Vanderbilt Institute of Chemical Biology



Discovery at the VICB







A New Tool for Mosquito Control


By: Carol A. Rouzer, VICB Communications
Published: December 14, 2016



Novel compounds that block inwardly rectifying potassium channels offer hope for new, more effective insecticides against disease-carrying mosquitoes.


One can easily argue that mosquitoes (Figure 1) are our most dangerous insects due to their ability to carry and transmit numerous diseases, including malaria, yellow fever, dengue fever, Zika, chikungunya, and filariasis. Annually, mosquito-borne diseases strike over 700 million people, killing as many as one million around the world. A major weapon against these diseases is the use of various insecticides to control mosquito populations; however, increasing insecticide resistance is reducing the effectiveness of this approach. The two major classes of insecticides are the pyrethrins, which block inactivation of voltage-gated sodium channels, and carbamates/organophosphates, which inhibit the enzyme acetylcholinesterase. Both of these target insects at all stages of their life cycle, thereby creating a high selection pressure to develop resistance. The need for new insecticides that act via novel mechanisms is great, leading Vanderbilt Institute of Chemical Biology member Jerod Denton and his collaborator Peter Piermarini (Ohio State) to explore the inward rectifier class of potassium channels (Kir) as potential insecticide targets [D. R. Swale, et al. (2016) Sci. Rep., 6, 36954].


FIGURE 1. Anopheles freeborni mosquito taking a blood meal.  Image reproduced from the Centers for Disease Control, CDC, public domain.



Kir channels play a major role in stabilizing the resting membrane potential and are important for normal function of numerous tissues including heart, skeletal muscle, kidney, brain, and vasculature in mammals. Prior studies had shown that deletion of the genes encoding Kir1, Kir2, or Kir3 in the fruit fly (Drosophila melanogaster) led to death or abnormal wing development. Knockdown of Kir1 and Kir2 expression in the heart and renal tissues of adult flies altered immune responses and fluid secretion, respectively. Prior studies in the Denton and Piermarini labs had shown that small molecule inhibitors of Kir1, such as VU625 (Figure 2), disrupt renal function, impairing fluid balance and urine production in adult Aedes aegypti mosquitoes, which carry yellow fever, dengue, Zika, and other diseases. These findings led to their hypothesis that Kir1 channel inhibitors could be developed as a new class of insecticides for mosquito control. However, the compounds they initially identified did not penetrate the insect cuticle, and therefore could not be administered topically. Thus, their goal was to discover the next generation of compounds in order to overcome this barrier.



FIGURE 2. Structures of active (VU625, VU041, and VU730) and inactive (VU937) Kir1 inhibitiors. IC50 values are those obtained in the whole cell patch clamp assay).



The investigators screened 26,000 compounds for their ability to modulate the channel activity of Kir1 from the malaria vector Anopheles gambiae (AnKir1). The screen was based on the ability of potassium channels to conduct thallium ions nearly as well as potassium ions. By preloading HEK293 cells expressing AnKir1 with a Tl+-sensitive dye and then exposing the cells to medium containing Tl+, the investigators could measure channel activity – and the effects of compounds on that activity – by the change in fluorescence emission of the dye. The screen identified 121 confirmed AnKir1 inhibitors. Among them was VU041 (Figure 2), a compound of particular interest due to its high partition coefficient, a property that facilitates penetrance of the insect cuticle. Patch clamp assays in intact cells confirmed the activity of VU041 (IC50 = 496 nM). The compound was not as potent as the previously discovered VU625, but the investigators were willing to accept some loss in potency in exchange for cuticle penetrance. Further Tl+ flux assays confirmed VU041's ability to block both AnKir1 and AeKir1 (the channel from Ae. aegypti) with IC50 values of 2.5 μM and 1.7 μM, respectively. Notably, the compound also exhibited very poor activity against most mammalian Kir channels. The one important exception was its ability to block mammalian Kir2.1 with reasonable potency  (IC50 = 12.7 μM).


Extensive lead optimization efforts failed to yield a compound with significantly improved potency or penetrance than VU041. However, these experiments did produce VU730, a compound with a partition coefficient and potency similar to those of VU041 (IC50 = 2.5 μM in Tl+ flux assay and 717 nM in patch clamp assay) but lacking activity against Kir2.1. The investigators also identified VU937, a compound structurally similar to VU041 and VU730 that lacked activity against AnKir1 or AeKir1, to serve as a negative control.


Toxicity studies evaluated the effects of VU041 following topical application using death or flightlessness as an endpoint for An. gambiae and Ae. aegypti, respectively. In both cases, the compound was equally active against strains of mosquitoes that were resistant or sensitive to conventional insecticides. Exposure of the mosquitoes to an inhibitor of cytochromes P450 had only a moderate effect on potency, suggesting that VU041 is not rapidly or extensively metabolized by these enzymes.


As prior studies had shown that Kir1 inhibitors impair urine production in insect renal tissues (Malpighian tubules), the investigators evaluated the ability of VU041 to block the elimination of excess fluid and electrolytes taken in during a blood meal. They found that the normal abdominal swelling that occurs after a blood meal did not recede in An. gambaie mosquitoes treated with a sublethal dose of VU041 (Figure 3). Further tests confirmed that VU041 blocks diuresis (formation of urine in response to a fluid stress) in Ae. aegypti. These effects, which were not observed in mosquitoes treated with VU937, provide additional evidence that Kir1 channel blockade interferes with renal function in mosquitoes.


FIGURE 3. Photographs of the abdomens of female An. gambaie mosquitoes 24 h after the following treatments: NBF, no blood meal, no inhibitor control; Vehicle, blood meal-fed and vehicle-treated; VU937, blood meal-fed and UV937-treated; VU041, blood meal-fed and VU041-treated. Figure reproduced under a Creative Commons Attribution 4.0 International License from D. R. Swale, et al. (2016) Sci. Rep., 6, 36954.



Female mosquitoes require a blood meal prior to egg laying. The interference of blood meal processing by VU041 suggested that the compound might also reduce fertility in female mosquitoes. Indeed, this proved to be the case. Treatment with sublethal doses of VU041 following a blood meal significantly reduced the number of eggs laid by An. gambiae and Ae. aegypti mosquitoes.


A major challenge to developing insecticides with novel mechanisms is finding ones that are selective for mosquitoes over beneficial insects such as pollinator bees. For this reason, the finding that treatment of the honey bee A. mellifera with doses of VU041 that would be toxic to mosquitoes had no significant effect on bee survival is particularly interesting.


In summary, VU041 and VU730 are prototypes of a new class of insecticide that targets Kir1 channels. The compounds are topically active and exhibit selectivity for mosquitoes over both mammals and at least some beneficial insects. These exciting results establish the foundation for further optimization of the compounds in the hopes of obtaining an effective new means to control life-threatening mosquito populations.




View Scientific Reports article: An insecticide resistance-breaking mosquitocide targeting inward rectifier potassium channels in vectors of Zika virus and malaria










The Vanderbilt Institute of Chemical Biology, 896 Preston Building, Nashville, TN 37232-6304, phone 866.303 VICB (8422), fax 615 936 3884
Vanderbilt University is committed to principles of equal opportunity and affirmative action. Copyright © 2014 by Vanderbilt University Medical Center