Development of an automated screen for Kv7.2 potassium channels and discovery of a new agonist chemotype

Abstract

To identify pore domain ligands on Kv7.2 potassium ion channels, we compared wild-type (WT) and W236L mutant Kv7.2 channels in a series of assays with previously validated and novel agonist chemotypes. Positive controls were retigabine, flupirtine, and RL-81; i.e. Kv7.2 channel activators that significantly shift voltage-dependent activation to more negative potentials (ΔV₅₀) at 5 µM. We identified 6 new compounds that exhibited differential enhancing activity between WT and W236L mutant channels. Whole cell patch-clamp electrophysiology studies were conducted to identify Kv7.2. Kv7.2/3, Kv7.4, and Kv7.5 selectivity. Our results validate the SyncroPatch platform and establish new structure activity relationships (SAR). Specifically, in addition to selective Kv7.2, Kv7.2/3, Kv7.4. and Kv7.5 agonists, we identified a novel chemotype, ZK-21, a 4-aminotetrahydroquinoline that is distinct from any of the previously described Kv7 channel modifiers. Using flexible receptor docking, ZK-21 was predicted to be stabilized by W236 and bind perpendicular to retigabine, burying the benzyl carbamate group into a tunnel reaching the core of the pore domain.

Keywords: Agonists; Potassium channels; Quinolines; SyncroPatch screen; Voltage-dependent activation.

Figure 1.

(A) Topology of single voltage-gated potassium ion channel subunit (Kv); the retigabine/W236 site is labeled in the S5 helix of the pore domain. (B) A heterotetrameric channel assembly.

Figure 2.

Structures of representative Kv7.2–5 channel activators.

Figure 3.

Structures of the aniline (“RL”), thiazine 1,1-dioxide (“CH”), 3-aminoisoquinolinone (“DP”), 4-aminotetrahydroquinoline (“ZK”, “MS”), and 2-phenylindole (“PM”) series of screening samples.

Figure 4.

Functional characterization of activators on human wild type (WT) and mutant W236L Kv7.2 channels. (A) Differential effect on Kv7.2 ΔV₅₀ of 22 analogs measured at 5 μM. Variation is expressed as ±SD, and values are listed in Table 1. (B) Concentration–response curves for six Kv7.2 channel hits. The ΔV₅₀ represent the half-activation voltage (V₅₀) of G-V curve shifts in the presence and in the absence of the compound. ΔV₅₀ (mV) = V₅₀ in control − V₅₀ in the presence of analogs was plotted against the compound concentration. Values are the mean ± SEM.

Figure 5.

Docking studies at the K_v7.2 retigabine (A) and ztz240 (B) binding sites. Dots represent experimentally tested ligands in shown in Figure 3 with Docking Energy <−10, where those with experimental Kv7.2 ΔV₅₀ <−20 mV at 5 μM concentration are labeled and shown in red.

Figure 6.

ZK-21 binding mode predicted by RosettaLigand. (A) Top-down view of the Kv7.2 homotetramer clipped at the retigabine binding site between the S5 and S6 helices of the pore domain. (B) Overlay of the experimental retigabine and predicted ZK-21 binding modes and (C,D) schematics of the receptor contacts. ZK-21 is predicted to bind perpendicular to retigabine allowing the benzyl carbamate to fill the tunnel towards the pore core. Binding modes for (E) ZK-21, (F) ZK-94, (G) ZK-29, and (H) ZK-16.

Scheme 1.

Synthesis of CH-02.

Scheme 2.

Synthesis of PM-17, PM-24, PM-28, PM-29, PM-44, PM-57, PM-63, and PM-91.