Pharmacological Validation of an Inward-Rectifier Potassium (Kir) Channel as an Insecticide Target in the Yellow Fever Mosquito <i>Aedes aegypti</i>

<div><p>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 (<i>Ae</i>Kir1) in the yellow fever mosquito <i>Aedes aegypti</i>. We show that VU590, a small-molecule inhibitor of mammalian Kir1.1 and Kir7.1 channels, potently inhibits <i>Ae</i>Kir1 but not another mosquito Kir channel (<i>Ae</i>Kir2B) in vitro. Moreover, we show that a previously identified inhibitor of <i>Ae</i>Kir1 (VU573) elicits an unexpected agonistic effect on <i>Ae</i>Kir2B 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 <i>Ae</i>Kir1 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 <i>Ae</i>Kir1.</p></div

Compositions (in mM) of solutions used in oocyte electrophysiology.

<p>The pH of all solutions was adjusted to 7.5 with NMDG-OH.</p><p>The osmolality of each solution was verified to be 190 mOsm kg⁻¹ H₂O (± 5 mOsm kg⁻¹ H₂O) by vapor pressure osmometry.</p><p>NMDG  =  N-methyl-D-glucamine.</p

Effects of VU590, VU608, VU573, and VU342 on the in vivo excretory capacity of adult female mosquitoes (<i>A. aegypti</i>).

<p>(A) 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-cyclodextrane, and 0.008% Solutol), the vehicle containing VU590 (0.77 mM), or the vehicle containing VU608 (0.77 mM). Values are means ± SEM; <i>n</i> = 11 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 (<i>P</i><0.05). (B) Same as ‘A’, but with VU573 and VU342. <i>n</i> = 9 trials of 5 mosquitoes per treatment.</p

Effects of VU590 on the survival of adult female mosquitoes (<i>A. aegypti</i>).

<p>(A) Dose-response curve of the toxic effects of VU590 on mosquitoes (R² = 0.87). Mortality was assessed 24 h after injecting the hemolymph with 69 nl of the vehicle (K⁺-PBS₅₀ with 15% DMSO, 1% β-cyclodextran, and 0.1% Solutol) containing appropriate concentrations of VU590 to deliver the doses indicated. The calculated LD₅₀ is 1.56 nmol (95% CI: 1.29–1.88 nmol). Values are means ± SEM; <i>n</i> = 4 trials of 10 mosquitoes per dose. (B) Comparison of the toxic effects of the vehicle, VU590, and VU608. Mortality was assessed 24 h after injecting the hemolymph with the vehicle (K⁺-PBS₇₅ with 15% DMSO, 1% β-cyclodextran, and 0.1% Solutol) or the vehicle containing a small molecule (2.8 nmol). Values are means ± SEM; <i>n</i> = 4 trials of 10 mosquitoes. Lower-case letters indicate statistical categorization of the means as determined by a one-way ANOVA with a Newman-Keuls posttest (<i>P</i><0.05).</p

Effects of VU590 and VU608 on <i>Ae</i>Kir1 channels expressed heterologously in TREx-HEK293 cells.

<p>(A) Chemical structures of the <i>Ae</i>Kir1 inhibitor VU590 and its ‘inactive’ analog VU608. Gray shading highlights the chemical differences between the molecules. (B) Concentration-response curves of VU590 (filled circles) and VU608 (open circles) derived from Tl⁺-flux assays. Values are means ± SEM. <i>n</i> = 2 independent experiments, each performed in triplicate. The calculated IC₅₀ values for VU590 and VU608 are 5.6 µM (95% CI: 4.3–7.2 µM) and >100 µM, respectively.</p

Effects of VU590 and VU573 on <i>Ae</i>Kir1 and <i>Ae</i>Kir2B channels expressed heterologously in <i>Xenopus</i> oocytes.

<p>Current-voltage (I–V) relationships of representative <i>Ae</i>Kir1 (A, C) and <i>Ae</i>Kir2B (B, D) oocytes bathed consecutively in solutions containing 0.5 mM K⁺ (open boxes), 10 mM K⁺ (filled circles), and 10 mM K⁺ + 50 µM of a small molecule (gray circles). The small molecule is VU590 in panels ‘A’ and ‘B’, and VU573 in panels ‘C’ and ‘D’. (E) Summary of the percent changes of inward currents at −140 mV in <i>Ae</i>Kir1 and <i>Ae</i>Kir2B oocytes elicited by VU590 and VU573. Positive and negative percent changes indicate activation and inhibition, respectively. <i>P</i> values indicate significant activation or inhibition as determined by a one sample t test (on arcsine transformed values). Values are non-transformed means ± SEM. For VU590 experiments, <i>n</i> = 3 oocytes each for <i>Ae</i>Kir1 and <i>Ae</i>Kir2B. For VU573 experiments, <i>n</i> = 5 and 8 oocytes each for <i>Ae</i>Kir1 and <i>Ae</i>Kir2B, respectively.</p

MDM2-ALT1 over-expression leads to G1 phase cell cycle arrest in a p53-dependent and p21-dependent manner.

<p>A. HCT116 wild-type (wt), B. HCT116 <i>p53</i>^−/− and C. HCT116 <i>p21</i>^−/− cells were transfected with myc-tagged GFP or MDM2-ALT1 (2Alt1) or MDMX-ALT2 (XAlt2), harvested 24 hours post-transfection and stained with propidium iodide solution and sorted for DNA content. The bar graphs represent the percentage of cells in the various phases of the cell cycle. Error bars represent the standard error mean from at least 3 independent experiments. HCT116 cells that are wildtype (A) show significantly higher percentage of cells in G1-phase upon MDM2-ALT1 expression (31.44% ±2.45 SEM) compared to GFP-expressing cells (24.40% ±1.40 SEM; n = 5, <i>p</i> = 0.0369). In case of MDMX-ALT2 over-expression in HCT116 wildtype cells, there is no significant change in percentage of cells in G1 phase compared to negative control GFP-expressing cells (<i>p</i> =  0.4389). HCT116 cells that are null for <i>p53</i> (B) or <i>p21</i> (C) do not show any differences in the percentage of cells in any of the cell cycle phases upon over-expression of GFP or MDM2-ALT1 or MDMX-ALT2. D. Representative immuno blot showing expression of p53, p21 and loading control GAPDH in HCT116 <i>wt</i>, <i>p53</i>^−/− and <i>p21</i>^−/− cells.</p

MDM2-ALT1 and MDMX-ALT2 interact with full-length MDM2 and MDMX.

<p>A. Full-length MDM2 is encoded by exons 3 to 12 of the <i>MDM2</i> gene and consists of the N-terminal p53-binding domain, the nuclear localization (NLS) and export signals (NES), the central ARF binding and Zinc finger domains and the C-terminal RING domain. <i>MDM2-ALT1</i> comprises only exons 3 and 12 spliced together and the protein lacks the p53-binding domain. However, it retains the RING domain. <b>B.</b> Full-length MDMX, a close family member of MDM2, also comprises an N-terminal p53-binding domain, a central Zinc finger domain and a C-terminal RING domain and is encoded by exons 2 to 11 of the <i>MDMX</i> gene. <i>MDMX-ALT2</i> consists of exons 2,3,10 and 11 and the protein is architecturally similar to MDM2-ALT1 in that it lacks the p53-binding domain but retains the RING domain. <b>C.</b> Myc-tagged constructs of LacZ, MDM2-ALT1 or MDMX-ALT2 were transfected into MCF7 cells. Immunoprecipitation of the myc-tagged proteins revealed the specific binding of full-length MDM2 to MDM2-ALT1 and MDMX-ALT2 and not to negative control protein myc-LacZ (compare lanes 2 and 3 to lane 1). Experiments were repeated a minimum of three times and consistent results were observed. Representative gel images are presented in the figure. <b>D.</b> Myc-tagged MDM2-ALT1 and MDMX-ALT2 co-immunoprecipitate with full-length MDMX while the negative control protein myc-LacZ does not interact with MDMX (compare lanes 2 and 3 to lane 1). These results were observed in two independent trials and representative images are shown.</p

Proposed model: MDM2-ALT1 and MDMX-ALT2 antagonize their full-length counterparts and lead to p53 stabilization.

<p>A. Under normal conditions, MDM2 and MDMX function to maintain low levels of p53 (via ubiquitination and subsequent degradation) and curb its transcriptional activity by binding p53. This helps maintain homeostasis and normal cellular functions including cell cycle progression. B. Under genotoxic stress, alternative splice forms MDM2-ALT1 and MDMX-ALT2 interact with the full-length MDM proteins and interfere in their p53-regulatory functions. This leads to the stabilization and upregulation of p53 levels and also the activation of p53 transcriptional targets leading to changes in cell cycle progression.</p

MDM2-ALT1 and MDMX-ALT2 expression causes upregulation of p53 and its downstream target p21.

<p><b>A.</b> The over-expression (O/E cell lysates) of the myc-tagged LacZ, MDM2-ALT1 or MDMX-ALT2 was confirmed using anti-myc tag antibody in the MCF7 cells that were transfected with the corresponding expression constructs (top panel). The level of p53 protein was examined in these samples using the anti-p53 antibody and an upregulation of p53 protein was observed upon MDM2-ALT1 or MDMX-ALT2 over-expression compared to LacZ expressing cells although the increase is more modest in MDMX-ALT2 over-expression (lanes 2 and 3 compared to lane 1). The positive control, UVC (50 J/m²) irradiated MCF7 cells show a strong upregulation of p53 protein levels in response to the stress when compared to untreated cells (lanes 4 and 5). β-actin was used as loading control. A minimum of three independent experiments was performed and representative gel images are shown. <b>B. </b><i>p21</i> expression at the mRNA level was examined using quantitative real-time PCR and <i>GAPDH</i> levels were used as the endogenous control. The ratio of <i>p21</i> to <i>GAPDH</i> is represented graphically and the error bars represent standard deviations from at least 3 independent experiments. MCF7 cells over-expressing MDM2-ALT1 (2Alt1) show statistically significant increase in <i>p21</i> transcript levels compared to LacZ expressing cells (p<0.01). The cells expressing MDMX-ALT2 (XAlt2) did not show statistically significant changes in <i>p21</i> expression at the mRNA level. <b>C.</b> The levels of p21 protein in the MCF7 cells transfected with myc-tagged LacZ, MDM2-ALT1 or MDMX-ALT2 was examined using anti-p21 antibody. Both MDM2-ALT1 and MDMX-ALT2 over-expression lead to upregulation of p21 protein levels compared to LacZ over-expression (compare lanes 2 and 3 with lane 1). A minimum of three independent experiments was performed and consistent results observed. Representative images are shown here. Additionally, UVC-irradiated MCF7 cells were used as positive control and show an upregulation of p21 compared to untreated cells (lanes 4 and 5).</p