BackgroundMany 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.Methodology/Principal FindingsWe 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 (AaDOP1 and AaDOP2) from the yellow fever mosquito, Aedes aegypti. Sequence analyses indicated that these receptors are orthologous to arthropod D1-like (Gαs-coupled) receptors, but share less than 55% amino acid identity in conserved domains with mammalian dopamine receptors. Heterologous expression of AaDOP1 and AaDOP2 in HEK293 cells revealed dose-dependent responses to dopamine (EC50: AaDOP1 = 3.1±1.1 nM; AaDOP2 = 240±16 nM). Interestingly, only AaDOP1 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 AaDOP2. Fifty-one compounds were identified as “hits,” and follow-up validation assays confirmed the antagonistic effect of selected compounds at AaDOP2. In vitro comparison studies between AaDOP2 and the human D1 dopamine receptor (hD1) revealed markedly different pharmacological profiles and identified amitriptyline and doxepin as AaDOP2-selective compounds. In subsequent Ae. aegypti larval bioassays, significant mortality was observed for amitriptyline (93%) and doxepin (72%), confirming these chemistries as “leads” for insecticide discovery.Conclusions/SignificanceThis 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 in vivo toxicity toward mosquitoes.

Phospholipase C (PLC) enzymes hydrolyze phosphatidylinositol lipids to produce second messengers, including inositol‐1,4,5‐triphosphate (IP3) and diacylgycerol (DAG), which increase intracellular calcium and activate protein kinase C (PKC), respectively. PLCɛ contributes to cardiac hypertrophy and contractility, as well as to oncogenic and inflammatory signaling pathways following activation of G protein‐coupled receptors and receptor tyrosine kinases. PLCɛ shares a conserved core with the PLC superfamily, but the roles of individual domains in regulation of activity and membrane binding have not been established. We used functional assays to show that the PLCɛ PH domain significantly increases basal lipase activity, but is dispensable for stability. We provide the first structural insights into domain organization of PLCɛ using small‐angle X‐ray scattering (SAXS) and electron microscopy (EM) to reveal that the PH domain is conformationally heterogeneous in solution. Comparisons of the PLCɛ solution structure to that of the closely‐related PLCβ enzyme demonstrate that the PLCβ PH domain is also mobile in solution, in contrast to previously reported crystal structures. We propose that the dynamic nature of the PLC PH domain and resulting conformational heterogeneity contributes to subfamily‐specific differences in activity and regulation by G proteins. We are now using cryo‐EM to expand on these findings and obtain higher resolution structures. Support or Funding Information This work is supported by the Purdue Center for Cancer Research, AHA grant 16SDG29930017, NIH NLHBI 1R01HL141076‐01 to A.M.L., and Purdue College of Science Staff and Administrative & Professional Staff Advisory Council Professional Development awards to E.E.G. SAXS experiments used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE‐AC02‐06CH11357. This project was supported by grant 9 P41 GM103622 from the National Institute of General Medical Sciences of the National Institutes of Health. Use of the Pilatus 3 1M detector was provided by grant 1S10OD018090‐01 from NIGMS. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phospholipase C (PLC) enzymes hydrolyze membrane phosphatidylinositol 4,5 bisphosphate (PIP 2 ) and regulate Ca 2+ and protein kinase signaling in virtually all mammalian cell types.

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Phospholipase Cε (PLCε) generates lipid-derived second messengers at the plasma and perinuclear membranes in the cardiovascular system. It is activated in response to a wide variety of signals, such as those conveyed by Rap1A and Ras, through a mechanism that involves its C-terminal Ras association (RA) domains (RA1 and RA2). However, the complexity and size of PLCε has hindered its structural and functional analysis. Herein, we report the 2.7 Å crystal structure of the minimal fragment of PLCε that retains basal activity. This structure includes the RA1 domain, which forms extensive interactions with other core domains. A conserved amphipathic helix in the autoregulatory X-Y linker of PLCε is also revealed, which we show modulates activity in vitro and in cells. The studies provide the structural framework for the core of this critical cardiovascular enzyme that will allow for a better understanding of its regulation and roles in disease.