The Acrylamide (S)-2 As a Positive and Negative Modulator of Kv7 Channels Expressed in Xenopus laevis Oocytes

BACKGROUND: Activation of voltage-gated potassium channels of the Kv7 (KCNQ) family reduces cellular excitability. These channels are therefore attractive targets for treatment of diseases characterized by hyperexcitability, such as epilepsy, migraine and neuropathic pain. Retigabine, which opens Kv7.2-5, is now in clinical trial phase III for the treatment of partial onset seizures. One of the main obstacles in developing Kv7 channel active drugs has been to identify compounds that can discriminate between the neuronal subtypes, a feature that could help diminish side effects and increase the potential of drugs for particular indications. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we have made a thorough investigation of the Bristol-Myers Squibb compound (S)-N-[1-(4-Cyclopropylmethyl-3,4-dihydro-2H-benzo[1], [4]oxazin-6-yl)-ethyl]-3-(2-fluoro-phenyl)-acrylamide [(S)-2] on human Kv7.1-5 channels expressed in Xenopus laevis oocytes. We found that the compound was a weak inhibitor of Kv7.1. In contrast, (S)-2 efficiently opened Kv7.2-5 by producing hyperpolarizing shifts in the voltage-dependence of activation and enhancing the maximal current amplitude. Further, it reduced inactivation, accelerated activation kinetics and slowed deactivation kinetics. The mechanisms of action varied between the subtypes. The enhancing effects of (S)-2 were critically dependent on a tryptophan residue in S5 also known to be crucial for the effects of retigabine, (S)-1 and BMS-204352. However, while (S)-2 did not at all affect a mutant Kv7.4 with a leucine in this position (Kv7.4-W242L), a Kv7.2 with the same mutation (Kv7.2-W236L) was inhibited by the compound, showing that (S)-2 displays a subtype-selective interaction with in the Kv7 family. CONCLUSIONS/SIGNIFICANCE: These results offer further insight into pharmacological activation of Kv7 channels, add to the understanding of small molecule interactions with the channels and may contribute to the design of subtype selective modulators

Clobazam and its active metabolite N-desmethylclobazam display significantly greater affinities for α₂- versus α₁-GABA(A)-receptor complexes.

Clobazam (CLB), a 1,5-benzodiazepine (BZD), was FDA-approved in October 2011 for the adjunctive treatment of seizures associated with Lennox-Gastaut syndrome (LGS) in patients 2 years and older. BZDs exert various CNS effects through allosteric modulation of GABAA receptors. The structurally distinct, 1,4-BZD clonazepam (CLN) is also approved to treat LGS. The precise mechanisms of action and clinical efficacy of both are unknown. Data show that the GABAA α₁-subunit-selective compound zolpidem [ZOL] exhibits hypnotic/sedative effects. Conversely, data from knock-in mice carrying BZD binding site mutations suggest that the α₂ subunit mediates anticonvulsant effects, without sedative actions. Hence, the specific pattern of interactions across the GABAA receptor complexes of BZDs might be reflected in their clinical efficacies and adverse effect profiles. In this study, GABAA-receptor binding affinities of CLB, N-desmethylclobazam (N-CLB, the major metabolite of CLB), CLN, and ZOL were characterized with native receptors from rat-brain homogenates and on cloned receptors from HEK293 cells transfected with combinations of α (α₁, α₂, α₃, or α₅), β₂, and γ₂ subtypes. Our results demonstrate that CLB and N-CLB have significantly greater binding affinities for α₂- vs. α₁-receptor complexes, a difference not observed for CLN, for which no distinction between α₂ and α₁ receptors was observed. Our experiments with ZOL confirmed the high preference for α₁ receptors. These results provide potential clues to a new understanding of the pharmacologic modes of action of CLB and N-CLB

Potency of (S)-2 on Kv7.2 and Kv7.4.

<p>Dose-response relationship for the effect of (S)-2 on (A) Kv7.2 (<i>n</i> = 6) and (B) Kv7.4 (<i>n</i> = 8) measured using an ⁸⁶Rb-flux assay.</p

Sensitivity of heteromeric Kv7.4/Kv7.4-W242L channels to (S)-2.

<p>(A) Current-voltage (I-V) relationship of Kv7.4 and Kv7.4/Kv7.4-W242L in the absence and presence of 15 µM (S)-2. The steady state peak current measured at potentials between –100 and +40 mV were normalized against the current at +40 mV in control recordings and plotted against the test potential. (B) Effect of (S)-2 on the voltage-dependence of activation of Kv7.4 and Kv7.4/Kv7.4-W242L. Tail currents measured after stepping back to –120 mV from potentials between –100 and +60 mV in the absence and presence of 15 µM (S)-2 were normalized and plotted against the preceding potential. The tail current-voltage relationship was then fitted to the Boltzmann equation to yield half-activation potentials (V_0.5): Kv7.4 (5.1±1.3 mV); Kv7.4+(S)-2 (−30.5±1.6 mV); Kv7.4/Kv7.4-W242L (4.1±1.6 mV); Kv7.4/Kv7.4-W242L+(S)-2 (−15.0±1.0 mV). (C) Dose-response relationship of (S)-2 on Kv7.4 and Kv7.4-W242L. The steady state peak currents elicited by a 5 s step to 0 mV in response to increasing concentrations of (S)-2 were normalized to the current in the absence of compound and plotted as a function of the concentration of (S)-2. The values were then analyzed by non-linear regression to fit a sigmoidal curve. The EC₅₀ values were determined to 0.46 µM and 0.72 µM and the Hill coefficients to 1.32±0.17 and 1.24±0.25 for Kv7.4 and Kv7.4/Kv7.4-W242L, respectively. Bars represent S.E.M. and <i>n</i> = 6–10.</p

Activation of Kv7 channels by (S)-2.

<p>Representative two-electrode voltage-clamp current traces in the absence (left) and presence (middle) of 10 µM (S)-2 and effect of (S)-2 on current-voltage (I-V) relationship (right) of Kv7.1 (A), Kv7.2 (B), Kv7.2/Kv7.3 (C), Kv7.4 (D) and Kv7.5 (E) channels expressed in <i>Xenopus laevis</i> oocytes. The channels were activated by 5 s voltage steps from −80 mV to potentials ranging from –100 to +40 mV in 10 mV increments followed by a 2 S step to –120 mV. The steady state peak current amplitudes in the absence and presence of 10 µM (S)-2 were normalized against the current at +40 mV in control recordings and plotted against the test potential to obtain I-V curves (left). Bars represent S.E.M. and <i>n</i> = 6-17. Please note that in some instances the S.E.M. is so small that the error bars are not visible.</p