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. 2017 Mar 1;140(3):669-683.
doi: 10.1093/brain/aww342.

Clemizole and modulators of serotonin signalling suppress seizures in Dravet syndrome

Aliesha Griffin  1 , Kyla R Hamling  1 , Kelly Knupp  2 , SoonGweon Hong  3 , Luke P Lee  3 , Scott C Baraban  1
Affiliations

Clemizole and modulators of serotonin signalling suppress seizures in Dravet syndrome

Aliesha Griffin et al. Brain. .

Abstract

Dravet syndrome is a catastrophic childhood epilepsy with early-onset seizures, delayed language and motor development, sleep disturbances, anxiety-like behaviour, severe cognitive deficit and an increased risk of fatality. It is primarily caused by de novo mutations of the SCN1A gene encoding a neuronal voltage-activated sodium channel. Zebrafish with a mutation in the SCN1A homologue recapitulate spontaneous seizure activity and mimic the convulsive behavioural movements observed in Dravet syndrome. Here, we show that phenotypic screening of drug libraries in zebrafish scn1 mutants rapidly and successfully identifies new therapeutics. We demonstrate that clemizole binds to serotonin receptors and its antiepileptic activity can be mimicked by drugs acting on serotonin signalling pathways e.g. trazodone and lorcaserin. Coincident with these zebrafish findings, we treated five medically intractable Dravet syndrome patients with a clinically-approved serotonin receptor agonist (lorcaserin, Belviq®) and observed some promising results in terms of reductions in seizure frequency and/or severity. Our findings demonstrate a rapid path from preclinical discovery in zebrafish, through target identification, to potential clinical treatments for Dravet syndrome.

Keywords: drug-screening; epilepsy; personalized medicine; serotonin; zebrafish.

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Figures

Figure 1
Figure 1
Confirmation of the antiepileptic activity of clemizole. (A) The chemical structure of clemizole. (B) Graph showing the change in mean velocity of 5 dpf scn1Lab mutant larvae treated with four concentrations of clemizole. Locomotion was recorded for 10 min after an exposure of 30 min (blue bars) and 90 min (yellow bars). Each bar represents the mean change in velocity ± SEM from three independent experiments of six treated larvae. The threshold for significant decrease in velocity is ≥40% (red line). Hatched bars indicate toxicity was observed. (C) Locomotion tracking plot for 5 dpf larvae from a scn1Laa heterozygous cross. Larvae were scored on their swim behaviour (Stage 0 to Stage III). (D) A representative local field potential recording from the forebrain of agar-embedded Stage III classified larva. Small and large amplitude spontaneous burst discharge were observed. (E) Graph showing the mean swim velocity of 12 larvae, Stage III ‘putative scn1Laa mutants’ and ‘putative sibling controls’. Putative scn1Laa mutants were confirmed by PCR. Significance was determined by one-way ANOVA followed by Holm-Sidak test. (F) Graph showing the velocity of untreated scn1Laa mutants (blue bars) and subsequent treatment with 250 µM of stiripentol (stp), diazepam (dzp), clemizole (clem) and lamotrigine (ltg) (yellow bars). Each bar represents the mean velocity ± SEM. Student’s paired t-test was used to determine significance. *P < 0.05; **P < 0.01.
Figure 2
Figure 2
Radioligand binding assay to identify binding targets of clemizole. Clemizole was subjected to radioligand binding assay against 132 targets. The functional agonist activity of clemizole against 67 targets is shown. Compound binding was calculated as % inhibition of the binding of a radioactively-labelled ligand specific for each target. Inhibition or stimulation higher than 50% and are represented in yellow and are considered to represent significant effects of clemizole.
Figure 3
Figure 3
Summary of behavioural locomotion library screening using scn1Lab mutant zebrafish larvae. Plots of locomotor seizure behaviour for 5 dpf scn1Lab mutants screened against (A) 52 ion channel ligands, (B) 254 compound GPCR ligands, and (C) 65 5-HT modulating compounds. Threshold for inhibition of seizure activity (positive hits) was determined as a reduction in mean swim velocity of ≥40% (red line). Blue data points represent compounds that were classified as toxic as treated larvae have no visible heartbeat or movement in response to touch after 90-min exposure.
Figure 4
Figure 4
Heat map of positive compounds identified from the three targeted libraries. The % change in velocity is shown for six individual larva from the first pass trial (1–6). Mean velocity data from six fish is shown for trial one and trial two. Drugs that reduced the mean swim velocity above threshold and were non-toxic in third trial using separately sourced compound are highlighted in bold. These positive compounds were considered for additional testing. Note: Lorcaserin was identified positive in both the GPCR and 5-HT libraries so it was also considered for further testing.
Figure 5
Figure 5
Electrophysiological assay to identify drugs that rescue the scn1Lab mutant epilepsy phenotype. Bar graphs showing the (A) number, and (B) duration of epileptiform events in a 10-min recording epoch for scn1Lab larvae exposed to lorcaserin (n = 8), trazodone (n = 10), MK-801 (n = 4), TCB-2 (n = 9), pancuronium (n = 8), tetracaine (n = 4), lidocaine (n = 6), loperamide (n = 8), detomidine (n = 5), rotundine (n = 4), or scn1Lab mutants (n = 20). Graph represent mean ± SEM. Student’s unpaired t-test or Mann–Whitney rank sum test were used *P < 0.05. (C) Representative field electrode recording epochs (10 min) are shown for four compounds with significant changes in the frequency of events compared to untreated scn1Lab mutant zebrafish (red). Recordings were obtained with an electrode placed in the forebrain of agar-immobilized scn1Lab larvae that had previously showed suppressed seizure-like behaviour in the locomotion assay.
Figure 6
Figure 6
Dose response evaluation of putative antiepileptic drugs in scn1Lab mutant zebrafish. Putative antiepileptic compounds trazodone and lorcaserin were tested for efficacy in 5 dpf scn1Lab mutant zebrafish. Chemical structure for each compound is shown (A and B). Graphs show the change in mean velocity over five concentrations of (C) trazodone and (D) lorcaserin. Locomotion was recorded for 10 min after an exposure of 30 min (blue bars) and 90 min (yellow bars). Toxicity is indicated by dashed bars. Each bar represents the mean change in velocity ± SEM from three independent experiments. The threshold for a decrease in velocity is ≥ 40% (red line). Representative tracking plots are shown from a single experiment of six individual 5 dpf scn1Lab zebrafish at baseline and following a 30 min and 90 min exposure of 250 µM (E) trazodone or (F) lorcaserin. Total movement is shown for a 10 min recording epoch.
Figure 7
Figure 7
iZAP EEG measurements of scn1Lab during treatment and washout with trazodone and lorcaserin. (A) Time-domain and (B) frequency-domain graphs of a representative field potential measured from one 5 dpf scn1Lab mutant treated with 250 µM trazodone. The inset photograph shows the larva positioned underneath the integrated surface electrodes of the iZAP, the reference electrode, and the trapping channel. (C) Representative zoomed field potential plots of baseline, trazodone and washing phase. The same data are shown for a representative individual scn1Lab mutant larva treated with 250 µM lorcaserin. During the 2-h treatment window there was a trend toward decreased efficacy with prolonged lorcaserin exposure (D–F).
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