The leucokinin-like peptide receptor from the cattle fever tick, Rhipicephalus microplus, is localized in the midgut periphery and receptor silencing with validated double-stranded RNAs causes a reproductive fitness cost

Brock, Christina M.; Temeyer, Kevin B.; Tidwell, Jason; Yang, Yunlong; Blandon, Maria Adylia; Carreón-Camacho, Diana; Longnecker, Michael T.; Almazán, Consuelo; León, Adalberto A. Pérez de; Pietrantonio, Patricia V.

doi:10.1016/j.ijpara.2018.11.006

Cited by 19 publications

(47 citation statements)

References 93 publications

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“…In the tick R. microplus LKR was immunolocalized on the midgut outer surface, and LKR-silenced females displayed variations in gut discoloration, had a reduction in body weight of ∼30%, reduced weight of their egg masses, and experienced decreased egg hatching. Thus, LKR silencing in female ticks caused a reproductive fitness cost, perhaps related to defects in heme metabolism because some guts from silenced females were completely white (Brock et al, 2019). In agreement, the LKR transcript of the tick R. sanguineous was apparently expressed at higher levels in the gut and salivary glands than in other tissues, as inferred from RT-PCR analyses (Lees et al, 2010).…”

Section: Discussionmentioning

confidence: 99%

“…Transcripts for tick GPCRs are often expressed in vivo at very low levels, which complicates their physiological characterization. For example, results of qRT-PCR after gene silencing is often highly variable in diverse tick tissues due to their normal low expression levels (Brock et al, 2019). To address this challenge, additional future approaches to elucidate the physiological function of the tick kinin system through loss-of-function experiments include the knockdown of the endogenous kinin neuropeptide transcript and a pharmacological approach to block receptor function.…”

Section: Discussionmentioning

confidence: 99%

“…scapularis and O. turicata (Egekwu et al, 2014, 2016; Guerrero et al, 2016). As the kinin receptor transcript level is low in ticks (as well as the level of receptor protein (Brock et al, 2019), it is tempting to speculate that kinin signaling may be regulated by changes in the expression and release of their expanded kinin peptide repertoire. A detailed temporal quantitative analysis of receptor and kinin precursor mRNA transcripts is still lacking.…”

Section: Discussionmentioning

confidence: 99%

“…aegypti (Kersch and Pietrantonio, 2011; Kwon et al, 2016). The most recent RNAi-mediated silencing of the kinin receptor in the cattle fever tick caused a reproductive fitness cost (Brock et al, 2019). While our cloning of this receptor represented the first known neuropeptide GPCR in the Acari, there has been no urgent need in establishing the endogenous ligand of the R. microplus tick.…”

Section: Introductionmentioning

confidence: 99%

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

2019

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

2019

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“…25,27,30 The tick KR appears to be a promising target because knocking-down its expression in female ticks results in significant reduction of their reproductive fitness. 31 However, the natural peptides cannot be directly used, as they are susceptible to degradation by endogenous peptidases 12,[32][33][34] and may not penetrate the cuticle. Knowledge of both chemical and conformational requirements responsible for endogenous neuropeptide biological activity can aid in the design of analogs containing unnatural moieties that can overcome these limitations.…”

Section: Introductionmentioning

confidence: 99%

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

Xiong

Kaczmarek

Zabrocki

et al. 2020

Pest Management Science

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BACKGROUND Kinins are multifunctional neuropeptides that regulate key insect physiological processes such as diuresis, feeding, and ecdysis. However, the physiological roles of kinins in ticks are unclear. Furthermore, ticks have an expanded number of kinin paracopies in the kinin gene. Silencing the kinin receptor (KR) in females of Rhipicephalus microplus reduces reproductive fitness. Thus, it appears the kinin signaling system is important for tick physiology and its disruption may have potential for tick control. RESULTS We determined the activities of endogenous kinins on the KR, a G protein‐coupled receptor, and identified potent peptidomimetics. Fourteen predicted R. microplus kinins (Rhimi‐K), and 11 kinin analogs containing aminoisobutyric acid (Aib) were tested. The latter incorporated tick kinin sequences and/or were modified for enhanced resistance to arthropod peptidases. A high‐throughput screen using a calcium fluorescence assay in 384‐well plates was performed. All tested kinins and Aib analogs were full agonists. The most potent kinin and two kinin analogs were equipotent. Analogs 2414 ([Aib]FS[Aib]WGa) and 2412 ([Aib]FG[Aib]WGa) were the most active with EC50 values of 0.9 and 1.1 nM, respectively, matching the EC50 of the most potent tick kinin, Rhimi‐K‐14 (QDSFNPWGa) (EC50 = 1 nM). The potent analog 2415 ([Aib]FR[Aib]WGa, EC50 = 6.8 nM) includes both Aib molecules for resistance to peptidases and a positively charged residue, R, for enhanced water solubility and amphiphilic character. CONCLUSION These tick kinins and pseudopeptides expand the repertoire of reagents for tick physiology and toxicology towards finding novel targets for tick management. © 2019 Society of Chemical Industry

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A random small molecule library screen identifies novel antagonists of the kinin receptor from the cattle fever tick, Rhipicephalus microplus (Acari: Ixodidae)

Xiong

Baker

Pietrantonio

2021

Pest Management Science

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BACKGROUND The southern cattle tick, Rhipicephalus microplus, is a primary vector of the deadly bovine disease babesiosis. Worldwide populations of ticks have developed resistance to acaricides, underscoring the need for novel target discovery for tick control. The arthropod‐specific R. microplus kinin receptor is such a target, previously validated by silencing, which resulted in female reproductive fitness costs, including a reduced percentage of eggs hatching. RESULTS In order to identify potent small molecules that bind and activate or inhibit the kinin receptor, a high‐throughput screening (HTS) assay was developed using a CHO‐K1 cell line expressing the recombinant tick kinin receptor (BMLK3). A total of ~20 000 molecules from a random in‐house small molecule library were screened in a ‘dual‐addition’ calcium fluorescence assay. This was followed by dose–response validation of the hit molecules identified both from HTS and an in silico screen of ~390 000 molecules. We validated 29 antagonists, 11 of them were full antagonists with IC50 values between 0.67 and 8 μmol L–1. To explore the structure–activity relationships (SAR) of the small molecules, we tested the activities of seven analogs of the most potent identified antagonist, additionally discovering three full antagonists and four partial antagonists. These three potent antagonists (IC50 < 3.2 μmol L–1) were validated in vitro using the recombinant mosquito kinin receptor and showed similar antagonistic activities. In vivo, these three compounds also inhibited the mosquito hindgut contraction rate induced by a myotropic kinin agonist analog 1728. CONCLUSION Antagonists identified in this study could become pesticide leads and are reagents for probing the kinin signaling system. © 2021 Society of Chemical Industry

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The leucokinin-like peptide receptor from the cattle fever tick, Rhipicephalus microplus, is localized in the midgut periphery and receptor silencing with validated double-stranded RNAs causes a reproductive fitness cost

Cited by 19 publications

References 93 publications

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

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

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

A random small molecule library screen identifies novel antagonists of the kinin receptor from the cattle fever tick, Rhipicephalus microplus (Acari: Ixodidae)

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