), with an emphasis on dopamine (DAR), octopamine/tyramine (OAR/TAR) and serotonin (5HT) receptors (Table 1). Biogenic amine receptors have proven amenable to cloning and expression in heterologous cell-based systems and functional assays are available to investigate receptor response to a variety of ligands. Notable studies include work on receptors from species of locust, silkworm, honey bee, fruit fly, cockroach, crickets, mosquitoes and ticks (38, 41, 45–52). Biogenic amines are derivatives of aromatic amino acids, and regulate a variety of behavioral and physiological processes, including locomotion, aggression, circadian rhythm, cardio-vascular control, learning and memory in invertebrates (53, 54). In contrast, the systems biology of biogenic-amine signaling in arthropods is a field in its infancy. Biogenic amines are also important 61 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


chemical messengers during embryonic and larval development, and synaptic organization of the brain. Dopamine and serotonin play fundamental roles in modulation of salivary gland function, reproduction, developmental (55–57), learning and memory (58), and diuresis (59). Octopamine and tyramine, the decarboxylation products of tyrosine, are functional counterparts of the vertebrate adrenergic transmitters epinephrine and norepinephrine (54). Octopamine plays key roles in modulating muscle activity in locusts, learning and memory in honeybees and fruit flies and stress response in crickets (60). The invertebrate octopaminergic system has received considerable research attention; detection of trace amounts of octopamine in mammals (58) has lead to speculation that the pathway is unique to arthropods. Notable studies include work on membrane preparations, neurons and cloned receptors in a number of invertebrate species (41, 44, 46, 49, 61–63). Octopamine functions in insects as a neurotransmitter, neurohormone and neuromodulator (20) and interacts with the invertebrate “α-adrenergic-like” (OctaR), “β-adrenergic-like” (OctbR), and “octopamine/tyramine” receptors (Oct/TyrR) which were identified based on their similarities in structure and in signaling properties to vertebrate adrenergic receptors (64). Pharmacological characterization of OARs and TARs is performed using agonists and antagonists identified from studies of the mammalian adrenergic system. Some progress has been made towards the identification of novel chemical entities active at vector OAR targets. Six candidate OAR/TAR genes were identified in the genome of the malaria mosquito, Anopheles gambiae and two OARs were cloned and functionally characterized by Kastner et al (49). Subsequent virtual screening using a homology model for one OAR enabled the identification of several agonist and antagonists, some of which exhibited toxicity to mosquito larvae. While octopamine receptor(s) are considered the primary target of formamidine metabolites, questions remain as to the role of OAR and TAR receptors in determining insecticidal effect. The interplay between OARs and TARs in response to formamidines has been investigated in the Bombyx mori (silkworm) system (41, 45, 46, 65–68). Functional assays involving the B. mori α-adrenergic-like OAR suggest that elevation of intracellular cAMP rather than Ca2+ mobilization might account for the insecticidal effect of formamidine insecticides (68). The B. mori β-adrenergic-like OAR showed a concentration-dependent increase in cellular cAMP in response to octopamine and a biphasic response to dimethylchlordimeform (46) in functional assays, while the tyramine receptor, TYR expressed in HEK-293 revealed an effect of BTS-27271 and demethylchlordimeform on forskolin-stimulated cAMP when the receptor was activated by tyramine (53). Similarly, pharmacological characterization of a putative OAR from the southern cattle tick, Rhipicephalus (Boophilus) microplus suggested that the receptor was likely a type-1 tyramine receptor (TAR-1) (69). Functional assays using heterologous expression of the receptor in Chinese hamster ovary cells (CHO-K1) showed strong fold-potency of tyramine versus octopamine at the receptor, antagonism by the α2-adrenergic antagonists, yohimbine and cyproheptadine, and agonistic effect of the amitraz metabolite BTS-27271 in the presence of tyramine. 62 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Receptor Family

Sub-Family

Anopheles gambiae23

Aedes aegypti24

Culex quinque fasciatus25

Drosophila melanogaster22

Ixodes scap ularis26

Biogenic amine

Dopamine

4

6

3

2

6

Serotonin

6

11

2

6

4

Muscarinic acetylcholine

2

3

1

2

2

Histamine

1

1

-

-

-

Melatonin

1

1

Octopamine/ Tyramine

4

6

4

2

4

Orphan

-

-

5

9

-

Total

18

28

15

21

16

12

11

11

7

7

1

2

1

1

3

3

-

4

-

63


Table 1. Summary of Invertebrate Class A (rhodopsin-like) G Protein-Coupled Receptors Showing Numbers of Predicted Receptors Identified in Each Family and Sub-family

Opsins Purine Glycoprotein hormone

Adenosine

3

-

Continued on next page.

Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

64


Table 1. (Continued). Summary of Invertebrate Class A (rhodopsin-like) G Protein-Coupled Receptors Showing Numbers of Predicted Receptors Identified in Each Family and Sub-family Receptor Family

Sub-Family

Anopheles gambiae23

Aedes aegypti24




Peptide

Allatostatin/ ACP/Allotropin/ Galanin

3

-

3

2

14

Leukokinin/ Neurokinin/ Tachykinin

5

9

5

5

22

GH secretagoge /Neurotensin/ TRHR/LH/ FSH/TRH

2

2

-

7

-

Gastrin/ Bombesin/CCK

4

8

1

4

-

GnRH

3

3

1

2

-

Neuropeptide Y/F

4

11

2

5

2

Bursicon/Opioid

1

-

1

1

Somatostatin

1

1

2

2

-

Vasopressin

2

2

1

1

-

Unclassified

-

3

10

4

22

Total

25

39

25

33

61


Anopheles gambiae23

Aedes aegypti24




Orphan Class A

21

20

-

14

43

TOTAL

80

103

52

80

130

Sub-Family

ACP, adipokinetic/corazonin-related peptide; CCK, cholecystokinin; FSH, follicle stimulating hormone; GH, growth hormone; GnRH, gonadotropin-releasing hormone; 5HT, 5-hydroxytryptamine; LH, luteinizing hormone; TRHR, thyroid releasing hormone receptor; TSH, thyroid stimulating hormone; MAchR, muscarinic Acetylcholine.

65


Receptor Family



Vertebrate DARs are classified as either D1-like (Gαs-coupled) or D2-like (Gαi-coupled) according to G protein coupling mechanisms (Figure 1). The neurotransmitter dopamine has important functions both in the central and peripheral nervous systems of vertebrates (70). Research has emphasized understanding and treatment of multiple neurological afflictions involving dopaminergic processes, including drug addiction, Parkinson’s disease and schizophrenia (71, 72). DAR-mediated regulation of intracellular cAMP is important for controlling signal transduction cascades triggered by phosphorylation activities of cAMP-dependent protein kinase (37). There are numerous reports involving cloning-based pharmacological characterization of invertebrate GPCRs, including D1- like and D2-like DARs from insects (38, 48, 50, 66, 73–76). Interestingly, D1-like DARs, and elevated cAMP levels have been implicated in tick salivation, a process linked to pathogen transmission during blood feeding (77, 78). Mustard et al (74) showed that inhibition of dopamine signaling decreased activity level in honeybees. This study provides a foundation for future work examining the importance of dopamine signaling in regulating distinct behaviors and elucidating the roles of specific dopamine receptors. Progress has been made towards an understanding of the pharmacology of mosquito and tick dopamine-binding receptors (DARs). Molecular and pharmacological studies identified two D1-like DARs in each of the mosquitoes Aedes aegypti, An. gambiae and Culex quinquefasciatus and the tick Ixodes scapularis (38, 39, 48, 50, 76). The DARs were identified based on genome assemblies available at NCBI and curated by VectorBase (35). Pharmacological studies revealed Gαs-coupling of receptors in vitro, and receptor agonism by dopamine and inhibition via a variety of small molecule antagonists (Figure 2). Importantly, lead elaboration combined with in vitro and in vivo SAR identified potent antagonists of the Ae. aegypti DOP2 DAR (79), enabling development of a preliminary pharmacophore. Some of these molecules exhibited two-fold selectivity for mosquito DARs versus the human ortholog, hD1 (Table 2). Additionally, Hill et al (76) showed pleiotropic coupling of the mosquito DARs expressed in a heterologous system to both Gαs and Gαq, highlighting possible plasticity in signaling modes. The conserved sequence and pharmacology of DARs between culicine (Aedes and Culex species) and anopheline (Anopheles species) mosquitoes suggests potential to develop products effective against multiple vectors and residual transmission by multiple anopheline vectors (76). Parallel characterization of dopamine receptors is an important step toward understanding the biological roles of dopaminergic processes in mosquitoes and ticks and generation of a pipeline for high throughput chemical screening of stably expressed vector GPCRs.

66 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 2. Pharmacological characterization of the Aedes aegypti AaDOP1 and AaDOP2 receptors. The mosquito receptors were stably expressed in HEK 293-CRELuc cells for dose-response assays. A, C: AaDOP1, B, D: AaDOP2. Representative curves for A, B: biogenic amines; C, D: synthetic dopamine receptor agonists; E: Inhibitory effect of 10 µM SCH23390 in the presence of 1 µM dopamine (n=4) shown for both mosquito dopamine receptors. ** p

Potential of GPCR-Targeting Insecticides for Control of Arthropod

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