Large-Scale Phenotype-Based Antiepileptic Drug Screening in a Zebrafish Model of Dravet Syndrome

Mutations in a voltage-gated sodium channel (SCN1A) result in Dravet Syndrome (DS), a catastrophic childhood epilepsy. Zebrafish with a mutation in scn1Lab recapitulate salient phenotypes associated with DS, including seizures, early fatality, and resistance to antiepileptic drugs. To discover new drug candidates for the treatment of DS, we screened a chemical library of ∼1000 compounds and identified 4 compounds that rescued the behavioral seizure component, including 1 compound (dimethadione) that suppressed associated electrographic seizure activity. Fenfluramine, but not huperzine A, also showed antiepileptic activity in our zebrafish assays. The effectiveness of compounds that block neuronal calcium current (dimethadione) or enhance serotonin signaling (fenfluramine) in our zebrafish model suggests that these may be important therapeutic targets in patients with DS. Over 150 compounds resulting in fatality were also identified. We conclude that the combination of behavioral and electrophysiological assays provide a convenient, sensitive, and rapid basis for phenotype-based drug screening in zebrafish mimicking a genetic form of epilepsy.

Significance Statement

Dravet syndrome is a catastrophic childhood epilepsy that is resistant to available medications. Current animal models for this disease are not amenable to high-throughput drug screening. We used a zebrafish model for Dravet syndrome and screened >1000 compounds. We report the identification of compounds with the ability to suppress seizure behavior and electrographic seizure activity. This approach provides an example of precision medicine directed to pediatric epilepsy.

Introduction

Dravet syndrome (DS) is a devastating genetic epileptic encephalopathy that has been linked to more than >300 de novo mutations in a neuronal voltage-gated sodium channel (SCN). Children with DS are at a higher risk for sudden unexplained death in epilepsy and episodes of uncontrolled status epilepticus (Dravet et al., 2005; Ceulemans et al., 2012). Delayed language development, disruption of autonomic function, and motor and cognitive impairment are also associated with this disease. Seizure management includes treatment with benzodiazepines, valproate, and/or stiripentol (Caraballo et al., 2005; Chiron and Dulac, 2011). Some reduction in seizure activity has been reported with the use of bromides and topiramate, or a ketogenic diet (Lotte et al., 2012; Wilmshurst et al., 2014; Dressler et al., 2015). Despite these options, available antiepileptic drugs (AEDs) do not achieve adequate seizure control in most DS patients (Dravet et al., 2005; Chiron and Dulac, 2011; Dressler et al., 2015), making the identification of new drugs a critical unmet need. High-throughput screening offers a powerful tool to identify new drug candidates for these patients. However, commonly available screening approaches rely on in vitro cell-based assays (Masimirembwa et al., 2001; Snowden and Green, 2008; Ko and Gelb, 2014) and do not recapitulate the complicated neural networks that generate seizures in vivo. Given the need for new treatments for children with DS, and the growing number of genetic epileptic encephalopathies that are medically intractable (Leppert, 1990; Epi4K Consortium, 2012; Ottman and Risch, 2012), we developed an alternative phenotype-based in vivo drug-screening strategy. While cell-based in vitro screening assays can efficiently identify compounds that bind specific targets, whole-organism-based screens are more likely to reliably predict therapeutic outcomes as they maintain the complex neural circuitry involved in the underlying disease process. Whole-organism screens do not require well validated targets to identify compounds that yield a desirable phenotypic outcome, but can be prohibitively costly and time consuming in mammals. As a simple vertebrate with significant genetic similarity to human, zebrafish are now recognized as an ideal cost-effective alternative to achieve rapid in vivo phenotype-based screening (Ali et al., 2011).

Using scn1a mutant zebrafish larvae with a gene homologous to human and spontaneously occurring seizures (Baraban et al., 2013), we screened, in a blinded manner, a repurposed library of ∼1000 compounds for drugs that inhibit unprovoked seizure events. We also screened two compounds (huperzine A and fenfluramine) that were discovered in rodent-based assays using acquired seizure protocols and that were recently suggested as potential treatments for DS (Boel and Casaer, 1996; Coleman et al., 2008; Ceulemans et al., 2012; Bialer et al., 2015). Only 20 compounds in the repurposed drug library reduced swim behavior to control levels. However, many of these compounds were toxic or were not confirmed on retesting, and only four compounds advanced to a second-stage in vivo electrophysiology assay. Of these compounds (cytarabine, dimethadione, theobromine, and norfloxacin) only dimethadione, a T-type calcium channel antagonist previously reported to have anticonvulsant activity (Lowson et al., 1990; Zhang et al., 1996), reduced ictal-like electrographic discharges seen in scn1Lab mutant larvae. This two-stage phenotype-based screening approach, using a genetic DS model with >75% genomic similarity to human, is a sensitive, rapid means to successfully identify compounds with antiepileptic activity.

Materials and Methods

Zebrafish

Zebrafish were maintained in a light- and temperature-controlled aquaculture facility under a standard 14:10 h light/dark photoperiod. Adult zebrafish were housed in 1.5 L tanks at a density of 5-12 fish per tank and fed twice per day (dry flake and/or flake supplemented with live brine shrimp). Water quality was continuously monitored: temperature, 28-30º C; pH 7.4-8.0; conductivity, 690-710 mS/cm. Zebrafish embryos were maintained in round Petri dishes (catalog #FB0875712, Fisher Scientific) in “embryo medium” consisting of 0.03% Instant Ocean (Aquarium Systems, Inc.) and 000002% methylene blue in reverse osmosis-distilled water. Larval zebrafish clutches were bred from wild-type (WT; TL strain) or scn1Lab (didys552) heterozygous animals that had been backcrossed to TL wild-type for at least 10 generations. Homozygous mutants (n = 6544), which have widely dispersed melanosomes and appear visibly darker as early as 3 d postfertilization (dpf; Fig. 1), or WT larvae (n = 71) were used in all experiments at 5 or 6 dpf. Embryos and larvae were raised in plastic petri dishes (90 mm diameter, 20 mm depth) and density was limited to ∼60 per dish. Larvae between 3 and 7 dpf lack discernible sex chromosomes. The care and maintenance protocols comply with requirements outlined in the Guide for the Care and Use of Animals (ebrary Inc., 2011) and were approved by the Institutional Animal Care and Use Committee (protocol #AN108659-01D).

Figure 1.

Locomotion assay to identify drugs that rescue the scn1Lab mutant epilepsy phenotype. , Schematic of the phenotype-based screening process. Chemical libraries can be coded and aliquoted in small volumes (75 µL) into individual wells containing one mutant fish. The 96-well microplate is arranged so that six fish are tested per drug; with one row of six fish maintained as an internal control (red circles) on each plate. , Representative images for WT and scn1Lab mutant zebrafish larvae at 5 dpf. Note the morphological similarity but darker pigmentation in mutant larvae. , Box plot of mean velocity (in millimeters per second) for two consecutive recordings of mutant larvae in embryo media. Experiments were performed by first placing the mutant larvae in embryo media and obtaining a baseline locomotion response; embryo media was then replaced with new embryo media (to mimic the procedure used for test compounds), and a second locomotion response was obtained. The percentage change in velocity from baseline (recording 1) versus experimental model (recording 2) is shown. In the box plot, the bottom and top of the box represent the 25th percentile and the 75th percentile, respectively. The line across the box represents the median value, and the vertical lines encompass the entire range of values. This plot represents normal changes in tracking activity in the absence of a drug challenge. , Plot of locomotor seizure behavior for scn1Lab mutants at 5 dpf for the 1012 compounds tested. Threshold for inhibition of seizure activity (positive hits) was set as a reduction in mean swim velocity of ≥44%; the threshold for a proconvulsant or hyperexcitable effect was set at an increase in the mean swim velocity of ≥44% (green dashed lines).

Seizure monitoring

Zebrafish larvae were placed individually into 1 well of a clear flat-bottomed 96-well microplate (catalog #260836, Fisher Scientific) containing embryo media. Microplates were placed inside an enclosed motion-tracking device and acclimated to the dark condition for 10-15 min at room temperature. Locomotion plots were obtained for one fish per well at a recording epoch of 10 min using a DanioVision system running EthoVision XT software (DanioVision, Noldus Information Technology); threshold detection settings to identify objects darker than the background were optimized for each experiment. Seizure scoring was performed using the following three-stage scale (Baraban et al., 2005): Stage 0, no or very little swim activity; Stage I, increased, brief bouts of swim activity; Stage II, rapid “whirlpool-like” circling swim behavior; and Stage III, paroxysmal whole-body clonus-like convulsions, and a brief loss of posture. WT fish are normally scored at Stage 0 or I. Plots were analyzed for distance traveled (in millimeters) and mean velocity (in millimeters per second). As reported previously (Winter et al., 2008; Baraban et al., 2013), velocity changes were a more sensitive assay of seizure behavior. For electrophysiology studies, zebrafish larvae were briefly paralyzed with α-bungarotoxin (1 mg/ml) and immobilized in 1.2% agarose; field recordings were obtained from forebrain structures. Epileptiform events were identified post hoc in Clampfit (Molecular Devices) and were defined as multispike or polyspike upward or downward membrane deflections greater than three times the baseline noise level and >500 ms in duration. During electrophysiology experiments zebrafish larvae were continuously monitored for the presence (or absence) of blood flow and heart beat by direct visualization on an Olympus BX51WI upright microscope equipped with a CCD camera and monitor.

Drugs

Compounds for drug screening were purchased from MicroSource Discovery Systems, Inc. (PHARMAKON 1600) and were provided as 10 mm DMSO solutions (Table 1). Test compounds for locomotion or electrophysiology studies were dissolved in embryo media and were tested at an initial concentration of 100 µm, with a final DMSO concentration of <2%. In all drug library screen studies, compounds were coded and experiments were performed by investigators who were blind to the nature of the compound. Baseline recordings of seizure behavior were obtained from mutants bathed in embryo media, as described above; a second locomotion plot was then obtained following a solution change to a test compound and an equilibration period of 15–30 min. Criteria for a positive hit designation were as follows: (1) a decrease in mean velocity of ≥44% (e.g., a value based on the trial-to-trial variability measured in control tracking studies; Fig. 1); and (2) a reduction to Stage 0 or Stage I seizure behavior in the locomotion plot for at least 50% of the test fish. Each test compound classified as a “positive hit” in the locomotion assay was confirmed, under direct visualization on a stereomicroscope, as the fish being alive based on movement in response to external stimulation and a visible heartbeat following a 60 min drug exposure. Toxicity (or mortality) was defined as no visible heartbeat or movement in response to external stimulation in at least 50% of the test fish. Hyperexcitability was defined as a compound causing a ≥44% increase in swim velocity and/or Stage III seizure activity in at least 50% of the test fish. Hits identified in the primary locomotion screen were selected from the PHARMAKON 1600 library and rescreened using the method described above. Select compound stocks that were successful in two primary locomotion assays, and were not classified as toxic in two independent clutches of zebrafish, were then purchased separately from Sigma-Aldrich for further testing. Drug concentrations between 0.5 and 1 mm were used for electrophysiology assays to account for more limited diffusion in agar-embedded larvae.

Table 1.

List of compounds from the PHARMAKON 1600 library used in this screen.

ABACAVIR SULFATE

ABAMECTIN (avermectin B1a shown)

ACADESINE

ACARBOSE

ACEBUTOLOL HYDROCHLORIDE

ACECLIDINE

ACECLOFENAC

ACENOCOUMAROL

ACETAMINOPHEN

ACETOHYDROXAMIC ACID

ACETOPHENAZINE MALEATE

ACETRIAZOIC ACID

ACETYLCHOLINE CHLORIDE

ACETYLCYSTEINE

ACIPIMOX

ACONITINE

ACRIFLAVINIUM HYDROCHLORIDE

ACRISORCIN

ACTARIT

ACYCLOVIR

ADAPALENE

ADELMIDROL

ADENINE

ADENOSINE

ADENOSINE PHOSPHATE

ADIPHENINE HYDROCHLORIDE

AKLOMIDE

ALAPROCLATE

ALBENDAZOLE

ALBUTEROL (+/-)

ALENDRONATE SODIUM

ALEXIDINE HYDROCHLORIDE

ALLANTOIN

ALLOPURINOL

ALMOTRIPTAN

alpha-TOCHOPHEROL

alpha-TOCHOPHERYL ACETATE

ALPRAZOLAM

ALRESTATIN

ALTHIAZIDE

ALTRETAMINE

ALVERINE CITRATE

AMANTADINE HYDROCHLORIDE

AMCINONIDE

AMIFOSTINE

AMIKACIN SULFATE

AMILORIDE HYDROCHLORIDE

AMINACRINE

AMINOCAPROIC ACID

AMINOGLUTETHIMIDE

AMINOHIPPURIC ACID

AMINOLEVULINIC ACID HYDROCHLORIDE

AMINOSALICYLATE SODIUM

AMITRIPTYLINE HYDROCHLORIDE

AMLEXANOX

AMLODIPINE BESYLATE

AMODIAQUINE DIHYDROCHLORIDE

AMOROLFINE HYDROCHLORIDE

AMOXICILLIN

AMPHOTERICIN B

AMPICILLIN SODIUM

AMPROLIUM

AMSACRINE

ANASTROZOLE

ANCITABINE HYDROCHLORIDE

ANETHOLE

ANIRACETAM

ANISINDIONE

ANTAZOLINE PHOSPHATE

ANTHRALIN

ANTIPYRINE

APOMORPHINE HYDROCHLORIDE

APRAMYCIN

ARGININE HYDROCHLORIDE

ARMODAFINIL

ARTEMETHER

ARTEMOTIL

ARTESUNATE

ASCORBIC ACID

ASPIRIN

ATENOLOL

ATORVASTATIN CALCIUM

ATOVAQUONE

ATROPINE SULFATE

AUROTHIOGLUCOSE

AVOBENZONE

AZACITIDINE

AZASERINE

AZATADINE MALEATE

AZATHIOPRINE

AZELAIC ACID

AZITHROMYCIN

AZLOCILLIN SODIUM

AZTREONAM

BACAMPICILLIN HYDROCHLORIDE

BACITRACIN

BACLOFEN

BALSALAZIDE DISODIUM

BECLOMETHASONE DIPROPIONATE

BEKANAMYCIN SULFATE

BEMOTRIZINOL

BENAZEPRIL HYDROCHLORIDE

BENDROFLUMETHIAZIDE

BENORILATE

BENSERAZIDE HYDROCHLORIDE

BENZALKONIUM CHLORIDE

BENZETHONIUM CHLORIDE

BENZOCAINE

BENZOIC ACID

BENZONATATE

BENZOYL PEROXIDE

BENZTHIAZIDE

BENZYL ALCOHOL

BENZYL BENZOATE

BEPRIDIL HYDROCHLORIDE

BERGAPTEN

beta-CAROTENE

BETAHISTINE HYDROCHLORIDE

BETAINE HYDROCHLORIDE

BETAMETHASONE

BETAMETHASONE 17,21-DIPROPIONATE

BETAMETHASONE VALERATE

BETAMIPRON

beta-NAPHTHOL

BETAZOLE HYDROCHLORIDE

BETHANECHOL CHLORIDE

BEZAFIBRATE

BICALUTAMIDE

BIOTIN

BISACODYL

BISOCTRIZOLE

BISORCIC

BITHIONATE SODIUM

BLEOMYCIN (bleomycin B2 shown)

BRETYLIUM TOSYLATE

BRINZOLAMIDE

BROMHEXINE HYDROCHLORIDE

BROMOCRIPTINE MESYLATE

BROMPHENIRAMINE MALEATE

BROXYQUINOLINE

BUDESONIDE

BUMETANIDE

BUPIVACAINE HYDROCHLORIDE

BUPROPION

BUSULFAN

BUTACAINE

BUTAMBEN

BUTOCONAZOLE

CAFFEINE

CAMPHOR (1R)

CANDESARTAN

CANDESARTAN CILEXTIL

CANDICIDIN

CANRENOIC ACID, POTASSIUM SALT

CANRENONE

CAPECITABINE

CAPREOMYCIN SULFATE

CAPSAICIN

CAPTAMINE

CAPTOPRIL

CARBACHOL

CARBENICILLIN DISODIUM

CARBENOXOLONE SODIUM

CARBETAPENTANE CITRATE

CARBIDOPA

CARBINOXAMINE MALEATE

CARBOPLATIN

CARISOPRODOL

CARMUSTINE

CARNITINE (dl) HYDROCHLORIDE

CARPROFEN

CARVEDILOL

CEFACLOR

CEFADROXIL

CEFAMANDOLE NAFATE

CEFAMANDOLE SODIUM

CEFAZOLIN SODIUM

CEFDINIR

CEFEPIME HYDROCHLORIDE

CEFMENOXIME HYDROCHLORIDE

CEFMETAZOLE SODIUM

CEFOPERAZONE

CEFORANIDE

CEFOTAXIME SODIUM

CEFOTETAN

CEFOXITIN SODIUM

CEFPIRAMIDE

CEFSULODIN SODIUM

CEFTIBUTEN

CEFTIOFUR HYDROCHLORIDE

CEFTRIAXONE SODIUM TRIHYDRATE

CEFUROXIME AXETIL

CEFUROXIME SODIUM

CELECOXIB

CEPHALEXIN

CEPHALOTHIN SODIUM

CEPHAPIRIN SODIUM

CEPHRADINE

CETYLPYRIDINIUM CHLORIDE

CHENODIOL

CHLORAMBUCIL

CHLORAMPHENICOL

CHLORAMPHENICOL HEMISUCCINATE

CHLORAMPHENICOL PALMITATE

CHLORCYCLIZINE HYDROCHLORIDE

CHLORHEXIDINE

CHLOROCRESOL

CHLOROGUANIDE HYDROCHLORIDE

CHLOROQUINE DIPHOSPHATE

CHLOROTHIAZIDE

CHLOROXINE

CHLOROXYLENOL

CHLORPHENIRAMINE (S) MALEATE

CHLORPROMAZINE

CHLORPROPAMIDE

CHLORPROTHIXENE HYDROCHLORIDE

CHLORTETRACYCLINE HYDROCHLORIDE

CHLORTHALIDONE

CHLORZOXAZONE

CHOLECALCIFEROL

CHOLESTEROL

CHOLINE CHLORIDE

CICLOPIROX OLAMINE

CILOSTAZOL

CIMETIDINE

CINCHOPHEN

CINNARAZINE

CINOXACIN

CINTRIAMIDE

CIPROFIBRATE

CIPROFLOXACIN

CISPLATIN

CITALOPRAM HYDROBROMIDE

CITICOLINE

CLARITHROMYCIN

CLAVULANATE LITHIUM

CLEMASTINE

CLIDINIUM BROMIDE

CLINAFOXACIN HYDROCHLORIDE

CLINDAMYCIN HYDROCHLORIDE

CLIOQUINOL

CLOBETASOL PROPIONATE

CLOFARABINE

CLOFIBRATE

CLOMIPHENE CITRATE

CLONIDINE HYDROCHLORIDE

CLOPIDOGREL SULFATE

CLORSULON

CLOSANTEL

CLOTRIMAZOLE

CLOXACILLIN SODIUM

CLOXYQUIN

CLOZAPINE

COENZYME B12

COLCHICINE

COLFORSIN

COLISTIMETHATE SODIUM

CORTISONE ACETATE

COTININE

CRESOL

CROMOLYN SODIUM

CRYOFLURANE

CYCLAMIC ACID

CYCLIZINE

CYCLOBENZAPRINE HYDROCHLORIDE

CYCLOHEXIMIDE

CYCLOPENTOLATE HYDROCHLORIDE

CYCLOPHOSPHAMIDE HYDRATE

CYCLOSERINE (D)

CYCLOSPORINE

CYCLOTHIAZIDE

CYPERMETHRIN

CYPROTERONE ACETATE

CYSTEAMINE HYDROCHLORIDE

CYTARABINE

DACARBAZINE

DACTINOMYCIN

DANAZOL

DANTHRON

DANTROLENE SODIUM

DAPSONE

DAPTOMYCIN

DASATINIB

DAUNORUBICIN

DECIMEMIDE

DEFEROXAMINE MESYLATE

DEFLAZACORT

DEHYDROACETIC ACID

DEHYDROCHOLIC ACID

DEMECLOCYCLINE HYDROCHLORIDE

DERACOXIB

DESIPRAMINE HYDROCHLORIDE

DESOXYCORTICOSTERONE ACETATE

DESVENLAFAXINE SUCCINATE

DEXAMETHASONE

DEXAMETHASONE ACETATE

DEXAMETHASONE SODIUM PHOSPHATE

DEXIBUPROFEN

DEXLANSOPRAZOLE

DEXPROPRANOLOL HYDROCHLORIDE

DEXTROMETHORPHAN HYDROBROMIDE

DIAVERIDINE

DIBENZOTHIOPHENE

DIBUCAINE HYDROCHLORIDE

DICHLOROPHENE

DICHLORVOS

DICLAZURIL

DICLOFENAC SODIUM

DICLOXACILLIN SODIUM

DICUMAROL

DICYCLOMINE HYDROCHLORIDE

DIENESTROL

DIETHYLCARBAMAZINE CITRATE

DIETHYLSTILBESTROL

DIFLOXACIN HYDROCHLORIDE

DIFLUNISAL

DIGITOXIN

DIGOXIN

DIHYDROERGOTAMINE MESYLATE

DIHYDROSTREPTOMYCIN SULFATE

DILAZEP DIHYDROCHLORIDE

DIMENHYDRINATE

DIMERCAPROL

DIMETHADIONE

DIOXYBENZONE

DIPHENHYDRAMINE HYDROCHLORIDE

DIPHENYLPYRALINE HYDROCHLORIDE

DIPYRIDAMOLE

DIPYRONE

DIRITHROMYCIN

DISOPYRAMIDE PHOSPHATE

DISULFIRAM

DOBUTAMINE HYDROCHLORIDE

DOCETAXEL

DONEPEZIL HYDROCHLORIDE

DOPAMINE HYDROCHLORIDE

DOXEPIN HYDROCHLORIDE

DOXOFYLLINE

DOXYCYCLINE HYDROCHLORIDE

DOXYLAMINE SUCCINATE

DROFENINE HYDROCHLORIDE

DROPERIDOL

DROSPIRENONE

DYCLONINE HYDROCHLORIDE

DYPHYLLINE

ECAMSULE TRIETHANOLAMINE

ECONAZOLE NITRATE

EDETATE DISODIUM

EDITOL

EDOXUDINE

EMETINE

ENALAPRIL MALEATE

ENALAPRILAT

ENOXACIN

ENROFLOXACIN

ENTACAPONE

EPHEDRINE (1R,2S) HYDROCHLORIDE

EPINEPHRINE BITARTRATE

EPRINOMECTIN

ERGOCALCIFEROL

ERGONOVINE MALEATE

ERYTHROMYCIN

ERYTHROMYCIN ESTOLATE

ERYTHROMYCIN ETHYLSUCCINATE

ESCITALOPRAM OXALATE

ESOMEPRAZOLE POTASSIUM

ESTRADIOL

ESTRADIOL BENZOATE

ESTRADIOL CYPIONATE

ESTRADIOL DIPROPIONATE

ESTRADIOL VALERATE

ESTRAMUSTINE

ESTRIOL

ESTRONE

ESTROPIPATE

ETHACRYNIC ACID

ETHAMBUTOL HYDROCHLORIDE

ETHAVERINE HYDROCHLORIDE

ETHINYL ESTRADIOL

ETHIONAMIDE

ETHISTERONE

ETHOPROPAZINE HYDROCHLORIDE

ETHYL PARABEN

ETODOLAC

ETOPOSIDE

EUCALYPTOL

EUCATROPINE HYDROCHLORIDE

EUGENOL

EVANS BLUE

EXEMESTANE

EZETIMIBE

FAMCICLOVIR

FAMOTIDINE

FAMPRIDINE

FASUDIL HYDROCHLORIDE

FEBUXOSTAT

FENBENDAZOLE

FENBUFEN

FENDILINE HYDROCHLORIDE

FENOFIBRATE

FENOPROFEN

FENOTEROL HYDROBROMIDE

FENSPIRIDE HYDROCHLORIDE

FEXOFENADINE HYDROCHLORIDE

FIPEXIDE HYDROCHLORIDE

FIROCOXIB

FLOXURIDINE

FLUCONAZOLE

FLUDROCORTISONE ACETATE

FLUFENAMIC ACID

FLUINDAROL

FLUMEQUINE

FLUMETHASONE

FLUMETHAZONE PIVALATE

FLUNARIZINE HYDROCHLORIDE

FLUNISOLIDE

FLUNIXIN MEGLUMINE

FLUOCINOLONE ACETONIDE

FLUOCINONIDE

FLUOROMETHOLONE

FLUOROURACIL

FLUOXETINE

FLUPHENAZINE HYDROCHLORIDE

FLURANDRENOLIDE

FLURBIPROFEN

FLUROFAMIDE

FLUTAMIDE

FLUVASTATIN

FOLIC ACID

FOSCARNET SODIUM

FOSFOMYCIN CALCIUM

FTAXILIDE

FULVESTRANT

FURAZOLIDONE

FUROSEMIDE

FUSIDIC ACID

GABOXADOL HYDROCHLORIDE

GADOTERIDOL

GALANTHAMINE

GALLAMINE TRIETHIODIDE

GANCICLOVIR

GATIFLOXACIN

GEFITINIB

GEMFIBROZIL

GENTAMICIN SULFATE

GENTIAN VIOLET

GLIMEPIRIDE

GLUCONOLACTONE

GLUCOSAMINE HYDROCHLORIDE

GLUTAMINE (D)

GRAMICIDIN

GRANISETRON HYDROCHLORIDE

GRISEOFULVIN

GUAIFENESIN

GUANABENZ ACETATE

GUANETHIDINE SULFATE

HALAZONE

HALCINONIDE

HALOPERIDOL

HEPTAMINOL HYDROCHLORIDE

HETACILLIN POTASSIUM

HEXACHLOROPHENE

HEXYLRESORCINOL

HISTAMINE DIHYDROCHLORIDE

HOMATROPINE BROMIDE

HOMATROPINE METHYLBROMIDE

HOMOSALATE

HYCANTHONE

HYDRALAZINE HYDROCHLORIDE

HYDRASTINE (1R, 9S)

HYDROCHLOROTHIAZIDE

HYDROCORTISONE

HYDROCORTISONE ACETATE

HYDROCORTISONE BUTYRATE

HYDROCORTISONE HEMISUCCINATE

HYDROCORTISONE PHOSPHATE TRIETHYLAMINE

HYDROFLUMETHIAZIDE

HYDROQUINONE

HYDROXYAMPHETAMINE HYDROBROMIDE

HYDROXYCHLOROQUINE SULFATE

HYDROXYPROGESTERONE CAPROATE

HYDROXYTOLUIC ACID

HYDROXYUREA

HYDROXYZINE PAMOATE

HYOSCYAMINE

IBANDRONATE SODIUM

IBUPROFEN

IDOXURDINE

IDOXURIDINE

IMIPRAMINE HYDROCHLORIDE

IMIQUIMOD

INAMRINONE

INDAPAMIDE

INDOMETHACIN

INDOPROFEN

INOSITOL

IODIPAMIDE

IODIXANOL

IODOQUINOL

IOHEXOL

IOPANIC ACID

IOTHALAMIC ACID

IOVERSOL

IOXILAN

IPRATROPIUM BROMIDE

IRBESARTAN

ISONIAZID

ISOPROPAMIDE IODIDE

ISOPROTERENOL HYDROCHLORIDE

ISOSORBIDE DINITRATE

ISOSORBIDE MONONITRATE

ISOTRETINON

ISOXICAM

ISOXSUPRINE HYDROCHLORIDE

ITOPRIDE HYDROCHLORIDE

IVERMECTIN

KANAMYCIN A SULFATE

KETOCONAZOLE

KETOPROFEN

KETOROLAC TROMETHAMINE

KETOTIFEN FUMARATE

LABETALOL HYDROCHLORIDE

LACTULOSE

LAMIVUDINE

LANATOSIDE C

LANSOPRAZOLE

LEFLUNOMIDE

LETROZOLE

LEUCOVORIN CALCIUM

LEVAMISOLE HYDROCHLORIDE

LEVOCETIRIZINE DIHYDROCHLORIDE

LEVOFLOXACIN

LEVOMENTHOL

LEVONORDEFRIN

LEVONORGESTREL

LEVOSIMENDAN

LEVOTHYROXINE

LIDOCAINE HYDROCHLORIDE

LINCOMYCIN HYDROCHLORIDE

LINDANE

LINEZOLID

LIOTHYRONINE

LIOTHYRONINE (L- isomer) SODIUM

LISINOPRIL

LITHIUM CITRATE

LOBELINE HYDROCHLORIDE

LOFEXIDINE HYDROCHLORIDE

LOMEFLOXACIN HYDROCHLORIDE

LOMERIZINE HYDROCHLORIDE

LOMUSTINE

LORATADINE

LORNOXICAM

LOSARTAN

LOVASTATIN

LUMIRACOXIB

MAFENIDE HYDROCHLORIDE

MALATHION

MANGAFODIPIR TRISODIUM

MANIDIPINE HYDROCHLORIDE

MANNITOL

MAPROTILINE HYDROCHLORIDE

MEBENDAZOLE

MEBEVERINE HYDROCHLORIDE

MEBHYDROLIN NAPHTHALENESULFONATE

MECAMYLAMINE HYDROCHLORIDE

MECHLORETHAMINE

MECLIZINE HYDROCHLORIDE

MECLOCYCLINE SULFOSALICYLATE

MECLOFENAMATE SODIUM

MECLOFENOXATE HYDROCHLORIDE

MEDROXYPROGESTERONE ACETATE

MEDRYSONE

MEFENAMIC ACID

MEFEXAMIDE

MEFLOQUINE

MEGESTROL ACETATE

MEGLUMINE

MELOXICAM SODIUM

MELPERONE HYDROCHLORIDE

MELPHALAN

MEMANTINE HYDROCHLORIDE

MENADIONE

MEPARTRICIN

MEPENZOLATE BROMIDE

MEPHENESIN

MEPHENTERMINE SULFATE

MEPIVACAINE HYDROCHLORIDE

MEQUINOL

MERBROMIN

MERCAPTOPURINE

MEROPENEM

MESNA

MESO-ERYTHRITOL

MESTRANOL

METAPROTERENOL

METARAMINOL BITARTRATE

METAXALONE

METHACHOLINE CHLORIDE

METHACYCLINE HYDROCHLORIDE

METHAPYRILENE HYDROCHLORIDE

METHAZOLAMIDE

METHENAMINE

METHICILLIN SODIUM

METHIMAZOLE

METHOCARBAMOL

METHOTREXATE(+/-)

METHOXAMINE HYDROCHLORIDE

METHOXSALEN

METHSCOPOLAMINE BROMIDE

METHYCLOTHIAZIDE

METHYLBENZETHONIUM CHLORIDE

METHYLDOPA

METHYLERGONOVINE MALEATE

METHYLPREDNISOLONE

METHYLPREDNISOLONE SODIUM SUCCINATE

METHYLTHIOURACIL

METOCLOPRAMIDE HYDROCHLORIDE

METOPROLOL TARTRATE

METRONIDAZOLE

MEXILETINE HYDROCHLORIDE

MICONAZOLE NITRATE

MIDODRINE HYDROCHLORIDE

MIGLITOL

MILNACIPRAN HYDROCHLORIDE

MINAPRINE HYDROCHLORIDE

MINOCYCLINE HYDROCHLORIDE

MINOXIDIL

MITOMYCIN C

MITOTANE

MITOXANTRONE HYDROCHLORIDE

MOLSIDOMINE

MONENSIN SODIUM (monensin A is shown)

MONOBENZONE

MORANTEL CITRATE

MOXALACTAM DISODIUM

MOXIFLOXACIN HYDROCHLORIDE

MYCOPHENOLATE MOFETIL

MYCOPHENOLIC ACID

NABUMETONE

NADIDE

NADOLOL

NAFCILLIN SODIUM

NAFRONYL OXALATE

NALBUPHINE HYDROCHLORIDE

NALIDIXIC ACID

NALOXONE HYDROCHLORIDE

NALTREXONE HYDROCHLORIDE

NAPHAZOLINE HYDROCHLORIDE

NAPROXEN(+)

NAPROXOL

NATEGLINIDE

NEFAZODONE HYDROCHLORIDE

NEFOPAM

NELARABIN

NEOMYCIN SULFATE

NEOSTIGMINE BROMIDE

NEVIRAPINE

NIACIN

NICARDIPINE HYDROCHLORIDE

NICERGOLINE

NICLOSAMIDE

NICOTINYL ALCOHOL TARTRATE

NIFEDIPINE

NIFURSOL

NILUTAMIDE

NIMODIPINE

NITAZOXANIDE

NITRENDIPINE

NITROFURANTOIN

NITROFURAZONE

NITROMIDE

NOCODAZOLE

NOMIFENSINE MALEATE

NOREPINEPHRINE

NORETHINDRONE

NORETHINDRONE ACETATE

NORETHYNODREL

NORFLOXACIN

NORGESTREL

NORTRIPTYLINE

NOSCAPINE HYDROCHLORIDE

NOVOBIOCIN SODIUM

NYLIDRIN HYDROCHLORIDE

NYSTATIN

OCTOPAMINE HYDROCHLORIDE

OFLOXACIN

OLMESARTAN

OLMESARTAN MEDOXOMIL

OLSALAZINE SODIUM

OLSELTAMIVIR PHOSPHATE

OMEGA-3-ACID ESTERS (EPA shown)

ONDANSETRON

ORLISTAT

ORPHENADRINE CITRATE

OUABAIN

OXACILLIN SODIUM

OXALIPLATIN

OXCARBAZEPINE

OXETHAZAINE

OXIBENDAZOLE

OXIDOPAMINE HYDROCHLORIDE

OXOLINIC ACID

OXYBENZONE

OXYMETAZOLINE HYDROCHLORIDE

OXYPHENBUTAZONE

OXYPHENCYCLIMINE HYDROCHLORIDE

OXYQUINOLINE HEMISULFATE

OXYTETRACYCLINE

PACLITAXEL

PALIPERIDONE

PAPAVERINE HYDROCHLORIDE

PARACHLOROPHENOL

PARAROSANILINE PAMOATE

PARGYLINE HYDROCHLORIDE

PAROMOMYCIN SULFATE

PAROXETINE HYDROCHLORIDE

PEMETREXED

PENCICLOVIR

PENICILLAMINE

PENICILLIN G POTASSIUM

PENICILLIN V POTASSIUM

PENTOLINIUM TARTRATE

PENTOXIFYLLINE

PERGOLIDE MESYLATE

PERHEXILINE MALEATE

PERICIAZINE

PERINDOPRIL ERBUMINE

PERPHENAZINE

PHENACEMIDE

PHENAZOPYRIDINE HYDROCHLORIDE

PHENELZINE SULFATE

PHENINDIONE

PHENIRAMINE MALEATE

PHENOLPHTHALEIN

PHENTOLAMINE HYDROCHLORIDE

PHENYL AMINOSALICYLATE

PHENYLBUTAZONE

PHENYLEPHRINE HYDROCHLORIDE

PHENYLMERCURIC ACETATE

PHENYLPROPANOLAMINE HYDROCHLORIDE

PHENYTOIN SODIUM

PHTHALYLSULFATHIAZOLE

PHYSOSTIGMINE SALICYLATE

PILOCARPINE NITRATE

PIMOZIDE

PINDOLOL

PIOGLITAZONE HYDROCHLORIDE

PIPERACETAZINE

PIPERACILLIN SODIUM

PIPERAZINE

PIPERIDOLATE HYDROCHLORIDE

PIPERINE

PIPOBROMAN

PIRACETAM

PIRENPERONE

PIRENZEPINE HYDROCHLORIDE

PIROCTONE OLAMINE

PIROXICAM

PITAVASTATIN CALCIUM

PIZOTYLINE MALATE

POLYMYXIN B SULFATE

POTASSIUM p-AMINOBENZOATE

PRAMIPEXOLE DIHYDROCHLORIDE

PRAMOXINE HYDROCHLORIDE

PRASUGREL

PRAZIQUANTEL

PRAZOSIN HYDROCHLORIDE

PREDNICARBATE

PREDNISOLONE

PREDNISOLONE ACETATE

PREDNISONE

PRILOCAINE HYDROCHLORIDE

PRIMAQUINE DIPHOSPHATE

PRIMIDONE

PROADIFEN HYDROCHLORIDE

PROBENECID

PROBUCOL

PROCAINAMIDE HYDROCHLORIDE

PROCAINE HYDROCHLORIDE

PROCARBAZINE HYDROCHLORIDE

PROCHLORPERAZINE EDISYLATE

PROCYCLIDINE HYDROCHLORIDE

PROGESTERONE

PROGLUMIDE

PROMAZINE HYDROCHLORIDE

PROMETHAZINE HYDROCHLORIDE

PRONETALOL HYDROCHLORIDE

PROPAFENONE HYDROCHLORIDE

PROPANTHELINE BROMIDE

PROPIOLACTONE

PROPOFOL

PROPYLTHIOURACIL

PSEUDOEPHEDRINE HYDROCHLORIDE

PUROMYCIN HYDROCHLORIDE

PYRANTEL PAMOATE

PYRAZINAMIDE

PYRETHRINS

PYRIDOSTIGMINE BROMIDE

PYRILAMINE MALEATE

PYRIMETHAMINE

PYRITHIONE ZINC

PYRONARIDINE TETRAPHOSPHATE

PYRVINIUM PAMOATE

QUETIAPINE

QUINACRINE HYDROCHLORIDE

QUINAPRIL HYDROCHLORIDE

QUINESTROL

QUINETHAZONE

QUINIDINE GLUCONATE

QUININE SULFATE

QUIPAZINE MALEATE

RACEPHEDRINE HYDROCHLORIDE

RACTOPAMINE HYDROCHLORIDE

RAMIPRIL

RAMOPLANIN [A2 shown; 2mm]

RANITIDINE

RASAGILINE

RESERPINE

RESORCINOL

RESORCINOL MONOACETATE

RETAPAMULIN

RETINOL

RETINYL PALMITATE

RIBAVIRIN

RIFAMPIN

RITANSERIN

RITODRINE HYDROCHLORIDE

RITONAVIR

RIZATRIPTAN BENZOATE

ROFECOXIB

RONIDAZOLE

ROPINIROLE

ROSIGLITAZONE

ROSUVASTATIN CALCIUM

ROXARSONE

ROXATIDINE ACETATE HYDROCHLORIDE

ROXITHROMYCIN

RUFLOXACIN HYDROCHLORIDE

SACCHARIN

SALICIN

SALICYL ALCOHOL

SALICYLAMIDE

SALICYLANILIDE

SALINOMYCIN, SODIUM

SALSALATE

SANGUINARINE SULFATE

SCOPOLAMINE HYDROBROMIDE

SELAMECTIN

SEMUSTINE

SENNOSIDE A

SERATRODAST

SERTRALINE HYDROCHLORIDE

SEVOFLURANE

SIBUTRAMINE HYDROCHLORIDE

SILDENAFIL CITRATE

SIMVASTATIN

SIROLIMUS

SISOMICIN SULFATE

SODIUM DEHYDROCHOLATE

SODIUM NITROPRUSSIDE

SODIUM OXYBATE

SODIUM PHENYLACETATE

SODIUM PHENYLBUTYRATE

SODIUM SALICYLATE

SPARFLOXACIN

SPARTEINE SULFATE

SPECTINOMYCIN HYDROCHLORIDE

SPIPERONE

SPIRAMYCIN

SPIRAPRIL HYDROCHLORIDE

SPIRONOLACTONE

STAVUDINE

STREPTOMYCIN SULFATE

STREPTOZOSIN

SUCCINYLSULFATHIAZOLE

SULBACTAM

SULCONAZOLE NITRATE

SULFABENZAMIDE

SULFACETAMIDE

SULFACHLORPYRIDAZINE

SULFADIAZINE

SULFADIMETHOXINE

SULFAMERAZINE

SULFAMETER

SULFAMETHAZINE

SULFAMETHIZOLE

SULFAMETHOXAZOLE

SULFAMETHOXYPYRIDAZINE

SULFAMONOMETHOXINE

SULFANILATE ZINC

SULFANITRAN

SULFAPYRIDINE

SULFAQUINOXALINE SODIUM

SULFASALAZINE

SULFATHIAZOLE

SULFINPYRAZONE

SULFISOXAZOLE

SULINDAC

SULMAZOLE

SULOCTIDIL

SULPIRIDE

SUPROFEN

SURAMIN

TACROLIMUS

TAMOXIFEN CITRATE

TANDUTINIB

TANNIC ACID

TAZOBACTAM

TEGASEROD MALEATE

TELMISARTAN

TEMEFOS

TEMOZOLAMIDE

TENIPOSIDE

TENOXICAM

TERBUTALINE HEMISULFATE

TERCONAZOLE

TERFENADINE

TESTOSTERONE

TESTOSTERONE PROPIONATE

TETRACAINE HYDROCHLORIDE

TETRACYCLINE HYDROCHLORIDE

TETRAHYDROZOLINE HYDROCHLORIDE

TETROQUINONE

THALIDOMIDE

THEOBROMINE

THEOPHYLLINE

THIABENDAZOLE

THIAMPHENICOL

THIMEROSAL

THIOGUANINE

THIORIDAZINE HYDROCHLORIDE

THIOSTREPTON

THIOTEPA

THIOTHIXENE

THIRAM

THONZONIUM BROMIDE

THONZYLAMINE HYDROCHLORIDE

TIAPRIDE HYDROCHLORIDE

TIBOLONE

TIGECYCLINE

TILARGININE HYDROCHLORIDE

TILETAMINE HYDROCHLORIDE

TILMICOSIN

TIMOLOL MALEATE

TINIDAZOLE

TOBRAMYCIN

TODRALAZINE HYDROCHLORIDE

TOLAZAMIDE

TOLAZOLINE HYDROCHLORIDE

TOLBUTAMIDE

TOLMETIN SODIUM

TOLNAFTATE

TOLPERISONE HYDROCHLORIDE

TOSYLCHLORAMIDE SODIUM

TRANEXAMIC ACID

TRANYLCYPROMINE SULFATE

TRAZODONE HYDROCHLORIDE

TRETINOIN

TRIACETIN

TRIAMCINOLONE

TRIAMCINOLONE ACETONIDE

TRIAMCINOLONE DIACETATE

TRIAMTERENE

TRICHLORMETHIAZIDE

TRIFLUOPERAZINE HYDROCHLORIDE

TRIFLUPROMAZINE HYDROCHLORIDE

TRIFLURIDINE

TRIHEXYPHENIDYL HYDROCHLORIDE

TRILOSTANE

TRIMEPRAZINE TARTRATE

TRIMETHOBENZAMIDE HYDROCHLORIDE

TRIMETHOPRIM

TRIMETOZINE

TRIMIPRAMINE MALEATE

TRIOXSALEN

TRIPELENNAMINE CITRATE

TRIPROLIDINE HYDROCHLORIDE

TRISODIUM ETHYLENEDIAMINE TETRACETATE

TROLEANDOMYCIN

TROPICAMIDE

TROPISETRON HYDROCHLORIDE

TRYPTOPHAN

TUAMINOHEPTANE SULFATE

TUBOCURARINE CHLORIDE

TYROTHRICIN

URACIL

URAPIDIL HYDROCHLORIDE

UREA

URETHANE

URSODIOL

VALDECOXIB

VALGANCICLOVIR HYDROCHLORIDE

VALPROATE SODIUM

VALSARTAN

VANCOMYCIN HYDROCHLORIDE

VENLAFAXINE

VIDARABINE

VINBLASTINE SULFATE

VINORELBINE

VINPOCETINE

VIOMYCIN SULFATE

VORICONAZOLE

VORINOSTAT

WARFARIN

XYLAZINE

XYLOMETAZOLINE HYDROCHLORIDE

YOHIMBINE HYDROCHLORIDE

ZALCITABINE

ZAPRINAST

ZIDOVUDINE [AZT]

ZIPRASIDONE MESYLATE

ZOMEPIRAC SODIUM

ZOPICLONE

Data analysis

Data are presented as the mean and SEM, unless stated otherwise. Pairwise statistical significance was determined with a Student’s two-tailed unpaired t test, ANOVA, or Mann–Whitney rank sum test, as appropriate, unless stated otherwise. Results were considered significant at p < 0.05, unless otherwise indicated.

Results

A first-stage behavioral screen for antiepileptic activity

Locomotion tracking is a reliable and rapid strategy with which to monitor behavioral seizures in freely swimming larval zebrafish (Baraban et al., 2005, 2013; Winter et al., 2008). In these locomotion plots, high-velocity movements of ≥20 mm/s correspond to paroxysmal whole-body convulsions, referred to as Stage III, and are consistently observed in unprovoked scn1Lab mutant larvae but not in age-matched wild-type siblings. Using automated locomotion tracking, we performed a phenotype-based screen to identify compounds that significantly reduce mutant swim behavior to levels associated with Stage 0 or Stage I (e.g., activity equivalent to that seen in normal untreated WT zebrafish). In a 96-well format, we tracked mutant swim activity at baseline, and then again after addition of a test compound (100 µm); each compound was tested on six individual mutant larvae (Fig. 1), and larvae were sorted based on pigmentation differences (Fig. 1). Mutant swim activity between two consecutive recording epochs in embryo media is tracked on every plate as an internal control. A box plot showing the change in swim velocity in untreated mutants is shown in Figure 1 (n = 112) and defined as the control. Based on an SD of 21.8 for these data, we set the detection threshold as any compound that inhibits movement (measured as a change in mean velocity) by >2 SDs (or ≥44%). This approach was previously validated using standard antiepileptic drugs in this model (Baraban et al., 2013). Next, we screened a repurposed library in which all compounds have reached the clinical evaluation stage (PHARMAKON 1600 Collection; http://www.msdiscovery.com/pharma.html). Among the 1012 compounds screened (Fig. 1) only 20 (or 1.97%) were found to significantly inhibit spontaneous seizure behavior in scn1Lab mutants. All 20 compounds were subsequently retested in a separate clutch of scn1Lab mutants at a concentration of 100 µm (Fig. 2, trial 2; N = 6 fish/compound). A total of 154 compounds were classified as “toxic” (Table 2); 55 compounds were classified as “hyperexcitable” (Table 3). Representative locomotion tracking raw data plots for gemfibrozil, a toxic nonsteroid nuclear receptor ligand, and mepivacaine, a hyperexcitable proconvulsant anesthetic, are shown in Figure 2.

Figure 2.

Positive hits identified in the locomotion assay. , Heat map showing the results of individual zebrafish trials (1-6) for compounds tested at a concentration of 100 µm in the locomotion-tracking assay. Raw data values for individual fish are shown within the color-coded boxes for one sample trial. Mean velocity data are shown at right for “trial 1” and “trial 2”; six fish per trial. Note: only drugs highlighted in bold type were classified as positive nontoxic hits in two independent trials and moved on to further testing. , Representative raw locomotion data plots for six individual scn1Lab mutant larvae at baseline (top) and following the addition of a compound resulting in fatality (bottom, gemfibrozil) or hyperactivity (bottom, mepivacaine). Movement is color coded, with low-velocity movements shown in yellow, and high velocity movements shown in red.

Table 2:

List of compounds exhibiting toxicity.

ABACAVIR SULFATE

ACIPIMOX

ADENOSINE PHOSPHATE

ALAPROCLATE

AMLEXANOX

AMOROLFINE HYDROCHLORIDE

ANTAZOLINE PHOSPHATE

ARTEMETHER

ASCORBIC ACID

ATORVASTATIN CALCIUM

AUROTHIOGLUCOSE

AZELAIC ACID

BENORILATE

BENZONATATE

BETAINE HYDROCHLORIDE

BETAMIPRON

BROMHEXINE HYDROCHLORIDE

BUDESONIDE

BUPIVACAINE HYDROCHLORIDE

BUSULFAN

BUTOCONAZOLE

CAPSAICIN

CARPROFEN

CEFORANIDE

CEFOTAXIME SODIUM

CEFOXITIN SODIUM

CEPHALEXIN

CHLORAMBUCIL

CHLORAMPHENICOL HEMISUCCINATE

CHLOROGUANIDE HYDROCHLORIDE

CHLORPHENIRAMINE (S) MALEATE

CINCHOPHEN

CINNARAZINE

CINTRIAMIDE

CIPROFLOXACIN

CLIDINIUM BROMIDE

CLOZAPINE

COLISTIMETHATE SODIUM

CRYOFLURANE

CYCLOPHOSPHAMIDE HYDRATE

CYCLOTHIAZIDE

CYPERMETHRIN

DAUNORUBICIN

DECIMEMIDE

DEXTROMETHORPHAN HYDROBROMIDE

DICHLOROPHENE

DIETHYLCARBAMAZINE CITRATE

DIOXYBENZONE

DIRITHROMYCIN

DISOPYRAMIDE PHOSPHATE

DISULFIRAM

ECONAZOLE NITRATE

EDETATE DISODIUM

EMETINE

ENALAPRILAT

ERYTHROMYCIN

ETHINYL ESTRADIOL

ETHIONAMIDE

ETHOPROPAZINE HYDROCHLORIDE

ETHYL PARABEN

EUGENOL

FIPEXIDE HYDROCHLORIDE

FLUMETHASONE

FLUNISOLIDE

FLUVASTATIN

GEMFIBROZIL

GENTAMICIN SULFATE

GLUCONOLACTONE

HALAZONE

HALCINONIDE

HETACILLIN POTASSIUM

HEXACHLOROPHENE

HOMATROPINE METHYLBROMIDE

HYDRASTINE (1R, 9S)

HYDROXYAMPHETAMINE HYDROBROMIDE

HYDROXYCHLOROQUINE SULFATE

IODIXANOL

IOHEXOL

IRBESARTAN

LEVOSIMENDAN

LISINOPRIL

LOMERIZINE HYDROCHLORIDE

MANGAFODIPIR TRISODIUM

MECLOFENOXATE HYDROCHLORIDE

MESTRANOL

METHACHOLINE CHLORIDE

METHYLERGONOVINE MALEATE

METRONIDAZOLE

MIGLITOL

MONENSIN SODIUM (monensin A is shown)

MONOBENZONE

MOXALACTAM DISODIUM

NADOLOL

NALBUPHINE HYDROCHLORIDE

NALTREXONE HYDROCHLORIDE

NAPHAZOLINE HYDROCHLORIDE

NAPROXEN(+)

NEOMYCIN SULFATE

NIFEDIPINE

NITAZOXANIDE

NITROMIDE

NORETHINDRONE

OLMESARTAN MEDOXOMIL

OXYMETAZOLINE HYDROCHLORIDE

PARACHLOROPHENOL

PAROMOMYCIN SULFATE

PERHEXILINE MALEATE

PHENTOLAMINE HYDROCHLORIDE

PHENYLBUTAZONE

PHENYLMERCURIC ACETATE

PHYSOSTIGMINE SALICYLATE

PIMOZIDE

PIPERACILLIN SODIUM

PIPERAZINE

PIRACETAM

PIRENZEPINE HYDROCHLORIDE

PIROCTONE OLAMINE

PITAVASTATIN CALCIUM

PRIMAQUINE DIPHOSPHATE

PROBENECID

PROCARBAZINE HYDROCHLORIDE

PROGLUMIDE

PROMETHAZINE HYDROCHLORIDE

PUROMYCIN HYDROCHLORIDE

QUININE SULFATE

RETINYL PALMITATE

RIFAMPIN

RITONAVIR

ROFECOXIB

RUFLOXACIN HYDROCHLORIDE

SACCHARIN

SALICIN

SENNOSIDE A

STAVUDINE

STREPTOMYCIN SULFATE

SULFADIAZINE

SULINDAC

SULOCTIDIL

TANNIC ACID

TELMISARTAN

TENOXICAM

THEOPHYLLINE

TILETAMINE HYDROCHLORIDE

TILMICOSIN

TIMOLOL MALEATE

TOLBUTAMIDE

TOLNAFTATE

TRAZODONE HYDROCHLORIDE

TRETINOIN

TRIFLUPROMAZINE HYDROCHLORIDE

TROPISETRON HYDROCHLORIDE

VALDECOXIB

VORINOSTAT

ZALCITABINE

Table 3:

List of compounds exhibiting hyperexcitable or proconvulsant activity.

ADENOSINE PHOSPHATE

ALBUTEROL (+/-)

ALEXIDINE HYDROCHLORIDE

AMANTADINE HYDROCHLORIDE

AMINOHIPPURIC ACID

AMINOLEVULINIC ACID HYDROCHLORIDE

AUROTHIOGLUCOSE

AZACITIDINE

BENZOYL PEROXIDE

BETAZOLE HYDROCHLORIDE

BROMHEXINE HYDROCHLORIDE

BUSULFAN

CEFSULODIN SODIUM

CEFUROXIME AXETIL

CHLOROGUANIDE HYDROCHLORIDE

CYSTEAMINE HYDROCHLORIDE

ECAMSULE TRIETHANOLAMINE

ECONAZOLE NITRATE

EDOXUDINE

ENROFLOXACIN

ESTRADIOL CYPIONATE

ETHINYL ESTRADIOL

ETHOPROPAZINE HYDROCHLORIDE

ETOPOSIDE

FASUDIL HYDROCHLORIDE

FEBUXOSTAT

FLUMETHASONE

FLUOROMETHOLONE

FURAZOLIDONE

GANCICLOVIR

GLUCONOLACTONE

GRANISETRON HYDROCHLORIDE

HALAZONE

HEXACHLOROPHENE

IODIPAMIDE

LABETALOL HYDROCHLORIDE

MEPIVACAINE HYDROCHLORIDE

MITOXANTRONE HYDROCHLORIDE

MORANTEL CITRATE

NOCODAZOLE

OFLOXACIN

PENTOLINIUM TARTRATE

PERINDOPRIL ERBUMINE

PIOGLITAZONE HYDROCHLORIDE

PRAMIPEXOLE DIHYDROCHLORIDE

PROGLUMIDE

RIFAMPIN

SERATRODAST

SERTRALINE HYDROCHLORIDE

SIBUTRAMINE HYDROCHLORIDE

SUCCINYLSULFATHIAZOLE

TACROLIMUS

TETROQUINONE

TIMOLOL MALEATE

URACIL

A second-stage electrophysiology assay for antiepileptic activity

Extracellular recording electrodes are a reliable, reproducible, and sensitive approach to monitor electroencephalographic activity in agar-immobilized larval zebrafish (Baraban et al., 2005; Baraban, 2013). Field electrodes offer high a signal-to-noise ratio and can be placed, using direct visualization in transparent larvae, into specific CNS structures (i.e., telencephalon or optic tectum). Using a local field electrode, we can efficiently monitor the occurrence of electrographic seizure events in the same zebrafish that were previously tested in the locomotion assay. Based on a positive nontoxic result in two independent locomotion assays, four drugs moved on to electrophysiology testing at concentrations between 500 µm and 1 mm (Fig. 3). Consistent with a “false-positive” classification, spontaneous epileptiform discharge activity was observed for three of these drugs: norfloxacin, theobromine, and cytarabine. Dimethadione, previously shown to inhibit spontaneous epileptiform discharges in thalamocortical slices at concentrations between 1 and 10 mm (Zhang et al., 1996), suppressed burst discharge activity in scn1Lab mutant larvae (Fig. 3). To identify whether any of these four compounds exert nonspecific effects on behavior, they were also tested on freely swimming WT zebrafish larvae (5 dpf) at a concentration of 500 µm. Comparing the total distance moved during a 10 min recording epoch before, and after, the application of a test compound failed to reveal any significant changes in locomotor activity (Fig. 3).

Figure 3.

Electrophysiology assay to identify drugs that rescue the scn1Lab mutant epilepsy phenotype. , Representative field electrode recording epochs (5 min in duration) are shown for the “positive” compounds identified in the locomotion assay. All recordings were obtained with an electrode placed in the forebrain of agar-immobilized scn1Lab larvae that was previously tested in the locomotion assay. A suppression of epileptiform electrographic discharge activity was noted in mutants exposed to dimethadione. , Bar plot showing the mean number of epileptiform events in a 10 min recording epoch for scn1Lab larvae exposed to cytarabine (N = 6), dimethadione (N = 6), theobromine (N = 6), and norfloxacin (N = 6). The mean ± SEM is shown. The fish shown were tested in the locomotion assay first. , Bar plot showing the total distance traveled before (black bars) and after (white bars) exposure to a test compound; 10 min recording epoch and six fish per drug. The mean ± SEM is shown.

Assessment of huperzine A and fenfluramine for antiepileptic activity

Next, we tested two additional compounds that were not in our drug library, but have recently been described as potential antiepileptic treatments for DS. Huperzine A, a small-molecule alkaloid isolated from Chinese club moss with NMDA-type receptor blocking and anticholinesterase activity, has purported antiepileptic actions against NMDA- or soman-induced seizures (Tonduli et al., 2001; Coleman et al., 2008). In the locomotion assay, huperzine A failed to significantly alter scn1Lab seizure behavior at any concentration tested (Fig. 4). In contrast, huperzine A was effective at 1 mm in the acute pentylenetetrazole (PTZ) assay (Fig. 4). Fenfluramine is an amphetamine-like compound that has been reported to successfully reduce seizure occurrence in children with DS as a low-dose add-on therapy (Ceulemans et al., 2012). In the locomotion assay, fenfluramine significantly reduced mutant mean swim velocity at concentrations between 100 and 500 μm (Fig. 4); 1 mm fenfluramine was toxic in the scn1Lab and PTZ assays (Fig. 4). The fenfluramine-treated scn1Lab mutant exhibited a suppression of spontaneous electrographic seizure discharge to levels similar to controls at 500 μm, but only a partial reduction in electrographic activity at 250 μm (Fig. 4).

Figure 4.

Evaluation of putative antiepileptic drugs in scn1Lab mutants. , Locomotion tracking plots for scn1Lab zebrafish at baseline and following huperzine A administration. Total movement is shown for a 10 min recording epoch. , Plot showing the change in mean velocity for three different huperzine A concentrations (blue bars). Each bar is the mean change for six fish. The threshold for a positive hit is shown as a dashed line. WT fish exposed to PTZ and huperzine A are shown in red (N = 7). , , Same for fenfluramine. Note that 1 mm fenfluramine was toxic, as indicated. , Representative field recordings from scn1Lab mutant larvae at 5 dpf. Electrographic activity is shown for a 5 min recording epoch (top traces); high-resolution traces are shown below, as indicated. Note that abnormal burst discharge activity persists in scn1Lab mutants exposed to 250 µm fenfluramine. The fish shown were tested in the locomotion assay first.

Discussion

Zebrafish and humans share extensive genomic similarity. With regard to disease, 84% of genes known to be associated with disease states in humans have a zebrafish homolog (Howe et al., 2013). This genetic similarity and the characteristic of zebrafish larvae to exhibit quantifiable seizure behaviors or electrographic seizure discharge that is fundamentally similar to that recorded in humans (Jirsa et al., 2014) make this an ideal system for drug discovery. Behavioral assays customized for automated evaluation of locomotion (Winter et al., 2008; Creton, 2009; Baxendale et al., 2012; Baraban et al., 2013; Raftery et al., 2014) make moderate-to-high-throughput phenotype-based drug screening in zebrafish possible. Using this approach and a zebrafish scn1 mutant (Baraban et al., 2013), we successfully identified antiepileptic compounds. Here we report results from screening ∼1000 compounds from a repurposed drug library and present data that will be periodically updated on-line using this open-access publishing mechanism.

As a model system, the scn1Lab mutant zebrafish has many advantages. First, in contrast to transient and variable knockdown of gene expression using antisense morpholino oligonucleotides (Teng et al., 2010; Finckbeiner et al., 2011; Mahmood et al., 2013), scn1Lab mutants carry a stable and heritable amino acid substitution at position 1208 in the third domain of SCN1A that shares 76% homology with humans (Schoonheim et al., 2010; Baraban et al., 2013). Mutations in this channel are one of the primary genetic causes underlying DS (Claes et al., 2003; Escayg and Goldin, 2010; De Jonghe, 2011; Saitoh et al., 2012). As zebrafish possess two scn1 genes (Novak et al., 2006), homozygous mutants for scn1Lab are comparable to the haploinsufficient clinical condition, and there is no variability from larvae to larvae, or clutch to clutch, with respect to gene inactivation, as is commonly observed with morpholino injections (Kok et al., 2015). Although crosses of heterozygotes produce only one-quarter homozygous scn1Lab mutants per mating, there are virtually no limitations on maintaining a large colony of healthy, adult breeders for these types of large-scale screens. Second, it is possible to observe and monitor seizure-like behavior consisting of rapid movements and whole-body convulsions in freely swimming scn1Lab mutants as early as 4 dpf that persist for the life of the larvae (∼12 dpf). These behaviors are comparable to those observed with exposure to a common convulsant agent (PTZ) and classified as Stage III (Baraban et al., 2005). In addition, clear evidence for epileptiform discharge generated in the CNS of immobilized scn1Lab mutant larvae has been obtained at ages between 4 and 8 dpf (Baraban et al., 2013). Both zebrafish measures of seizure activity are sensitive to inhibition by AEDs commonly prescribed to children with DS (e.g., valproate, benzodiazepines, and stiripentol), but are resistant to many antiepileptic compounds (e.g., phenytoin, carbamazepine, ethosuximide, decimemide, tiletamine, primidone, phenacemide, and vigabatrin). Pharmacoresistance is defined as the inability to control seizure activity with at least two different AEDs (Berg, 2009), and, with demonstrated resistance to eight AEDs, our model clearly fits this definition. This level of model validation has not been possible with morpholinos probably owing to the high degree of variability, or off-target effects, associated with this technique (Kok et al., 2015).

Our screening results highlight the stringency of our approach with a positive hit rate of only 1.97% on the first-stage locomotion assay, and successful identification of 1 compound (of 1012 compounds) with known antiepileptic activity (i.e., dimethadione, a T-type channel antagonist). In additional testing, we confirmed an antiepileptic action for fenfluramine (serotonin uptake inhibitor). Similar to ethosuximide, a reduction in regenerative burst discharges associated with neuronal T-type calcium currents could be the underlying mechanism for dimethadione in DS mutants; however, it is worth noting that T-type channel blockers ethosuximide and flunarizine were not similarly effective (Baraban et al. 2013; this article), and that dimethadione can cause arrhythmia owing to blockade of cardiac human ether-a-go-go-related gene potassium channels (Azarbayjani and Danielsson, 2002; Danielsson et al., 2007). Modulation of serotonin [5-hydroxytryptamine (5-HT)] signaling by blocking uptake or increasing release from neurons by acting as substrates for 5-HT transporter (sertraline) proteins (Fuller et al., 1988; Gobbi and Mennini, 1999; Baumann et al., 2000; Rothman et al., 2010) may be the mechanism of action for fenfluramine in patients with DS, though a detailed analysis of precisely how fenfluramine modulates excitability via this signaling pathway has not been performed. Nonetheless, both drugs probably exert a direct effect on network excitability (at neuronal or synaptic levels, respectively) to suppress electrographic discharge and the associated high-velocity seizure behavior seen in scn1Lab mutants, and may be potential targets for clinical use. In contrast, three other drugs identified in the primary locomotion assay were not effective in suppressing electrical events and were designated as false positives. This is not altogether surprising given that the xanthine alkaloid (theobromine), chemotherapeutic (cytarabine), and antibiotic (norfloxacin) mechanisms for these compounds would not be consistent with seizure inhibition. Moreover, the variability inherent in behavioral experiments performed on different zebrafish larvae, in different microplates, and on different days may contribute to these false-positive designations in locomotion assays, and is evident in the range of mean velocity values seen during tracking episodes from control studies (Fig. 1) or in the failure of many of the initial 20 lead compounds to be confirmed on subsequent retesting (see Fig. 2). This is a limitation of locomotion-based screening assays and is another reason why a secondary electrophysiology assay on the same zebrafish is a critical advantage of our approach.

An additional advantage of in vivo screening with zebrafish larvae is the simultaneous identification of compounds resulting in toxicity. Zebrafish-based anticonvulsant drug-screening assays based primarily on in situ hybridization detection of early gene expression at 2 dpf (Baxendale et al., 2012) do not routinely monitor spontaneous swim behavior, heart rate, or response to external stimuli. Lacking these real-time measures of toxicity, compounds observed to induce fatality in a freely swimming scn1Lab-based behavioral assay (e.g., gemfibrozil, suloctidil, pimozide, or dioxybenzone) were mistakenly classified as seizure-suppressing compounds in the PTZ-based c-Fos in situ hybridization assay. Indeed, 41% of the “anticonvulsant” compounds positively identified at 2 dpf in Baxendale et al. (2012) were toxic, proconvulsant, or simply not effective in scn1Lab mutant assays at 5-6 dpf. Similarly, it is critical to monitor blood flow and heart activity even in the agar-immobilized electrophysiology assay as compounds effective in suppressing electrical activity can also be toxic. These discrepancies highlight the potential problems associated with zebrafish drug-screening strategies that do not encompass multiple readouts and suggest the need for a note of caution when comparing screening results from different laboratory groups. While any lead compound identified in a zebrafish-based screening assay will, ultimately, need to be independently replicated and/or validated in additional mammalian model systems, the ability to rapidly identify such compounds, while simultaneously identifying potential negative side effects, makes genetically modified zebrafish a unique resource for drug discovery in an age of personalized medicine.