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

Abstract

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

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

“…The Rhimi-CAPA-PK2 is the only predicted PK from the cloned cDNA of R. microplus featuring the conserved motif in its sequence GT F V PRL a , ending in RLa while the other PKs end in RNa or RIa [ 25 ]. The Rhimi-CAPA-PK2 activates the recombinant R. microplus PK receptor with an EC 50 of 188 nM [ 30 ], therefore, it is expected to similarly activate the receptor in vivo. The identical sequence is present in R. sanguineus (Rhisa-CAPA-PK2), while this peptide in I. scapularis is GS F V PRL a , Ixosc-CAPA-PK2 [ 25 ].…”

“…Beyond a few neuropeptides and cognate GPCRs characterized in ticks [ 10 , 23 , 26 – 29 ], little is known about the physiological role of GPCRs in this taxa, but some have shown a potential for interventions after RNA interference (RNAi) experiments by decreasing tick fitness [ 28 ]. Subsequently, high-throughput screens were conducted with the aim to discover new chemistries of antagonists of the tick kinin receptor [ 30 ]. The significance of these approaches is underscored by the lack of recombinant vaccines to prevent deadly babesiosis of cattle caused by Babesia spp.…”

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

“…The success of amitraz as an acaricide that targets the tick octopamine/tyramine receptor is a proof-of-principle ( Kita et al, 2017 ). Efforts have been made in academic settings toward discovering new chemistries against tick GPCR targets such as the leucokinin and dopamine receptors ( Hill et al, 2013 ; Xiong et al, 2021a ). In contrast to the rapid advance in the identification of ligands and GPCRs by means of omics tools, studies on the physiological function of tick neuropeptides are still limited.…”

Identification of a copper-responsive small molecule inhibitor of uropathogenic Escherichia coli

“…This library comprised 51,098 unique small molecules and was assembled by randomly selecting compounds with a high structural diversity and medicinal properties. Details on the construction and characteristics of the screening library have been previously described ( 92 , 93 ). Each compound is dissolved to ~1 mM in DMSO using an average molecular weight of 250 g/mol for the entire library.…”