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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. [ABSTRACT FROM AUTHOR]

Malpighian tubules of adult female yellow fever mosquitoes Aedes aegypti express three inward rectifier K + (Kir) channel subunits: Ae Kir1, Ae Kir2B and Ae Kir3. Here we 1) elucidate the cellular and membrane localization of these three channels in the Malpighian tubules, and 2) characterize the effects of small molecule inhibitors of Ae Kir1 and Ae Kir2B channels (VU compounds) on the transepithelial secretion of fluid and electrolytes and the electrophysiology of isolated Malpighian tubules. Using subunit-specific antibodies, we found that Ae Kir1 and Ae Kir2B localize exclusively to the basolateral membranes of stellate cells and principal cells, respectively; Ae Kir3 localizes within intracellular compartments of both principal and stellate cells. In isolated tubules bathed in a Ringer solution containing 34 mM K + , the peritubular application of VU590 (10 μM), a selective inhibitor of Ae Kir1, inhibited transepithelial fluid secretion 120 min later. The inhibition brings rates of transepithelial KCl and fluid secretion to 54% of the control without a change in transepithelial NaCl secretion. VU590 had no effect on the basolateral membrane voltage (V bl ) of principal cells, but it significantly reduced the cell input conductance (g in ) to values 63% of the control within ∼90 min. In contrast, the peritubular application of VU625 (10 μM), an inhibitor of both Ae Kir1 and Ae Kir2B, started to inhibit transepithelial fluid secretion as early as 60 min later. At 120 min after treatment, VU625 was more efficacious than VU590, inhibiting transepithelial KCl and fluid secretion to ∼35% of the control without a change in transepithelial NaCl secretion. Moreover, VU625 caused the V bl and g in of principal cells to respectively drop to values 62% and 56% of the control values within only ∼30 min. Comparing the effects of VU590 with those of VU625 allowed us to estimate that Ae Kir1 and Ae Kir2B respectively contribute to 46% and 20% of the transepithelial K + secretion when the tubules are bathed in a Ringer solution containing 34 mM K + . Thus, we uncover an important role of Ae Kir1 and stellate cells in transepithelial K + transport under conditions of peritubular K + challenge. The physiological role of Ae Kir3 in intracellular membranes of both stellate and principal cells remains to be determined. [ABSTRACT FROM AUTHOR]