Discovery and Characterization of a Potent and Selective Inhibitor of Aedes aegypti Inward Rectifier Potassium Channels

Abstract

Vector-borne diseases such as dengue fever and malaria, which are transmitted by infected female mosquitoes, affect nearly half of the world's population. The emergence of insecticide-resistant mosquito populations is reducing the effectiveness of conventional insecticides and threatening current vector control strategies, which has created an urgent need to identify new molecular targets against which novel classes of insecticides can be developed. We previously demonstrated that small molecule inhibitors of mammalian Kir channels represent promising chemicals for new mosquitocide development. In this study, high-throughput screening of approximately 30,000 chemically diverse small-molecules was employed to discover potent and selective inhibitors of Aedes aegypti Kir1 (AeKir1) channels heterologously expressed in HEK293 cells. Of 283 confirmed screening ‘hits’, the small-molecule inhibitor VU625 was selected for lead optimization and in vivo studies based on its potency and selectivity toward AeKir1, and tractability for medicinal chemistry. In patch clamp electrophysiology experiments of HEK293 cells, VU625 inhibits AeKir1 with an IC50 value of 96.8 nM, making VU625 the most potent inhibitor of AeKir1 described to date. Furthermore, electrophysiology experiments in Xenopus oocytes revealed that VU625 is a weak inhibitor of AeKir2B. Surprisingly, injection of VU625 failed to elicit significant effects on mosquito behavior, urine excretion, or survival. However, when co-injected with probenecid, VU625 inhibited the excretory capacity of mosquitoes and was toxic, suggesting that the compound is a substrate of organic anion and/or ATP-binding cassette (ABC) transporters. The dose-toxicity relationship of VU625 (when co-injected with probenecid) is biphasic, which is consistent with the molecule inhibiting both AeKir1 and AeKir2B with different potencies. This study demonstrates proof-of-concept that potent and highly selective inhibitors of mosquito Kir channels can be developed using conventional drug discovery approaches. Furthermore, it reinforces the notion that the physical and chemical properties that determine a compound's bioavailability in vivo will be critical in determining the efficacy of Kir channel inhibitors as insecticides.

An insecticide resistance-breaking mosquitocide targeting inward rectifier potassium channels in vectors of Zika virus and malaria

“…A signature feature of inhibiting Kir channels in mosquitoes is impairment of fluid secretion/urine production in Malpighian tubules, which reduces the mosquito’s capacity for diuresis15161718. Diuresis plays an especially important role in adult female mosquitoes after a blood meal by excreting the excess fluid and electrolytes that are absorbed into the hemolymph2627.…”

“…aegypti ), pharmacological inhibition of Kir1 with structurally-distinct small molecules (i.e. VU573, VU590, or VU625) disrupts the secretion of fluid and K + in isolated Malpighian tubules, and impairs flight, urine production, and K + homeostasis in intact females15161718. A major limitation of the potential use of these inhibitors as adulticides is their inability to penetrate the mosquito cuticle, thereby requiring microinjection to induce toxicity.…”

Two inwardly rectifying potassium channels,Irk1andIrk2, play redundant roles inDrosophilarenal tubule function

“…In addition, transcripts for inwardly rectifying K ϩ channels are expressed in the Malpighian tubules of Aedes aegypti, the vector for yellow fever, dengue, and chikungunya (29); Anopheles gambiae, the malaria vector (30); and the bed bug Cimex lectularius (21). Drugs targeting renal tubule inwardly rectifying K ϩ channels are currently being developed as mosquitocidal insecticides in Aedes aegypti (31,32,38). These channels may represent targets for the control of other insect pests as well, although killing of benign or beneficial insects will need to be avoided.…”

“…In contrast, compounds that inhibit AeKir1 and AeKir2B have inhibitory effects on whole-mosquito urine excretion (31,32,38) and on transepithelial fluid secretion and K ϩ flux in the Ramsay assay (28,36). This is broadly consistent with our results that both Irk1 and Irk2 play roles in the fly tubule although there are interesting differences; in Aedes, AeKir1 is located in the stellate cell, whereas AeKir2B is in the principal cell, and inhibition of both AeKir1 and AeKir2 has additive effects on K ϩ flux.…”

“…For example, in mosquitoes, pharmacological inhibition of inwardly rectifying K ϩ channels decreases renal tubule fluid secretion and alters both transepithelial ion fluxes in the tubule as well as whole-body ion clearances and urine excretion (28,31,32,36,38,41). This results in decreased viability and flight capacity (31,32,38). As such, inwardly rectifying K ϩ channels are potential targets for insecticidal agents.…”