The Cattle Fever Tick, Rhipicephalus microplus, as a Model for Forward Pharmacology to Elucidate Kinin GPCR Function in the Acari

Abstract

The success of the acaricide amitraz, a ligand of the tick tyramine/octopamine receptor (a G protein-coupled receptor; GPCR), stimulated interest on arthropod-specific GPCRs as targets to control tick populations. This search advances tick physiology because little is known about the pharmacology of tick GPCRs, their endogenous ligands or their physiological functions. Here we explored the tick kinin receptor, a neuropeptide GPCR, and its ligands. Kinins are pleiotropic insect neuropeptides but their function in ticks is unknown. The endogenous tick kinins are unknown and their cDNAs have not been cloned in any species. In contrast, more than 271 insect kinin sequences are available in the DINeR database. To fill this gap, we cloned the kinin cDNA from the cattle fever tick, Rhipicephalus microplus , which encodes 17 predicted kinins, and verified the kinin gene structure. We predicted the kinin precursor sequences from additional seven tick species, including Ixodes scapularis . All species showed an expansion of kinin paracopies. The “kinin core” (minimal active sequence) of tick kinins FX 1 X 2 WGamide is similar to those in insects. Pro was predominant at the X 2 position in tick kinins. Toward accelerating the discovery of kinin function in ticks we searched for novel synthetic receptor ligands. We developed a dual-addition assay for functional screens of small molecules and/or peptidomimetics that uses a fluorescent calcium reporter. A commercial library of fourteen small molecules antagonists of mammalian neurokinin (NK) receptors was screened using this endpoint assay. One acted as full antagonist (TKSM02) with inhibitory concentration fifty (IC 50 ) of ∼45 μM, and three were partial antagonists. A subsequent calcium bioluminescence assay tested these four antagonists through kinetic curves and confirmed TKSM02 as full antagonist and one as partial antagonist (TKSM14). Antagonists of NK receptors displayed selectivity (>10,000-fold) on the tick kinin receptor. Three peptidomimetic ligands of the mammalian NK receptors (hemokinin 1, antagonist G, and spantide I) were tested in the bioluminescence assay but none were active. Forward approaches may accelerate discovery of kinin ligands, either as reagents for tick physiological research or as lead molecules for acaricide development, and they demonstrate that selectivity is achievable between mammalian and tick neuropeptide systems.

Comparative morphological and transcriptomic analyses reveal novel chemosensory genes in the poultry red mite,Dermanyssus gallinaeand knockdown by RNA interference

“…Identification of G proteins are also relevant for the understanding of acari biology in general, as these proteins could be targeted by novel compounds. Most information on acari GPCRs were obtained from tick transcriptome datasets and recombinant receptor systems (Gross et al, 2015; Xiong et al, 2019). Previous experiments in ticks indicated that prediction of GPCRs using transcriptome analysis might be a challenging task.…”

Activity of native tick kinins and peptidomimetics on the cognate target G protein‐coupled receptor from the cattle fever tick, Rhipicephalus microplus (Acari: Ixodidae)

“…To help visualize the sequence conservation of tick kinins across species in relation to the herein determined potency of the Rhimi kinins, the kinin peptide precursor sequences of seven hard tick species were aligned. 39 Sequences were aligned with MegaPro (DNASTAR, Lasergene v16.0) using the MAFFT 43 program with the iterative refinement algorithm E-INS-I because of the occurrence of known insertions and deletions during the evolution of neuropeptide genes. 39 The kinin conserved motifs are represented by Logos obtained with the default alignment settings of MegaPro.…”

“…30 To fill this gap in knowledge for the tick KR, we cloned the cDNA of the kinin neuropeptide precursor from R. microplus. Within the translated amino acid sequence, we predicted the sequences of 17 tick kinins, based on both the identification of the canonical conserved residues in the pentapeptide kinin core 'FXXWGa' (FXXWGG) and the putative dibasic enzyme cleavage cutting sites (KK/KR/RR) on the precursor, 38,39 but their activity on the receptor has not yet been tested. The current study determined the activities of the predicted R. microplus endogenous kinin ligands and modified analogs on the recombinant tick KR through a cellular functional assay using calcium fluorescence as a reporter and in high-throughput mode.…”

“…In this paper, we further explore the use of the sterically hindered Aib moiety in biostable analogs that also specifically incorporate residues from kinins native to the tick R. microplus. 39 We also included an analog of this motif with X 1 = A, found in a predicted R. microplus kinin, Rhimi-K-7, which was not tested (see the Discussion section). 39 In a few of these analogs the N-terminus is further protected from aminopeptidases with either acetyl (Ac) or pyroglutamate (pQ) groups.…”

“…39 We also included an analog of this motif with X 1 = A, found in a predicted R. microplus kinin, Rhimi-K-7, which was not tested (see the Discussion section). 39 In a few of these analogs the N-terminus is further protected from aminopeptidases with either acetyl (Ac) or pyroglutamate (pQ) groups. 12 A couple of additional analogs were evaluated on the tick receptor that feature either the strongly basic, positively charged R or dR placed at the N-terminal of the pentapeptide core in order to enhance the aqueous solubility of the generally hydrophobic insect kinin core sequence.…”