Gilchrist J et al. (2014), Nav1.1 modulation by a novel triazole compound ...

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ACS Chem Biol 2014 May 16;95:1204-12. doi: 10.1021/cb500108p.

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Nav1.1 modulation by a novel triazole compound attenuates epileptic seizures in rodents.

Gilchrist J, Dutton S, Diaz-Bustamante M, McPherson A, Olivares N, Kalia J, Escayg A, Bosmans F.

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Here, we report the discovery of a novel anticonvulsant drug with a molecular organization based on the unique scaffold of rufinamide, an anti-epileptic compound used in a clinical setting to treat severe epilepsy disorders such as Lennox-Gastaut syndrome. Although accumulating evidence supports a working mechanism through voltage-gated sodium (Nav) channels, we found that a clinically relevant rufinamide concentration inhibits human (h)Nav1.1 activation, a distinct working mechanism among anticonvulsants and a feature worth exploring for treating a growing number of debilitating disorders involving hNav1.1. Subsequent structure-activity relationship experiments with related N-benzyl triazole compounds on four brain hNav channel isoforms revealed a novel drug variant that (1) shifts hNav1.1 opening to more depolarized voltages without further alterations in the gating properties of hNav1.1, hNav1.2, hNav1.3, and hNav1.6; (2) increases the threshold to action potential initiation in hippocampal neurons; and (3) greatly reduces the frequency of seizures in three animal models. Altogether, our results provide novel molecular insights into the rational development of Nav channel-targeting molecules based on the unique rufinamide scaffold, an outcome that may be exploited to design drugs for treating disorders involving particular Nav channel isoforms while limiting adverse effects.

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Species referenced: Xenopus
Genes referenced: acta4 nav1 scn1a

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	Figure 1. Effect of 100 μM rufinamide on human Nav channel isoforms involved in epilepsy. (A) Molecular organization of rufinamide consisting of the 1,2,3-triazole ring connecting the 2,6-difluorophenyl and the carboxamide. (B–E) The left column shows G/Gmax and I/Imax relationships, whereas the right column displays the recovery from fast inactivation. The figure shows that rufinamide inhibits hNav1.1 opening whereas the effect on hNav1.6 is not significant compared to the effect of DMSO treatment of this particular isoform (see Table 1). Furthermore, the recovery from fast inactivation slows for the four Nav channel isoforms tested here. All data are shown before (green, DMSO control) and after (red) addition of 100 μM rufinamide to hNav1.1 (B), hNav1.2 (C), hNav1.3 (D), and hNav1.6 (E). All fit values are listed in Table 1. n = 5–8, and error bars represent the standard error of the mean.
	Figure 2. Effect of rufinamide derivatives on hNav1.1. (A–D) The left column shows G/Gmax relationships before (green, DMSO control) and after (red) addition of 100 μM rufinamide derivatives fitted with the Boltzmann equation. Compounds A and B clearly inhibit hNav1.1 opening, whereas compound C no longer affects hNav1.1 opening (compound D is not significant when considering p < 0.005). Fit values are listed in Table 1. n = 5–8, and error bars represent the standard error of the mean. The right column displays the molecular organization of the four tested derivatives (compounds A–D).
	Figure 3. Effect of compound B on rat hippocampal neurons. (A) Representative recordings of cells treated with DMSO-containing vehicle (left) vs cells treated with 100 μM compound B (right). The inset shows the current clamp protocol of a series of 10 pA depolarizing current injections in which red and green indicate the current needed to elicit an action potential under control conditions (20 pA) and that of the treated cells (80 pA), respectively. (B) Average action potential thresholds are significantly higher in cells treated with compound B [−27.8 ± 1.6 mV (○); n = 7] than in cells treated with the DMSO-containing vehicle [−43.3 ± 2.4 mV (●); n = 7].
	Figure 4. Compound B increases latencies to picrotoxin- and fluorothyl-induced generalized seizures. (A) Picrotoxin (10 mg/kg) was administered to vehicle-treated (black) and compound B-treated (red) male Crl:CF1 mice (75 mg/kg) 15 min prior to seizure induction. The average latencies in seconds to the first myoclonic jerk (MJ), forelimb clonus (FC), and generalized tonic-clonic seizure (GTCS) are compared. Compound B increases the average latency to the first GTCS. p < 0.05. Error bars represent the standard error of the mean. (B) The latencies to the MJ, GTCS, and GTCS with hind limb extension (GTCS+) in the fluorothyl model are compared between vehicle-treated (black) and compound B-treated (red) male Crl:CF1 mice (75 mg/kg). The average latencies in seconds to the GTCS and GTCS+ are significantly longer in the compound B-treated mice. **p < 0.001. Error bars represent the standard error of the mean.
	Figure 5. Compound B reduces the severity of seizures induced by the 6 Hz psychomotor paradigm. A current intensity of 17 mA was used to induce partial seizures in vehicle-treated (black) and compound B-treated (red) male Crl:CF1 mice (75 mg/kg). Following treatment with compound B, there is a 63% reduction in the number of mice exhibiting seizures (A; p < 0.0001). In addition, the seizures that were observed in the compound B-treated mice are typically less severe than those seen in the vehicle-treated mice (black) (B; p < 0.0001). Error bars represent the standard error of the mean.

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