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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]

Mosquitoes are important disease vectors that transmit a wide variety of pathogens to humans, including those that cause malaria and dengue fever. Insecticides have traditionally been deployed to control populations of disease-causing mosquitoes, but the emergence of insecticide resistance has severely limited the number of active compounds that are used against mosquitoes. Thus, to improve the control of resistant mosquitoes there is a need to identify new insecticide targets and active compounds for insecticide development. Recently we demonstrated that inward rectifier potassium (Kir) channels and small molecule inhibitors of Kir channels offer promising new molecular targets and active compounds, respectively, for insecticide development. Here we provide pharmacological validation of a specific mosquito Kir channel (AeKir1) in the yellow fever mosquito Aedes aegypti. We show that VU590, a small-molecule inhibitor of mammalian Kir1.1 and Kir7.1 channels, potently inhibits AeKir1 but not another mosquito Kir channel (AeKir2B) in vitro. Moreover, we show that a previously identified inhibitor of AeKir1 (VU573) elicits an unexpected agonistic effect on AeKir2B in vitro. Injection of VU590 into the hemolymph of adult female mosquitoes significantly inhibits their capacity to excrete urine and kills them within 24 h, suggesting a mechanism of action on the excretory system. Importantly, a structurally-related VU590 analog (VU608), which weakly blocks AeKir1 in vitro, has no significant effects on their excretory capacity and does not kill mosquitoes. These observations suggest that the toxic effects of VU590 are associated with its inhibition of AeKir1. [ABSTRACT FROM AUTHOR]

The inward rectifier potassium (Kir) channels play key roles in the physiology of mosquitoes and other insects. Our group, among others, previously demonstrated that small molecule inhibitors of Kir channels are promising lead molecules for developing new insecticides to control adult female mosquitoes. However, the potential use of Kir channel inhibitors as larvicidal agents is unknown. Here we tested the hypothesis that pharmacological inhibition of Kir channels in the larvae of Aedes aegypti, the vector of several medically important arboviruses, induces lethality. We demonstrated that adding barium, a non-specific blocker of Kir channels, or VU041, a specific small-molecule inhibitor of mosquito Kir1 channels, to the rearing water (deionized H2O) of first instar larvae killed them within 48 h. We further showed that the toxic efficacy of VU041 within 24 h was significantly enhanced by increasing the osmolality of the rearing water to 100 mOsm/kg H2O with NaCl, KCl or mannitol; KCl provided the strongest enhancement compared to NaCl and mannitol. These data suggest: (1) the important role of Kir channels in the acclimation of larvae to elevated ambient osmolality and KCl concentrations; and (2) the disruption of osmoregulation as a potential mechanism of the toxic action of VU041. The present study provides the first evidence that inhibition of Kir channels is lethal to larval mosquitoes and broadens the potential applications of our existing arsenal of small molecule inhibitors of Kir channels, which have previously only been considered for developing adulticides. [ABSTRACT FROM AUTHOR]

Mosquitoes are important disease vectors that transmit a wide variety of pathogens to humans, including those that cause malaria and dengue fever. Insecticides have traditionally been deployed to control populations of disease-causing mosquitoes, but the emergence of insecticide resistance has severely limited the number of active compounds that are used against mosquitoes. Thus, to improve the control of resistant mosquitoes there is a need to identify new insecticide targets and active compounds for insecticide development. Recently we demonstrated that inward rectifier potassium (Kir) channels and small molecule inhibitors of Kir channels offer promising new molecular targets and active compounds, respectively, for insecticide development. Here we provide pharmacological validation of a specific mosquito Kir channel (AeKir1) in the yellow fever mosquito Aedes aegypti. We show that VU590, a small-molecule inhibitor of mammalian Kir1.1 and Kir7.1 channels, potently inhibits AeKir1 but not another mosquito Kir channel (AeKir2B) in vitro. Moreover, we show that a previously identified inhibitor of AeKir1 (VU573) elicits an unexpected agonistic effect on AeKir2B in vitro. Injection of VU590 into the hemolymph of adult female mosquitoes significantly inhibits their capacity to excrete urine and kills them within 24 h, suggesting a mechanism of action on the excretory system. Importantly, a structurally-related VU590 analog (VU608), which weakly blocks AeKir1 in vitro, has no significant effects on their excretory capacity and does not kill mosquitoes. These observations suggest that the toxic effects of VU590 are associated with its inhibition of AeKir1. [ABSTRACT FROM AUTHOR]

Inward rectifier potassium (Kir) channels play fundamental roles in neuromuscular, epithelial, and endocrine function in mammals. Recent research in insects suggests that Kir channels play critical roles in the development, immune function, and excretory physiology of fruit flies and/or mosquitoes. Moreover, our group has demonstrated that mosquito Kir channels may serve as valuable targets for the development of novel insecticides. Here we characterize the molecular expression of 5 mRNAs encoding Kir channel subunits in the yellow fever mosquito, Aedes aegypti : Kir1 , Kir2A-c , Kir2B , Kir2B’ , and Kir3 . We demonstrate that 1) Kir mRNA expression is dynamic in whole mosquitoes, Malpighian tubules, and the midgut during development from 4th instar larvae to adult females, 2) Kir2B and Kir3 mRNA levels are reduced in 4th instar larvae when reared in water containing an elevated concentration (50 mM) of KCl, but not NaCl, and 3) Kir mRNAs are differentially expressed in the Malpighian tubules, midgut, and ovaries within 24 h after blood feeding. Furthermore, we provide the first characterization of Kir mRNA expression in the anal papillae of 4th instar larval mosquitoes, which indicates that Kir2A-c is the most abundant. Altogether, the data provide the first comprehensive characterization of Kir mRNA expression in Ae. aegypti and offer insights into the putative physiological roles of Kir subunits in this important disease vector. [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]