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

Abstract

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 (IC(50) = 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.

Keywords: GIRK; electrophysiology; fluorescence; high throughput; pharmacology; screening; thallium flux.

Scheme 1

Synthesis of theVU573 scaffold.

Figure 1

VU573 inhibits mGluR8-activated Kir3.1/3.2 channel activity in thallium flux assays. (A) Representative FluoZin-2 fluorescence traces recorded from HEK-293 cells stably expressing mGluR8 and Kir3.1/3.2 before and after co-application of thallium and different doses of glutamate (shaded box). From an 11-point glutamate concentration–response curve (not shown), the glutamate concentration evoking approximately 80% (EC₈₀) of the maximal response (EC_max) was determined and used for subsequent experiments. (B) Representative traces for changes of Tl⁺-induced FluoZin-2 fluorescence following 20 min pre-treatment of cells with the indicated concentrations of VU573 and subsequent thallium and glutamate-EC₈₀ addition (shaded box). (C) Mean ± SEM concentration–response curve for VU573-dependent inhibition of Kir3.1/3.2 (n = 3). The chemical structure of VU573 is shown in the inset. (D) Representative FluoZin-2 fluorescence traces recorded from monoclonal Kir1.1-S44D expressing cells cultured overnight in absence (−Tet) or presence (+Tet) of Tetracycline. (E) Representative traces for changes of Tl⁺-induced FluoZin-2 fluorescence following pre-treatment of cells with the indicated concentrations of VU573 and then exposed to thallium (shaded box). (F) Mean ± SEM concentration–response curve for VU573-dependent inhibition of Kir1.1-S44D (n = 3).

Figure 2

Effect of VU573 on Kir channels expressed in oocytes. (A) Representative Kir3.1/3.2 current traces recorded from an oocyte using the two-electrode voltage-clamp technique. Oocytes were initially bathed in a potassium-free (0K) solution and then switched to one containing 90 mM potassium (90K) to activate Kir3.1/3.2. After reaching a steady-state, the oocyte was exposed to 10 μM VU573 (in 90K) bath to inhibit Kir3.1/3.2. Residual Kir3.1/3.2 currents were inhibited with 2 mM barium (Ba²⁺). A final switch back to 0K was used to measure leak current at the end of each experiment. Representative whole-cell current traces recorded from oocytes expressing respectively (B) Kir1.1, (C) Kir2.1, (D) Kir2.3, and (E) Kir7.1 before and after application of 50 μM VU573. Residual Kir currents were inhibited with 2 mM barium (Ba²⁺). (F) Mean ± SEM percent inhibition of current evoked by Kir3.1/3.2, Kir3.1/3.4, Kir1.1, Kir2.1, Kir2.3, and Kir7.1 with the indicated concentrations of VU573 () or Ba²⁺ (■) n = 4–6).

Figure 3

VU573-dependent inhibition of Kir3.1/3.2, Kir2.3, and Kir7.1 channel activity expressed in HEK-293 cells. (A,D,G) Representative whole-cell Kir3.1/3.2, Kir2.3, and Kir7.1 current traces recorded from transfected HEK-293 cells. The cell was voltage ramped every 5 s between −120 and 120 mV for 200 ms from a holding potential of −75 mV. Normalized Kir3.1/3.2, Kir2.3, and Kir7.1 currents recorded before (black line) and after reaching steady-state inhibition by 30 μM VU573 (gray line) are shown. (B,E,H) Representative time course traces of VU573-dependent inhibition of Kir3.1/3.2, Kir2.3, and Kir7.1 by 30 μM VU573 using the protocol described above. After achieving whole-cell access, the current was allowed to stabilize before adding 30 μM VU573 and then 2 mM barium (Ba²⁺) to inhibit channel activity. (C,F,I) Mean ± SEM concentration–response curves for Kir3.1/3.2 (n = 4–7), Kir2.3 (n = 4–6), and Kir7.1 (n = 4–7), respectively.

Figure 4

Development of a thallium flux assay for Kir2.3. (A) Thallium flux-dependent FluoZin-2 fluorescence recorded from monoclonal Kir2.3 T-REx-HEK-293 cells cultured overnight in absence (−Tet) or presence (+Tet) of Tetracycline. The fluorescence emission was recorded before and after the addition of extracellular thallium (shaded box). (B) Representative traces for changes of Tl⁺-induced FluoZin-2 fluorescence following 20 min pre-treatment of cells with the indicated concentrations of VU573. (C) CRC for VU573-dependent inhibition of Kir2.3 activity. Values are mean ± SEM (n = 3). A fit of the CRC with a single-site four-parameter logistic function yielded IC₅₀ of 4.7.

Figure 5

Development of a thallium flux assay for Kir7.1 (M125R). (A) Mean ± SEM % inhibition of wild type (closed bars; n = 6–7) or M125R mutant (open bars; n = 4–6) Kir7.1 by the indicated concentration of VU573.Note that the wild type data are reproduced from Figure 3. (B) Thallium flux-dependent FluoZin-2 fluorescence recorded from polyclonalKir7.1 (M125R) T-REx-HEK-293 cells cultured overnight in absence (−Tet) or presence (+Tet) of Tetracycline. The fluorescence emission was recorded before and after the addition of extracellular thallium (shaded box). (C) Representative traces for changes of Tl⁺-induced FluoZin-2 fluorescence following 20 min pre-treatment of cells with the indicated concentrations of VU573. (D) CRC for VU573-dependent inhibition of Kir2.3 activity. Values are mean ± SEM (n = 3). A fit of the CRC with a single-site four-parameter logistic function yielded IC₅₀ of 4.9 μM.

Figure 6

Lack of effect of R70 on wild type Kir7.1 activity. Average Kir7.1 current traces (n = 4 each) recorded in the absence (A) or presence of (B) 10 μM R70 or (C) 10 μM VU573. (D) Mean ± SEM (n = 4) current–voltage Kir7.1 relationships recorded in the indicated conditions using the voltage-clamp protocol shown in (E).