Re-invigorating the insecticide discovery pipeline for vector control: GPCRs as targets for the identification of next gen insecticides

https://doi.org/10.1016/j.pestbp.2013.02.008Get rights and content

Highlights

  • The PIDP was established to pursue target-based insecticide discovery.

  • Screens identified agonists and antagonists of mosquito and tick DARs.

  • “Hit-to-lead” studies identified AaDOP2 antagonists as next-gen insecticide leads.

  • TM domains of mosquito and tick DARs share 73-100% amino acid identity.

  • Invertebrate DARs have potential for target-based insecticide discovery.

Abstract

G protein-coupled receptors (GPCRs) comprise a large family of membrane-bound molecules that mediate critical physiological roles in both vertebrates and invertebrates. GPCRs are widely exploited targets of the pharmaceutical industry; approximately 50% of human drugs interact with these receptors. GPCRs are also candidate targets for next-generation insecticides and provide opportunities to discover new mode-of-action chemistries for insect control. We present an overview of the Purdue Insecticide Discovery Pipeline which employs a target-based “genome-to-lead” approach to identify novel insecticidal molecules. The pipeline is focused on invertebrate GPCRs, with an emphasis on mosquito and tick dopamine receptors (DARs). We summarize published studies describing the characteristics of D1-like (Gαs coupled) DARs from the yellow fever mosquito, Aedes aegypti (AaDOP1, AaDOP2) and Lyme disease tick, Ixodes scapularis (IsDOP1, IsDOP2), and review our ongoing cell-based chemical library screening efforts to discover small molecule ligands and modulators of AaDOP2 and IsDOP2. We discuss “hit-to-lead” optimization of AaDOP2 antagonists and present in vivo assay data demonstrating that lead antagonists cause rapid and high mortality of Ae. aegypti larvae. To expand capabilities of the pipeline, we developed an in vitro screen to identify small molecule agonists of AaDOP2. Twenty-five agonists were discovered in the screen that exhibited significant potency at AaDOP2, although a subset of the hits that were tested (SKF82958, SKF81297) showed no evidence of in vivo toxicity to Ae. aegypti larvae. Finally, we analyze the conceptual protein sequence of D1-like DARs from the malaria mosquito, Anopheles gambiae and the northern-house mosquito, Culex quinquefasciatus, and discuss the potential application of GPCR target-based insecticide discovery for other mosquito vectors of importance to human health.

Introduction

New mode-of-action chemistries are urgently sought for the management of arthropods that transmit disease-causing agents impacting human health. Continued vector control is threatened by the development of pest populations with resistance to commercial insecticides. Moreover, resistance has been documented to each of the major insecticide classes used in the past and present for vector control, including organochlorines, organophosphates and carbamates, and pyrethroids which target sodium channels, acetylcholinesterase, and GABA receptors, respectively, in the insect nervous system [1]. Resistance is mediated via several mechanisms such as target-site mutations, modified metabolic processes, and gene amplification/transcriptional regulation [1]. Management of resistance is complicated by the fact that there has not been a new public health pesticide developed for vector control in over 30 years [2].

While the perception of poor market potential has limited investments by the private sector in the development of products to control neglected infectious diseases, the public sector is making profound contributions to drug discovery research, particularly in the area of infectious disease control. A recent survey indicates that diseases of developing countries represent approximately 30% of the research portfolios of US Academic Drug Discovery (ADD) centers [3]. In the past 40 years, 153 new Food and Drug Administration (FDA)-approved drugs, vaccines, or new indications for existing drugs were discovered through research carried out in in the public sector, with an emphasis on products for infectious diseases [4].

Frye et al. [3] noted the increasingly important role of target-based drug discovery in pharmaceutical research – target-based approaches account for more than 70% of the pipeline in ADD centers – and revealed the diversity of human drug target classes under investigation by these institutions (Table 1). Existing pesticides primarily affect molecular targets associated with the nervous, digestive and endocrine systems of invertebrates. New molecular targets associated with critical physiological processes in invertebrates represent important prospects for diversification of insecticide discovery efforts. Given the availability of assembled genome sequence data for multiple arthropod vectors (see VectorBase; www.vectorbase.org), the feasibility of implementing target-based approaches for development of novel public health products has never been greater. In point of fact, presentations at the 244th ACS symposium (this issue) reveal an increasing emphasis on research toward discovery and characterization of unique targets identified from vector genome sequences.

The G protein-coupled receptors (GPCRs) are one class of molecules with high potential for insecticide discovery. GPCRs comprise a large family of membrane-bound receptors that mediate critical biological processes in vertebrates and invertebrates. These receptors have been widely exploited as drug targets in human pharmaceutical research. Approximately 50% of human drugs influence GPCR-mediated processes [5]. The potential around GPCRs as insecticidal targets is recognized [6], yet these targets have been relatively poorly exploited for insect control.

The Purdue Insecticide Discovery Pipeline (PIDP; Fig. 1) was created to reinvigorate pesticide discovery by emphasizing development of new mode-of-action insecticides for vector control [7]. Implementation of the PIDP has required multi-disciplinary collaboration and includes our expertise in arthropod genomics, drug target identification and evaluation, and pharmacology to establish a platform for continuous insecticide discovery research within the academic setting. The PIDP employs a target-based “genome-to-lead” approach to identify molecules that disrupt GPCR-mediated processes in invertebrates. Briefly, this entails identification of GPCR sequences from the assembled genomes of arthropod vectors, which are cloned and expressed in heterologous cell-based assays for functional characterization. GPCRs of interest are then screened against libraries of molecules to identify chemistries that impact their signaling as measured by either an antagonist or agonist effect. “Hit” chemistries are subsequently evaluated in a series of “hit-to-lead” experiments needed to determine if these compounds are toxic to live insects using in vivo bioassays, and to identify additional novel chemistries with properties suitable for insecticide development using structure activity relationship (SAR) studies. The SAR process includes a series of efforts focused on hit expansion, chemoinformatics, and lead-based focused libraries to thoroughly explore the chemical space around hit compounds.

Our initial insecticide discovery research has focused on biogenic amine-binding GPCRs from species of dipteran vectors including mosquitoes, sand flies and tsetse flies, as well as ticks, with an emphasis on the dopamine receptors (DARs). Dopamine is an essential neurotransmitter in arthropods and the disruption of dopaminergic processes has been shown to affect behavior, locomotion and development, among other important physiological processes (see [7] for overview). DARs are classified as either D1- or D2-like based on their functional activity. Agonist binding to the D1-like DARs causes Gαs-mediated stimulation of adenylyl cyclase (AC) resulting in increased production of cAMP. We previously identified novel antagonist chemistries of the D1-like DARs AaDOP2 and IsDOP2 from the yellow fever mosquito, Aedes aegypti [7], and Lyme disease tick, Ixodes scapularis [9], respectively (Table 2) and demonstrated that AaDOP2 antagonists have potential as leads for new mode-of-action insecticides for mosquito control [7]. In addition, analysis of the fold-selectivity between AaDOP2 and the orthologous human D1 receptor, hD1 (Table 3) suggests that there is potential to identify chemistries possessing significant selectivity for vector versus mammalian receptors.

Here we review recent progress within the PIDP and discuss ongoing “hit-to-lead” studies to enhance discovery of novel AaDOP2 antagonist molecules with potential for pesticide development. Given that several insecticide classes act as agonists at invertebrate neurological targets, and that an acaricide, Amitraz, is presumed to have partial agonistic activity at an octopamine-sensitive GPCR [10], we developed and performed a high-throughput screen to identify novel agonists of AaDOP2. We discuss research required to investigate the mode-of-action of AaDOP2 antagonist and agonist molecules in the mosquito, and realize development of next-gen insecticide leads. Lastly, we examine prospects for expanding DAR target-based insecticide discovery efforts to encompass the genera Anopheles and Culex, which include many mosquito species of importance to human and animal health.

Section snippets

Invertebrate receptor identification, cloning and analysis

Methods used for the identification, cloning, and molecular and pharmacological characterization of the D1-like DARs, AaDOP1 and AaDOP2 from the yellow fever mosquito, Ae. aegypti, and IsDOP1 and IsDOP2 from the Lyme disease tick, I. scapularis are described in [7], [8], [9]7-9].

High-throughput cell-based screen of the LOPAC1280 library for agonists of the AaDOP2 receptor

To identify novel AaDOP2 receptor agonists, the Library of Pharmacologically Active Compounds (LOPAC1280) was screened at the Integrated Screening Technologies Laboratory, Discovery Park, Purdue University, using

High-throughput cell-based screen of the LOPAC1280 library for agonists of the AaDOP2 receptor

Previously we reported the results of two antagonist screens of the LOPAC1280 library against the AaDOP2 [7] and IsDOP2 [9] DARs (Table 2). These screens identified 51 and 85 antagonist chemistries that represented “hit” rates of 4% and 7% for AaDOP2 and IsDOP2, respectively. Both screens identified several mammalian DAR antagonists, biogenic amine receptor ligands and transport inhibitors, and additional miscellaneous chemistries that antagonized the invertebrate DARs (Fig. 2A and B). The

Discussion

We have developed and implemented a “genome-to-lead” pipeline for insecticide discovery to explore arthropod GPCRs as targets for new mode-of-action chemistries for vector control. The PIDP (Fig. 1) is currently populated with multiple biogenic amine-binding GPCR targets from vectors of importance to human and animal health. Previously, we analyzed the molecular and pharmacological properties of four D1-like DARs; two from the yellow fever mosquito, Ae. aegypti (AaDOP1, AaDOP2) [7] and two from

Conclusions

This research provides further proof-of-concept for the GPCR-based “genome-to-lead” approach toward novel insecticide discovery and evidence for the value of iterative “hit-to-lead” optimization of leads for development of new vector-active insecticides. Sequence data suggest the insecticide discovery pipeline could be expanded to find chemistries broadly active against multiple mosquito species of importance to human health. Screen data show that the scope of the pipeline can be further

Funding

This research was supported by a Purdue University Seed Grant (#203606), a Department of Defense/TATRC sub-award (#201596-PU) and Deployed war Fighter Protection Program (#105922), an Indiana Clinical and Translational Sciences Institute Core Pilot Award and a Trask Innovation Fund award (#205766) to C.A.H and V.J.W.

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