ML418: The First Selective, Sub-Micromolar Pore Blocker of Kir7.1 Potassium Channels

Abstract

The inward rectifier potassium (Kir) channel Kir7.1 (KCNJ13) has recently emerged as a key regulator of melanocortin signaling in the brain, electrolyte homeostasis in the eye, and uterine muscle contractility during pregnancy. The pharmacological tools available for exploring the physiology and therapeutic potential of Kir7.1 have been limited to relatively weak and nonselective small-molecule inhibitors. Here, we report the discovery in a fluorescence-based high-throughput screen of a novel Kir7.1 channel inhibitor, VU714. Site-directed mutagenesis of pore-lining amino acid residues identified glutamate 149 and alanine 150 as essential determinants of VU714 activity. Lead optimization with medicinal chemistry generated ML418, which exhibits sub-micromolar activity (IC50 = 310 nM) and superior selectivity over other Kir channels (at least 17-fold selective over Kir1.1, Kir2.1, Kir2.2, Kir2.3, Kir3.1/3.2, and Kir4.1) except for Kir6.2/SUR1 (equally potent). Evaluation in the EuroFins Lead Profiling panel of 64 GPCRs, ion-channels, and transporters for off-target activity of ML418 revealed a relatively clean ancillary pharmacology. While ML418 exhibited low CLHEP in human microsomes which could be modulated with lipophilicity adjustments, it showed high CLHEP in rat microsomes regardless of lipophilicity. A subsequent in vivo PK study of ML418 by intraperitoneal (IP) administration (30 mg/kg dosage) revealed a suitable PK profile (Cmax = 0.20 μM and Tmax = 3 h) and favorable CNS distribution (mouse brain/plasma Kp of 10.9 to support in vivo studies. ML418, which represents the current state-of-the-art in Kir7.1 inhibitors, should be useful for exploring the physiology of Kir7.1 in vitro and in vivo.

Keywords: KCNJ13; comparative modeling; electrophysiology; melanocortin signaling; myometrium; thallium flux.

Figure 1. Kir7.1 Tl⁺ flux assay used for HTS

(A) Representative Thallos fluorescence traces recorded from T-REx-HEK-293-Kir7.1-M125R cells cultured overnight with (grey line) or without (black line) tetracycline. Thallium stimulus buffer was added to each well simultaneously as indicated with the arrow. (B) DMSO tolerance test indicating that DMSO has no effect on Kir7.1-M125R–mediated Tl⁺ flux as concentrations up to 1.3% (v/v). (C) Determination of assay reproducibility. Alternate wells of a 384-well plate were treated with DMSO (vehicle) or Kir7.1 inhibitor VU573 (30 µM) before initiating Tl⁺ flux. Mean fluorescence and 3 S.D. from the mean for each well population are indicated with a blue dashed line and solid black line, respectively. The mean ± SEM. Z’ for 3 plates assayed on 3 separate days was Z’ = 0.67 ± 0.03.

Figure 2. Discovery and characterization of VU714

(A) Chemical structure of VU714. (B) Dose-dependent inhibition of Kir7.1-M125R–dependent Tl⁺ flux by VU714. Cells were pre-treated with the indicated concentrations of VU714 for 10 min before adding Tl⁺ stimulus buffer (arrow). (C) Mean ± SEM % control fluorescence recorded in the indicated concentrations of VU714 (n = 4). (D) Representative whole-cell patch clamp experiment showing timecourse of VU714-dependent inhibition of Kir7.1 current recorded at −120 mV. VU714 concentrations (in µM) are indicated at the top. Experiments were terminated by bath application of 2 mM barium (Ba). (E) Current-voltage plot showing inhibition of Kir7.1 by 10 µM VU714 or 2 mM Ba. (F) Mean ± SEM % Kir7.1 inhibition at −120 mV. IC₅₀ values were derived by fitting CRC data with a 4-parameter logistical function.

Figure 3. Analysis of VU714 and ML418 selectivity for Kir7.1 over other Kir channels

(A) VU714 CRCs constructed for Kir7.1-M125R over Kir6.2/SUR1 (open diamonds), Kir1.1 (closed circles), Kir2.1 (closed squares), Kir4.1 (open squares) in Tl⁺ flux assays. Kir2.2,Kir2.3, Kir3.1/3.2 (IC₅₀s >30 µM) have been excluded for clarity. Data are means ± SEM % control fluorescence (n = 4–10 per concentration). (B) ML418 CRCs constructed for the same channels in Tl⁺ flux assays. (C) Representative whole-cell patch clamp experiment showing dose-dependent inhibition of Kir7.1 current at −120 mV by the indicated concentration of ML418. The experiment was terminated by bath application of 2 mM Ba. (D) Comparison of CRCs for VU714 (grey line, data from Fig. 2) and ML418 determined in patch clamp electrophysiology experiments.

Figure 4. Identification of pore-lining residues in Kir7.1 required for VU714 activity

(A) Alignment of pore-lining M2 helices from human Kir7.1, Kir2.1, and Kir1.1, with predicted pore-facing residues indicated with arrowheads. (B) Effects of pore mutations on Kir7.1-WT or Kir7.1-M125R sensitivity to 3 µM VU714. Data are mean ± SEM % inhibition at −120 mV. * P <0.05 compared to respective control. N.F., not functional. (C) VU714 CRC for Kir7.1-WT (closed squares; IC₅₀=1.4 µM ), Kir7.1-M125R (open squares; IC₅₀=1.6 µM), Kir7.1-M125R–E148Q (open circles; IC₅₀=18.1 µM), Kir7.1-WT-A150S (closed circles; IC₅₀=6.9 µM). (D) Kir7.1 homology model showing low-energy pose of VU714 near residues E149 and A150. (E) Higher-magnification view (from white box in D) of VU714 near E149 and A150.

Figure 5. Time course in vivo PK profile of ML418

ML418 was dosed at 30 mg/kg in 10% EtOH, 40% PEG 400, 50% saline vehicle. The dosing solution was administered by intraperitoneal injection and whole blood collections via the carotid artery were performed at 0.117, 0.25, 0.5, 1, 2, 4, 7, and 24 hours post dose.