Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
PLoS One
2014 Jan 01;911:e110772. doi: 10.1371/journal.pone.0110772.
Show Gene links
Show Anatomy links
Discovery and characterization of a potent and selective inhibitor of Aedes aegypti inward rectifier potassium channels.
Raphemot R
,
Rouhier MF
,
Swale DR
,
Days E
,
Weaver CD
,
Lovell KM
,
Konkel LC
,
Engers DW
,
Bollinger SR
,
Bollinger SF
,
Hopkins C
,
Piermarini PM
,
Denton JS
.
???displayArticle.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.
???displayArticle.pubmedLink???
25375326
???displayArticle.pmcLink???PMC4222822 ???displayArticle.link???PLoS One ???displayArticle.grants???[+]
Figure 2. VU625 is a potent inhibitor of AeKir1 in Tl+ flux assays.(A) Chemical structure of VU625. (B) Dose-dependent inhibition of the AeKir1-mediated Tl+ flux by VU625 with concentrations ranging from ≤0.12 to 30 µM. The arrow indicates when Tl+ was added to the extracellular bath. (C) Concentration-response curves of VU625 derived from Tl+ flux assays. The IC50 and Hill-coefficient (nH) values are 315 nM (95% CI: 254.4–390.2 nM) and 0.98 respectively. Data are mean ±SEM. n = 4 independent experiments performed in triplicate.
Figure 3. VU625 is a potent and preferential inhibitor of AeKir1 over AeKir2B in whole-cell electrophysiology.(A) Normalized AeKir1 current-voltage relationships obtained from heterologous expression in T-Rex-HEK293 cells, illustrating VU625-dependent inhibition before (control) and after addition of 0.9 µM VU625. Residual AeKir1 currents were inhibited with 2 mM barium. Cells were voltage clamped at −75 mV and ramped between −120 mV and +60 mV. (B) Concentration-response curve of VU625 derived from patch clamp experiments (n = 4–6). The IC50 of VU625 is 96.8 nM (95% CI: 75.4–124.2 nM). (C) Concentration-response curves of current inhibition mediated by heterologous expression in Xenopus oocytes of AeKir1 (filled circles) and AeKir2B (open circles) channels after bath application of VU625. n = 4–5 oocytes per concentration. The calculated IC50 values of VU625 for AeKir1 and AeKir2B current inhibition are 3.8 µM (95% CI: 2.3–6.3 µM) and 45.1 µM (95% CI: 31.7–64.2 µM), respectively.
Figure 4. Design and chemical lead optimization strategy for VU625.(A) Modular approach to assess three areas of diversification of VU625: sulfonamide (red shading), central core (green shading), and southern amide (blue shading) portions. (B) General synthetic approach to access VU625 and analogs around the amide and sulfonamide portions.
Figure 5. Summary of structure-activity relationship (SAR).Summary of observed SAR of over 100 analogs synthesized exploring all three regions of VU625.
Figure 6. Effects of probenecid and VU625 on survival of adult female mosquitoes (A. aegypti).Percent mortality of mosquitoes at 24 h post-injection. Each mosquito was injected with 69 nl of the vehicle containing VU625 (10 mM), probenecid (50 mM), or both, to deliver the desired doses: 690 pmol of VU625, 3.4 nmol probenecid. n = 6–7 trials of 10 mosquitoes each per treatment. Lower-case letters indicate statistical categorization of the means as determined by a one-way ANOVA with a Newman-Keuls post-test (P<0.05).
Figure 7. The dose-response curve of the toxic effects of VU625 on adult female mosquitoes (A. aegypti) is biphasic.Normalized percent mortality of mosquitoes at 24 h post-injection. Each mosquito was injected with 69 nL of the vehicle containing probenecid (50 mM) and an appropriate concentration of VU625 to deliver the doses of VU625 indicated and 3.4 nmol of probenecid. The ED25 and ED75 were determined by fitting a non-linear biphasic curve to the data. n = 3–4 trials of 10 mosquitoes each per dose.
Figure 8. Effects of probenecid and VU625 on the in vivo excretory capacity of adult female mosquitoes (A. aegypti).Amount of urine excreted by mosquitoes 1 h after injection with 900 nL of the vehicle (K+-PBS50 containing 1.8% DMSO, 0.077% β-cyclodextrin, and 0.008% Solutol), or the vehicle containing VU625 (0.77 mM), probenecid (3.85 mM), or both, to deliver the desired doses: 690 pmol of VU625, 3.4 nmol probenecid. Values are means ±SEM; n = 6–18 trials of 5 mosquitoes per treatment. Lower-case letters indicate statistical categorization of the means as determined by a one-way ANOVA with a Newman-Keuls posttest (P<0.05).
Figure 1. Tl+ flux assay of AeKir1 channel activity for high-throughput screening.(A) Representative Tl+-induced changes in Thallos fluorescence in T-Rex-HEK293-AeKir1 cells cultured overnight with (+Tet) or without (-Tet) tetracycline. The shaded box indicates the cell exposure to Tl+. (B) DMSO concentrations up to 1.3% v/v DMSO have no effect on Tl+ flux through AeKir1. Data are means ±SEM (n = 3). One-way ANOVA P<0.0001, and asterisks (**, ***) indicate P<0.01 or P<0.001 respectively, when compared to 0% DMSO (Tukey's test). (C) Representative checkerboard analysis using 100 µM VU573 or 0.1% v/v DMSO as the vehicle control. The mean peak fluorescence amplitude of each sample population is indicated with a solid line and alternating samples for DMSO (top) and VU573 (bottom) are graphed as individual points. The mean ±SD Z′ calculated over 6 plates on 3 separate days was 0.69±0.05.
,
Correction: Discovery and characterization of a potent and selective inhibitor of Aedes aegypti inward rectifier potassium channels.
2015, Pubmed
,
Correction: Discovery and characterization of a potent and selective inhibitor of Aedes aegypti inward rectifier potassium channels.
2015,
Pubmed
Akamatsu,
Importance of physicochemical properties for the design of new pesticides.
2011,
Pubmed
Asidi,
Loss of household protection from use of insecticide-treated nets against pyrethroid-resistant mosquitoes, benin.
2012,
Pubmed
Baker,
A comprehensive gene expression atlas of sex- and tissue-specificity in the malaria vector, Anopheles gambiae.
2011,
Pubmed
Bhave,
Development of a selective small-molecule inhibitor of Kir1.1, the renal outer medullary potassium channel.
2011,
Pubmed
,
Xenbase
Dahal,
An inwardly rectifying K+ channel is required for patterning.
2012,
Pubmed
Denton,
Novel diuretic targets.
2013,
Pubmed
Denton,
Channeling dysglycemia: ion-channel variations perturbing glucose homeostasis.
2012,
Pubmed
Dermauw,
The ABC gene family in arthropods: comparative genomics and role in insecticide transport and resistance.
2014,
Pubmed
Döring,
Inwardly rectifying K+ (Kir) channels in Drosophila. A crucial role of cellular milieu factors Kir channel function.
2002,
Pubmed
,
Xenbase
Eleftherianos,
ATP-sensitive potassium channel (K(ATP))-dependent regulation of cardiotropic viral infections.
2011,
Pubmed
Evans,
Sulphonylurea sensitivity and enriched expression implicate inward rectifier K+ channels in Drosophila melanogaster renal function.
2005,
Pubmed
Feller,
ATP-dependent efflux of calcein by the multidrug resistance protein (MRP): no inhibition by intracellular glutathione depletion.
1995,
Pubmed
Hibino,
Inwardly rectifying potassium channels: their structure, function, and physiological roles.
2010,
Pubmed
Hill,
The anti-influenza drug oseltamivir exhibits low potential to induce pharmacokinetic drug interactions via renal secretion-correlation of in vivo and in vitro studies.
2002,
Pubmed
Jaehde,
Effect of probenecid on the distribution and elimination of ciprofloxacin in humans.
1995,
Pubmed
Lewis,
High-throughput screening reveals a small-molecule inhibitor of the renal outer medullary potassium channel and Kir7.1.
2009,
Pubmed
Li,
Molecular mechanisms of metabolic resistance to synthetic and natural xenobiotics.
2007,
Pubmed
Meyer,
A "genome-to-lead" approach for insecticide discovery: pharmacological characterization and screening of Aedes aegypti D(1)-like dopamine receptors.
2012,
Pubmed
Niswender,
A novel assay of Gi/o-linked G protein-coupled receptor coupling to potassium channels provides new insights into the pharmacology of the group III metabotropic glutamate receptors.
2008,
Pubmed
O'Donnell,
Too much of a good thing: how insects cope with excess ions or toxins in the diet.
2009,
Pubmed
Pattnaik,
Genetic defects in the hotspot of inwardly rectifying K(+) (Kir) channels and their metabolic consequences: a review.
2012,
Pubmed
Piermarini,
Cloning and functional characterization of inward-rectifying potassium (Kir) channels from Malpighian tubules of the mosquito Aedes aegypti.
2013,
Pubmed
,
Xenbase
Ranson,
Pyrethroid resistance in African anopheline mosquitoes: what are the implications for malaria control?
2011,
Pubmed
Raphemot,
Development and validation of fluorescence-based and automated patch clamp-based functional assays for the inward rectifier potassium channel Kir4.1.
2013,
Pubmed
Raphemot,
Molecular and functional characterization of Anopheles gambiae inward rectifier potassium (Kir1) channels: a novel role in egg production.
2014,
Pubmed
,
Xenbase
Raphemot,
Discovery, characterization, and structure-activity relationships of an inhibitor of inward rectifier potassium (Kir) channels with preference for Kir2.3, Kir3.x, and Kir7.1.
2011,
Pubmed
,
Xenbase
Raphemot,
Direct activation of β-cell KATP channels with a novel xanthine derivative.
2014,
Pubmed
Raphemot,
Eliciting renal failure in mosquitoes with a small-molecule inhibitor of inward-rectifying potassium channels.
2013,
Pubmed
Raphemot,
High-throughput screening for small-molecule modulators of inward rectifier potassium channels.
2013,
Pubmed
Rouhier,
Identification of life-stage and tissue-specific splice variants of an inward rectifying potassium (Kir) channel in the yellow fever mosquito Aedes aegypti.
2014,
Pubmed
Rouhier,
Pharmacological validation of an inward-rectifier potassium (Kir) channel as an insecticide target in the yellow fever mosquito Aedes aegypti.
2014,
Pubmed
Tice,
Selecting the right compounds for screening: does Lipinski's Rule of 5 for pharmaceuticals apply to agrochemicals?
2001,
Pubmed