Caixing Xiong

High-throughput screening of a chemical library on the recombinant kinin receptor from the southern cattle tick, Rhipicephalus microplus (Acari: Ixodidae)


The southern cattle tick, Rhipicephalus microplus, is a primary vector transmitting the deadly bovine babesiosis, and an increasing threat to the Texas Cattle industry. Recent tick outbreaks in the north Texas quarantine zone emphasize the importance of tick control. Due to the wide-spread global resistance to acaricides in this tick species population, it is urgent to discover novel targets for tick control. The invertebrate-specific kinin receptor (KR) is one of such targets, previously validated by RNAi: silencing of the tick KR is associated with significant female reproductive costs. This study aimed to identify potent small molecules that bind and activate (agonists) or inhibit (antagonists) the kinin receptor to modulate tick physiology. 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 at 2 µM in a ‘dual-addition’ calcium fluorescence assay. Because hits in HTS could be false positives, it is necessary to perform dose-response validation. After verification of the potency of 18 antagonists, their structures were used for an in silico screen of a larger compound library. These two approaches yielded 29 validated antagonists, 11 of them were full antagonists with IC50 values lower than 10 µM. To explore the structure-activity relationships of the small molecules, we tested the activities of 7 analogs of one of the most potent identified antagonists, additionally discovering three full antagonists and four partial antagonists. These three potent antagonists (IC50 < 3.2 µM) were validated as antagonists on the recombinant mosquito kinin receptor in vitro. This is the first time HTS was used to screen for neuropeptide GPCR ligands in any tick species. These active molecules are promising tools to study mosquito and tick physiology and potentially useful for pest control.


Comments are closed.