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

The inward rectifier family of potassium (Kir) channels is comprised of at least 16 family members exhibiting broad and often overlapping cellular, tissue, or organ distributions. The discovery of disease-causing mutations in humans and experiments on knockout mice has underscored the importance of Kir channels in physiology and in some cases raised questions about their potential as drug targets. However, the paucity of potent and selective small-molecule modulators targeting specific family members has with few exceptions mired efforts to understand their physiology and assess their therapeutic potential. A growing body of evidence suggests that G protein-coupled inward rectifier K (GIRK) channels of the Kir3.X subfamily may represent novel targets for the treatment of atrial fibrillation. In an effort to expand the molecular pharmacology of GIRK, we performed a thallium (Tl+) flux-based high-throughput screen of a Kir1.1 inhibitor library for modulators of GIRK. One compound, termed VU573, exhibited 10-fold selectivity for GIRK over Kir1.1 (IC50 = 1.9 and 19 μM, respectively) and was therefore selected for further study. In electrophysiological experiments performed on Xenopus laevis oocytes and mammalian cells, VU573 inhibited Kir3.1/3.2 (neuronal GIRK) and Kir3.1/3.4 (cardiac GIRK) channels with equal potency and preferentially inhibited GIRK, Kir2.3, and Kir7.1 over Kir1.1 and Kir2.1.Tl+ flux assays were established for Kir2.3 and the M125R pore mutant of Kir7.1 to support medicinal chemistry efforts to develop more potent and selective analogs for these channels. The structure–activity relationships of VU573 revealed few analogs with improved potency, however two compounds retained most of their activity toward GIRK and Kir2.3 and lost activity toward Kir7.1. We anticipate that the VU573 series will be useful for exploring the physiology and structure–function relationships of these Kir channels

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

Discovery of Dual-Stage Malaria Inhibitors with New Targets

View of the dome and area over the altar; The chapel is in the left aisle of the Lateran Basilica. The original chapel, probably the work of Carlo Maderno, was pulled down due to Borromini's restoration and the present chapel built by Giovanni Antonio de Rossi probably between 1674 and 1686. "Ecclesiastical commissions in Rome were rare at this time, limited in many instances to restorations. His commission (after 1674) to construct a chapel for the Lancellotti family at S Giovanni in Laterano was therefore an important one. For this project he turned to Michelangelo's Cappella Sforza in S Maria Maggiore for his model, updating it with an ornamental idiom derived from Borromini." Source: Grove Art Online; http://www.groveart.com/ (accessed 1/20/2008

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 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

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, 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