A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...

Texas A&M University

Insect kinins modulate aspects of diuresis, digestion, development, and sugar taste perception in tarsi and labellar sensilla in mosquitoes. They are, however, subject to rapid biological degradation by endogenous invertebrate peptidases. A series of α-aminoisobutyric (Aib) acid-containing insect kinin analogs incorporating sequences native to the Aedes aegypti mosquito aedeskinins were evaluated on two recombinant kinin invertebrate receptors stably expressed in cell lines, discovering a number of highly potent and biostable insect kinin mimics. On the Ae. aegypti mosquito kinin receptor, three highly potent, biostable Aib analogs matched the activity of the Aib-containing biostable insect kinin analog 1728, which previously showed disruptive and/or aversive activity in aphid, mosquito and kissing bug. These three analogs are IK-Aib-19 ([Aib]FY[Aib]WGa, EC = 18 nM), IK-Aib-12 (pQKFY[Aib]WGa, EC = 23 nM) and IK-Aib-20 ([Aib]FH[Aib]WGa, EC = 28 nM). On the Rhipicephalus (Boophilus) microplus tick receptor, IK-Aib-20 ([Aib]FH[Aib]WGa, EC = 2 nM) is more potent than 1728 by a factor of 3. Seven other potentially biostable analogs exhibited an EC range of 5-10 nM, all of which match the potency of 1728. Among the multi-Aib hexapeptide kinin analogs tested the tick receptor has a preference for the positively-charged, aromatic H over the aromatic residues Y and F in the X variable position ([Aib]FX[Aib]WGa), whereas the mosquito receptor does not distinguish between them. In contrast, in a mono-Aib pentapeptide analog framework (FX[Aib]WGa), both receptors exhibit a preference for Y over H in the variable position. Among analogs incorporating polyethylene glycol (PEG) polymer attachments at the N-terminus that can confer enhanced bioavailability and biostability, three matched or surpassed the potency of a positive control peptide. On the tick receptor IK-PEG-9 (P-R[Aib]FF[Aib]WGa) was the most potent. Two others, IK-PEG-8 (P-RFFPWGa) and IK-PEG-6 (P-RFFPWGa), were most potent on the mosquito receptor, with the first surpassing the activity of the positive control peptide. These analogs and others in the IK-Aib series expand the toolbox of potent analogs accessible to invertebrate endocrinologists studying the structural requirements for bioactivity and the as yet unknown role of the insect kinins in ticks. They may contribute to the development of selective, environmentally friendly pest arthropod control agents.

Evaluation of Aib and PEG-polymer insect kinin analogs on mosquito and tick GPCRs identifies potent new pest management tools with potentially enhanced biostability and bioavailability.

Regulation of many physiological processes in animals, certainly those controlled by neuropeptide hormones, involves G protein-coupled receptors (GPCRs). Our work focusing on endocrine regulation of diuresis and water balance in mosquitoes and ticks started in 1997 with the kinin receptor, at the dawn of the omics era. After the genomic revolution, we began work on the endocrinology of reproduction in the red imported fire ant. We will use the template of this comparative work to summarize key points about GPCRs and signaling, and emphasize the most recent developments in the pharmacology of arthropod neuropeptide GPCRs. We will discuss omics' contributions to the advancement of this field, and its influence on peptidomimetic design while emphasizing work on blood feeding arthropods.

G protein-coupled receptors in arthropod vectors: omics and pharmacological approaches to elucidate ligand-receptor interactions and novel organismal functions.

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, , which encodes 17 predicted kinins, and verified the kinin gene structure. We predicted the kinin precursor sequences from additional seven tick species, including . All species showed an expansion of kinin paracopies. The "kinin core" (minimal active sequence) of tick kinins FXXWGamide is similar to those in insects. Pro was predominant at the X 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) 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.

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

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.

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

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.

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

Pyrokinins (PKs) are pleiotropic neuropeptides with significant roles in invertebrate physiology. Although functions of PKs are known in insects, there is a lack of knowledge of PK-encoding genes and PKs functions in ticks. Herein the first tick cDNAs of the capability (capa) gene were cloned from the southern cattle tick, Rhipicephalus microplus (Acari: Ixodidae), and the blacklegged tick, Ixodes scapularis. Each cDNA encoded one periviscerokinin and five different pyrokinins. Two PKs were identical in sequence in the two species. The three PKs unique to R. microplus (Rhimi-CAPA-PK1, -PK2, and -PK5) were tested on the recombinant R. microplus pyrokinin receptor using a calcium bioluminescence assay. The Rhimi-CAPA-PKs acted as agonists with EC ranging from 101-188 nM. Twenty PK analogs designed for enhanced bioavailability and biostability were tested on the receptor. Five of these were designed based on the sequences of the three unique Rhimi-CAPA-PKs. Eight PK analogs were also agonists; four of them were full agonists that exhibited comparable efficacy to the native Rhimi-CAPA-PKs, with EC ranging from 401 nM-1.9 μM. The structure-activity relationships (SAR) of all analogs were analyzed. Our results suggested that a positively charged, basic lysine at the variable position X of the PK active core (FXPRLamide) conferred enhanced affinity to the analogs in their interaction with the tick receptor. These analogs are promising tools to elucidate the pyrokinin function in ticks in vivo as these analogs are expected to have prolonged hemolymph residence time in comparison to the native peptides.

Tick CAPA propeptide cDNAs and receptor activity of endogenous tick pyrokinins and analogs: Towards discovering pyrokinin function in ticks.

Neuropeptides regulate many important physiological processes in animals. The G protein-coupled receptors of corresponding small neuropeptide ligands are considered promising targets for controlling arthropod pests. Pyrokinins (PKs) are pleiotropic neuropeptides that, in some insect species, stimulate muscle contraction and modulate pheromone biosynthesis, embryonic diapause, and feeding behavior. However, their function remains unknown in ticks. In this study, we reported the myotropic activity of tick endogenous PKs and a PK agonist analog, PK-PEG (MS[PEG]-YFTPRLa), on feeding tissues of two tick species representing the family Ixodidae lineages, namely, Prostriata () and Metastriata (). First, we predicted the sequences of two periviscerokinins (PVK), one with a derived ending RNa and five PKs encoded by the CAPA peptide precursor from  and found the encoded PKs were identical to those of  identified previously. The pharynx-esophagus of both tick species responded with increased contractions to 10 μM of the endogenous PK as well as to PK-PEG but not to the scrambled PK peptide, as expected. A dose-dependent myotropic activity of the PK-PEG was found for both tick species, validating the analog activity previously found in the pyrokinin recombinant receptor assay. In agreement with the tissue activity elicited, we quantified the relative transcript abundance of  PK receptor in unfed female ticks and found it was the highest in the feeding tissues extracted from the capitulum and lowest in the reproductive tissue. This is the first report of the activity of pyrokinins in ticks. These findings strongly indicate the potential role of PKs in regulating tick blood feeding and therefore, making the tick PK receptor a potential target for interference.

Myotropic Activities of Tick Pyrokinin Neuropeptides and Analog in Feeding Tissues of Hard Ticks (Ixodidae).

Rhipicephalus microplus is the vector of deadly cattle pathogens, especially Babesia spp., for which a recombinant vaccine is not available. Therefore, disease control depends on tick vector control. However, R. microplus populations worldwide have developed resistance to available acaricides, prompting the search for novel acaricide targets. G protein-coupled receptors (GPCRs) are involved in the regulation of many physiological processes and have been suggested as druggable targets for the control of arthropod vectors. Arthropod-specific signaling systems of small neuropeptides are being investigated for this purpose. The pyrokinin receptor (PKR) is a GPCR previously characterized in ticks. Myotropic activity of pyrokinins in feeding-related tissues of Rhipicephalus sanguineus and Ixodes scapularis was recently reported.

Pyrokinin receptor silencing in females of the southern cattle tick Rhipicephalus (Boophilus) microplus is associated with a reproductive fitness cost.

Caixing Xiong

Department of Entomology

Texas A&M University

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

Caixing Xiong

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Entomology

Texas A&M University

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

Department of Entomology