Blom SM et al. (2009), The acrylamide (S)-2 as a positive and negative...

XB-ART-40822

PLoS One 2009 Dec 11;412:e8251. doi: 10.1371/journal.pone.0008251.

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The acrylamide (S)-2 as a positive and negative modulator of Kv7 channels expressed in Xenopus laevis oocytes.

Blom SM , Schmitt N , Jensen HS .

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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.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.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.

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Species referenced: Xenopus laevis
Genes referenced: arfgap1 kcnq2 kcnq3 kcnq4 kcnq5

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	Figure 1. Chemical structure of (S)-2.
	Figure 2. Activation of Kv7 channels by (S)-2.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 Xenopus laevis 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 n = 6-17. Please note that in some instances the S.E.M. is so small that the error bars are not visible.
	Figure 3. Effect of (S)-2 on the voltage-dependence of activation.Tail currents measured for Kv7.1 (A), Kv7.2 (B), Kv7.2/Kv7.3 (C), Kv7.4 (D) and Kv7.5 (E) after stepping back to −120 mV from 5 s long potentials between –100 and +60 mV were normalized and plotted against the preceding potential in the absence and presence of 10 µM (S)-2. The tail current-voltage relationship was then fitted to the Boltzmann equation (Eq. 1) to yield half-activation potentials (V0.5). Bars represent S.E.M. and n = 5–11.
	Figure 4. Potency of (S)-2 on Kv7.2 and Kv7.4.Dose-response relationship for the effect of (S)-2 on (A) Kv7.2 (n = 6) and (B) Kv7.4 (n = 8) measured using an 86Rb-flux assay.
	Figure 5. Effect of (S)-2 on the activation kinetics of Kv7.2 and Kv7.4.For determination of activation kinetics, current traces recorded during 5 s voltage steps from –80 mV to potentials between –40 and +40 mV for Kv7.2 in the absence and presence of (S)-2 were fitted to a double exponential function and the time constants τfast (A) and τslow (B) obtained were plotted against the step potential. For Kv7.4 current traces recorded during 5 s voltage steps to potentials between –20 and +60 mV were fitted to a single exponential function and τ is plotted against the step potential (C). Asterisks indicate statistical significant difference between absence and presence of (S)-2 determined by two-way ANOVA followed by Bonferroni post-test. * P<0.05, P<0.01 and * P<0.001. Bars represent S.E.M. and n = 8-13.
	Figure 6. Effect of (S)-2 on the deactivation kinetics of Kv7.2 and Kv7.4.Normalized tail current traces for Kv7.2 (A) and Kv7.4 (E) obtained at the indicated potentials in the absence and presence of (S)-2 illustrating the pronounced effect of the compound on the deactivation kinetics. Deactivation kinetics for Kv7.2 were determined by fitting the tail currents measured at potentials between –110 mV and –90 mV after an activating step to +40 mV to a double exponential function. The time constants τfast (B) and τslow (C) were plotted against the potential. (D) Relative contribution of the slow component of the deactivation kinetics for Kv7.2. Tail currents measured between −110 and −80 mV for Kv7.4 were fitted to a double exponential function and τfast (F) and τslow (G) were plotted against the potential. (H) Relative contribution of the slow component of the deactivation kinetics for Kv7.4. Asterisks indicate statistical significant difference between absence and presence of (S)-2 determined by two-way ANOVA followed by Bonferroni post-test. * P<0.05, P<0.01 and * P<0.001. Bars represent S.E.M. and n = 5–14.
	Figure 7. Effect of (S)-2 on the inactivation of Kv7.4.Representative two-electrode voltage-clamp recordings elicited by the voltage protocol shown in the inset in the absence (A) and presence (B) of 10 µM (S)-2. (C) The degree of inactivation in the absence and presence of (S)-2 is revealed by plotting the current amplitude measured at +40 mV normalized to the level measured after a prepulse potential of –120 mV as a function of the preceding prepulse potential. Bars represent S.E.M. and n = 11–14.
	Figure 8. Dependency of (S)-2 on a tryptophan residue in S5.Representative two-electrode voltage-clamp current traces of Kv7.4-W242L (A) and Kv7.2-W236L (D) in the absence (left) and presence (right) of 10 µM (S)-2. Currents were elicited by 5 s depolarizing pulses between −100 and +40 mV in 10 mV increments from a holding potential of –80 mV followed by a 2 s step to –120 mV. The steady state peak current amplitudes of Kv7.4-W242L (B) and Kv7.2-W236L (E) 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 current-voltage (I-V) curves (n = 10). (C) Cells expressing Kv7.4-W242L were investigated for inactivation by the same voltage protocol as in fig. 7. The plot shows the current amplitude measured at +40 mV normalized to the level measured after a prepulse potential of –120 mV as a function of the preceding prepulse potential, n = 11. (F) Dose-response relationship of the effect of (S)-2 on Kv7.2-W236L measured using a Rubidium-flux assay (n = 4). Effect of (S)-2 on the fast (G) and slow (H) components of the activation kinetics of Kv7.2-W236L. Current traces recorded during voltage steps from –80 mV to potentials between –40 and +40 mV in the absence and presence of (S)-2 were fitted to a double exponential function and the time constants τfast and τslow obtained were plotted against the step potential (n = 7). * P<0.05, P<0.01 * P<0.001 determined by two-way ANOVA followed by Bonferroni post-test. Bars represent S.E.M.
	Figure 9. Sensitivity of heteromeric Kv7.4/Kv7.4-W242L channels to (S)-2.(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 (V0.5): 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 n = 6–10.

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