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Potassium Channel Inhibition as a New Insecticide Target for Mosquito Control

By: Carol A. Rouzer, VICB Communications
Published: June 10, 2013


A small molecule inhibitor of potassium flux disrupts renal function in mosquitoes, leading to incapacitation or death.

Mosquitoes are not only annoying, they are vectors for serious human diseases. Mosquitoes of the genus Anopheles carry malaria, while those of the genus Culex carry viruses, including the causative agents of West Nile, dengue, and yellow fevers, and chikungunya. Disease transmission occurs when a female mosquito takes a blood meal from an infected host and then carries the infectious agent to an uninfected host during a subsequent blood meal. Only female mosquitoes feed on blood, which they require as a source of nutrients critical for egg development (Figure 1).

Figure 1. Anopheles freeborni mosquito taking a blood meal. Image reproduced from the Centers for Disease Control http://www.cdc.gov/malaria/about/biology/mosquitoes/, CDC, public domain.


To date, insecticides used for mosquito control have almost exclusively targeted the insect's nervous system. The increasing development of resistance to these conventional insecticides poses a serious threat to our ability to control the spread of mosquito-borne diseases. This challenge led VICB member Jerod Denton and his colleagues Peter Piermarini at Ohio State University and Klaus Beyenbach at Cornell University to explore renal potassium transport as a novel target for the development of mosquito insecticides [R. Raphemot et al., (2013) PLoS One, 8, e64905].

The Malpighian tubules serve as the mosquito’s kidneys, generating urine that is emptied into the hindgut and then ejected to eliminate metabolic waste products. Formation of urine requires transport of NaCl, KCl, and other solutes, a process that must be carefully regulated to maintain fluid homeostasis and electrolyte balance. Disruption of this process would likely lead to death of the insect. Uptake of K+ into the epithelium of the Malpighian tubule is carried out, in part, by inwardly rectifying potassium channels (Kir). In Malpighian tubules from Ae. aegypti, genes for AeKir1, AeKir2B, and AeKir3 are all expressed. Of these three channels, expression of AeKir1 in Xenopus oocytes produced the most robust ion currents, so the investigators focused their intention on this mosquito protein.

Prior work had identified VU573, a small molecule inhibitor of several human potassium channels (Figure 2). Using the patch clamp method to measure ion flux, the investigators found that VU573 also inhibited AeKir1 with an IC50 of 5.14 μM. This level of potency was similar to that seen for human channels Kir2.3, Kir3.x, and Kir7.1. The researchers also evaluated the potency of VU573 in a high-throughput screen that measured Kir-dependent thallium flux using a thallium-sensitive fluorescent dye. In that assay, using cells expressing AeKir1, VU573 inhibited channel activity with an IC50 of 15 μM. This result was consistent with prior studies showing that most compounds are less potent in the thallium flux assay than in the patch clamp assay. Using the high-throughput thallium-based screen, the investigators evaluated a library of VU573 analogs in search of compounds with greater potency against AeKir1. Unfortunately, these efforts yielded only a less potent compound, VU342 (IC50 > 100 μM), which subsequently served as a negative control.

Figure 2. Structures of VU573 and VU342.

 

Injection of VU573 into female Ae. aegypti mosquitoes led to incapacitation (inability to fly) or death within 24 h. The effect was dose-dependent, with an ED50 of 53.6 pmol. Some of the incapacitated mosquitoes exhibited markedly distended abdomens, consistent with fluid imbalance due to renal failure (Figure 3). Similar results were observed with An. gambiae, Ae. albopictus, and C. pipiens, indicating that VU573’s effects were applicable to multiple mosquito species. VU342 was inactive in this assay.

 

Figure 3. Ae. aegypti mosquitoes treated with vehicle (top) or VU573 (bottom). Bloating was observed in some VU573-treated mosquitoes Image reproduced under the Creative Commons Attribution License from R. Raphemot et al., (2013) PLoS One, 8, e64905.


To determine if VU573’s in vivo effects were due to blockade of renal function, the investigators measured the rate of fluid secretion in isolated mosquito Malpighian tubules incubated in the presence or absence of the compound. They found that VU573, but not VU342, suppressed fluid secretion in the isolated tubules. VU573 also induced hyperpolarization of the basolateral membrane of the principal cells of the Malpighian tubules. These observations were all consistent with Kir channel blockade.

In their intial in vivo studies, the researchers injected VU573 in a Na+-containing buffer. Under these conditions, 92% of the mosquitoes incapacitated by the compound were rendered flightless, while only 8% died within 24 h. The observation that K+-containing buffers increased the efficacy of VU573 on isolated Malpighian tubules led the investigators to repeat their in vivo studies using a vehicle containing K+. They found that subjecting the mosquitoes to a K+ load substantially increased the lethality of VU573 to nearly 80% (Figure 4). These findings are of particular interest because the amount of K+ injected was similar to the amount that would be present in a blood meal. Thus, VU573 is expected to be most toxic in actively feeding female mosquitoes.



Figure 4. Treatment of mosquitoes with VU573 in K+-containing vehicle resulted in a much higher rate of lethality than administration in a Na+-containing vehicle. Image reproduced under the Creative Commons Attribution License from R. Raphemot et al., (2013) PLoS One, 8, e64905.

The results suggest that renal function disruption through potassium channel blockade is a viable target for the development of novel mosquito insecticides. The investigators note that the major challenge going forward will be the discovery of more potent compounds that inhibit mosquito Kir channels with no effect on K+ transport in mammalian cells. The compounds must also be amenable to distribution to the target insect population. These goals are currently the focus of ongoing research in the Denton, Piermarini, and Beyenback laboratories.




 

 


 


 

 

 


 

 
     

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