| dc.description.abstract | Due to the worldwide prevalence of pesticide resistance in disease vectors, novel targets and corresponding reagents for alternative pest control are needed. Invertebrate neuropeptide G protein-coupled receptors (GPCRs) are involved in regulating many important physiological processes and are promising insecticide targets. In this study, we used the invertebrate-specific kinin receptor as a proof-of-principle for novel ligand discovery of arthropod neuropeptide GPCRs. Kinins are pleiotropic neuropeptides that are known to modulate insect diuresis, hindgut contraction, pre-ecdysis, digestion and chemosensory perception.
We utilized both forward and reverse pharmacological approaches to identify peptidomimetic and small molecule ligands of the kinin receptors from the yellow fever mosquito, Aedes aegypti, and the cattle fever tick, Rhipicephalus microplus. In forward approaches, novel peptidomimetics with enhanced biostability and bioavailability designed based on insect kinins were tested on the recombinant receptors using a calcium bioluminescence assay and analyzed for their EC50 and efficacy. These experiments yielded potent agonists for both receptors. I cloned the cDNA of the kinin neuropeptide precursor from R. microplus, predicted the sequences of 17 tick kinins, and functionally characterized fourteen of them on the cognate receptor. In this process, I found the tick kinins have a slightly different sequence motif compared to insect kinins, with proline being conserved in the variable position two of the C-terminal pentapeptide core.
For the reverse approach, I developed a high-throughput screening (HTS) calcium fluorescence assay using the recombinant tick kinin receptor, reporting the first HTS on a neuropeptide GPCR of any tick species. Through experimental screens and virtual screens, 36 antagonists were identified. Three of them were validated as antagonists on the mosquito receptor. These molecules also inhibited the mosquito hindgut contractions in the isolated hindgut contraction inhibition assay. By analyzing the structure-activity relationships of peptidomimetic agonists and small molecule antagonists respectively, we improved the understanding of ligand structures which will facilitate the design of potent ligands.
In summary, this study identified potent peptidomimetic agonists, and for the first time, identified antagonists of mosquito and tick kinin receptors. These ligands are important reagents and tools for studying the role of kinin in arthropod physiology in arthropods and potential leads of novel pesticides. This study improved our understanding of the arthropod kinin signaling system. | |
