Phenotype-Based Screening of Synthetic Cannabinoids in a Dravet Syndrome

Home , ADBICA, Synthetic cannabinoids

bioRxiv preprint doi: https://doi.org/10.1101/815811; this version posted October 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Phenotype-based screening of synthetic cannabinoids in a Dravet Syndrome

zebrafish model

Aliesha Griffin1, Mana Anvar1, Kyla Hamling1, and Scott C. Baraban1*

1Epilepsy Research Laboratory and Weill Institute for Neuroscience, Department of

Neurological Surgery, University of California San Francisco, San Francisco, CA, USA, 94122.

*Correspondence: Scott C. Baraban, PhD. Box 0520, Department of Neurological Surgery, 513

Parnassus Ave., San Francisco, CA 94143. Email: [email protected]

1 Dravet syndrome (DS) is a catastrophic epilepsy of childhood, characterized by cognitive

2 impairment, severe seizures and increased risk for sudden unexplained death in epilepsy

3 (SUDEP). Although refractory to conventional antiepileptic drugs, emerging preclinical and

4 clinical evidence suggests that modulation of the endocanniboid system could be therapeutic in

5 these patients. Here we used a validated zebrafish model of DS, scn1lab homozygous mutants,

6 to screen a commercially available library containing 370 synthetic cannabinoid (SC)

7 compounds for compounds effective in reducing spontaneous seizures. Primary phenotype-

8 based screening was performed using a locomotion-based assay in 96-well plates, and a

9 secondary local field potential recording assay was then used to confirm suppression of

10 electrographic epileptiform events. Identified SCs with anti-seizure activity, in both assays,

11 included five SCs structurally classified as indole-based cannabinoids: JWH 018 N-(5-

12 chloropentyl) analog, JWH 018 N-(2-methylbutyl) isomer, 5-fluoro PB-22 5-

13 hydroxyisoquinoline isomer, 5-fluoro ADBICA, and AB-FUBINACA 3-fluorobenzyl isomer.

14 Our approach demonstrates that two-stage phenotype-based screening in a zebrafish model of DS

15 successfully identifies synthetic cannabinoids with anti-seizure activity, and supports further

16 investigation of SCs for refractory epilepsies.

23 bioRxiv preprint doi: https://doi.org/10.1101/815811; this version posted October 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

25 Introduction

26 Seizures, in some epilepsy patients, can be difficult to control using conventional antiepileptic

27 drugs (AEDs). In patients classified with catastrophic epilepsies in childhood, effective seizure

28 control using AEDs can be a significant problem and early developmental exposure to AEDs is

29 often associated with undesirable side effects. In the search for new treatment options for these

30 patients there is growing interest in drugs that modulate the endogenous cannabinoid system. The

31 endocannabinoid system, comprised of two G-protein coupled receptors (CB1 and CB2), has

32 been demonstrated to play a role in regulating seizure activity in the brain1-4. Recent data

33 suggests broad anticonvulsant activity for synthetic cannabanoids, phytocannabinoids and

34 Cannabis sativa (marijiuana) e.g., drugs targeting the endocannabinoid system. Studies on

35 medical marijuana largely focus on anecdotal patient experiences. Careful preclinical

36 examination of medical marijuana and related extracts such as cannabadiol are difficult as these

37 are designated as Schedule II compounds by the U.S. Food and Drug Administration (FDA).

38 Synthetic cannabinoids (SCs), which represent a variety of compounds engineered to bind

39 cannabinoid receptors and minimize the psychogenic effects of marijuana, have been examined

40 most extensively in adult animal models of acute seizures, with mixed results. In the

41 pentylenetetrazole (PTZ) model of generalized seizures, WIN 55,212-2 (a mixed CB1/CB2

42 receptor agonist), exerts pro- and anticonvulsant effects wherease rachidonyl-2’-

43 chloroethylamide (a CB1 receptor agonist) was shown to decrease acute PTZ seizure thresholds,

44 increase acute PTZ seizure thresholds or have no effect at all5-8. In the maximal dentate

45 activation model of limbic seizures or limbic kindling models, WIN 55,212-2 reduced seizure

46 thresholds1, 2, delayed epileptogenesis3, or failed to provide any seizure protection4. Recent bioRxiv preprint doi: https://doi.org/10.1101/815811; this version posted October 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

47 testing completed at the Epilepsy Therapy Screening Program reported anti-seizure properties for

48 CBD in mouse 6 Hz 44 mA and mouse/rat maximal electroshock seizure assays5. Although even

49 less is known about experimental models of childhood epilepsies, a recent study by6 examined

50 seven cannabinoid receptor agonists in young postnatal rat models of PTZ- hypoxia- or methyl-

51 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate(DMCM)-evoked seizures, at concentrations

52 that also induced sedative effects only WIN 55,212-2 exhibited anti-seizure activity in all three

53 models.

55 Interestingly, Dravet syndrome (DS) - a genetic epilepsy associated, in most cases, with a loss-

56 of-function de novo mutation in the SCN1A voltage-gated sodium channel subunit - is one

57 particular form of childhood epilepsy where SCs, phytocannabinoids and medical marijuana

58 have consistently shown anticonvulsant activity in preclinical and clinical studies. Reductions in

59 seizure activity were observed in Scn1a+/- mice at drug concentrations above 100 mg/kg with

60 amelioration of autistic-like behaviors seen at a lower concentration of 10 mg/kg7. In the first

61 open-label investigational trial of cannabidiol (CBD) in children with DS, Devinsky et al.8

62 reported a 37% median reduction in monthly seizure counts. Additional positive seizure

63 reductions with CBD treatment9-11, and a GW Pharmaceuticals sponsored open-label double-

64 blinded study reporting an adjusted 23% reduced in seizure frequency led to FDA approval for

65 CBD (Epidiolex®) as a treatment for seizures associated with DS.

67 In the present study, we used a scn1 zebrafish mutant model of DS to screen a library of SCs for

68 those that exhibit anti-seizure properties. The scn1lab mutant zebrafish line exhibits

69 spontaneous seizures that can be easily monitored using acute behavioral and bioRxiv preprint doi: https://doi.org/10.1101/815811; this version posted October 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

70 electrophysiological assays12, 13. These mutants exhibit metabolic deficit, early fatality, sleep

71 disturbances and a pharmacological profile similar to DS patients14-16. In addition to replicating

72 key aspects of DS, scn1lab mutants were shown to be a useful model system for large-scale

73 phenotype-based drug screening and drugs identified with this zebrafish-centric approach have

74 already shown efficacy in the clinic13, 17. Using scn1lab mutants, we screened 370 SCs at two

75 different concentrations. After repeated locomotion and electrophysiological assays with

76 multiple biological replicates, we identified five SCs that exert significant anti-seizure activity

77 (reducing convulsive swim behavior and suppressing abnormal electrographic discharge

78 frequency) during acute exposures. Additionally, analysis of structure-activity relationships

79 revealed these compounds are structurally similar and are indole-derived cannabinoids18. These

80 data suggest that specific classes of SCs can exert antiepileptic activity and provide further

81 justification for using larval zebrafish models to identify novel therapeutic targets.

83 Materials and Methods

84 Zebrafish maintenance

85 Zebrafish were maintained according to standard procedures19 and following guidelines

86 approved by the University of California, San Francisco Institutional Animal Care and Use

87 Committee. The zebrafish room was maintained on a light-dark cycle, with lights-on at 9:00 AM

88 and lights-off at 11:00 PM. An automated feedback control unit was used to maintain aquarium

89 water conditions in the following ranges: 29-30°C, pH 7.5-8.0, conductivity (EC) 690-710.

90 Zebrafish embryos and larvae were raised in an incubator maintained at 28.5°C, on the same

91 light-dark cycle as the fish facility. Water used for embryos and larvae was made by adding

92 0.03% Instant Ocean and 0.000002% methylene blue to reverse-osmosis distilled water. bioRxiv preprint doi: https://doi.org/10.1101/815811; this version posted October 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

93 Embryos and larvae were raised in plastic petri dishes (90 mm diameter, 20 mm depth) and

94 housing density was limited to approximately 50-60 individuals per dish. The sex of embryos

95 and larvae cannot be determined at these early stages.

97 Drugs

98 Compounds for drug screening were purchased from Cayman Chemicals and were provided as

99 10 mM DMSO solutions (Supplemental Table I).

100

101 Locomotion assay

102 At 5 days post fertilization (dpf) offspring from crossing adult scn1lab+/- zebrafish were sorted

103 by pigmentation to isolate scn1lab-/- larvae15 and placed individually in one well of a 96-well

104 plate (75 μL of E3 media). The plate was positioned in a DanioVision (Noldus) chamber under

105 dark light for a 20-min habituation period before obtaining a 10 min “Baseline Recording”

106 epoch. Fresh drug solutions were prepared on each day of experimentation in 1 mL of E3 media.

107 After baseline measurements, embryo media was carefully removed and 75 μL test drug (at a

108 concentration of 10 or 250 µM) or embryo media (control) were added. The plate was returned

109 to the chamber for another 20-min habituation period before beginning a 10 min “Experimental

110 Recording” epoch. Criteria for a positive hit designation were as follows: (1) a decrease in mean

111 velocity of ≥40%, and (2) a reduction to Stage 0 or Stage I seizure behavior (defined in Baraban

112 et al. 200528) in the locomotion plot for at least 50% of the test fish. Each test compound

113 classified as a “positive hit” in the locomotion assay was assessed for toxicity by direct

114 visualization on a stereomicroscope following a 60 min drug exposure. Acute toxicity (or

115 mortality) was defined as no visible heartbeat or movement in response to external stimulation in bioRxiv preprint doi: https://doi.org/10.1101/815811; this version posted October 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

116 at least 50% of the test fish. Compounds identified as successful in the first locomotion screen

117 were tested on an independent clutch of larvae using the method described above. Compounds

118 that were successful in two independent locomotion assays, and were not acutely toxic, were

119 tested a third time using fresh drug stock sourced from Cayman Chemical.

120

121 Electrophysiology assay

122 Zebrafish larvae were first monitored in the 96-well format locomotion assay described in 2.3.

123 Individual larvae were then briefly exposed to cold and immobilized in 1.2% agarose by an

124 investigator blinded to the locomotion assay parameter. Local field potential recordings (LFP)

125 were obtained from midbrain using a single-electrode recording, as previously described20, 21.

126 LFP recordings, 10 min in duration, were obtained using Axoclamp software (Molecular

127 Devices; Sunnyvale, CA) at an acquisition rate of 1 kHz. Abnormal electrographic seizure-like

128 events were analyzed post hoc as: (i) brief interictal-like events (0.47 ± 0.02 sec duration; n =

129 52) comprised of spike upward or downward membrane deflections greater than 3x baseline

130 noise level or (ii) long duration, large amplitude ictal-like multi or poly-spike events (3.09 ± 1.01

131 sec duration; n = 21) greater than 5x baseline noise level. Noise level was measured as 0.35 ±

132 0.03 mV (n = 18). All scn1lab mutants exhibit some form of spontaneous electrographic seizure

133 activity and no clustering of activity was observed even with prolonged electrophysiology

134 monitoring (see22). Both events were counted using “threshold” and/or “template” detection

135 settings in Clampfit (Molecular Devices; Sunnyvale, CA). All embedded larvae were

136 continuously monitored for blood flow and heart rate using an Axiocam digital camera. All

137 experiments were performed on at least two independent clutches of scn1lab zebrafish larvae (5-

138 6 dpf). bioRxiv preprint doi: https://doi.org/10.1101/815811; this version posted October 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

139

140 Statistics

141 Electrophysiological data was examined by one-way analysis of variance with subsequent

142 Dunnett multiple comparisons tests.

143

144 Results

145

146 Behavioral screening

147 To identify SC compounds that modify convulsive swim behavior, we performed an in vivo

148 primary screen using an automated locomotion tracking protocol12, 13. Mutants were placed

149 individually in a single well of a 96-well plate and a baseline locomotion tracking plot (10 min in

150 duration) was obtained in embryo media. Embryo media was removed and mutants were then

151 exposed to test SC compounds at a single screening concentration of 250 µM (n = 6 fish/drug).

152 All compounds remained coded by compound number provided by Cayman Chemical. Mutant

153 swim activity between two consecutive recording epochs in embryo media was tracked on every

154 plate as an internal control. Mean velocity was calculated for each well and the percent change

155 from baseline for all 370 test compounds is plotted in Figure 1a; drugs that were toxic at 60 min

156 are shown in red. On first pass locomotion screening at 250 µM, 27% of SC compounds were

157 identified as positive hits and 39% of SC compounds were identified as acutely toxic (Figure 1c).

158 To reduce the percent of compounds being identified as toxic and potential false negatives, a

159 second screen was perfomed on all 370 compounds at a concentration of 10 µM (n = 6

160 fish/drug), following the same protocol (Figure 1b). On first pass locomotion screening at 10

161 µM, 25% of SC compounds were identified as acutely toxic and 22% were identified as positive bioRxiv preprint doi: https://doi.org/10.1101/815811; this version posted October 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

162 hits (Figure 1c). There were 20 SC compounds which effectively decreased the high velocity

163 seizure-like swim behavior in both the 10 µM and 250 µM behavior screen (Fig 1d,e).

164

165 Next, all 20 SCs compounds which emerged from the two primary screens were uncoded and

166 sourced as individual drugs from Cayman Chemical for a concentration-response studies using

167 scn1lab mutant larvae. SCs were tested at 1, 10 and 100 µM (n = 6 fish/drug/concentration). A

168 threshold for positive hits was set as a decrease in mean velocity of 40% or more. Additionally, a

169 Stage 0 or I swim behavior and a dose response needed to be observed. Representative

170 locomotion tracking plots are shown in Figure 2b. Five SC compounds were identified as being

171 able to decrease the seizure-like swim behavior observed in the scn1lab mutant larvae in a dose

172 dependent manner (Figure 2). Aggregating data from the first stage of our zebrafish antiseizure

173 drug screening platform, these five SC compounds were classified as positive hits for reducing

174 seizure-like swim behavior and subsequently moved on to the electrophysiology assay for

175 confirmation.

176

177 Electrophysiology screening

178 To evaluate whether selected SCs inhibit seizures, we performed an in vivo secondary screen

179 using electrophysiology techniques12, 13, 23. Mutants were placed in a single well of a 96-well

180 plate and exposed to test SC compounds (#10521, #10690, #14550, #14766, #900799 and

181 #9001523; Table 2) at drug concentrations determined above or DMSO (vehicle). Compound

182 #900799 was selected here as a “control” SC that fell below the positive hit threshold described

183 above for the final locomotion-based assay. After a 30 min exposure, scn1lab mutant larvae were

184 immobilized in agar for LFP recording and post hoc analysis of both interictal- (range: 107-453 bioRxiv preprint doi: https://doi.org/10.1101/815811; this version posted October 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

185 events/10 min) and ictal-like (range: 1-10 events/10 min) activity. Exposure to compounds

186 #10521, #10690, #14500, #14766 and #9001523 significantly reduced the frequency of these

187 spontaneous epileptiform events (F(6,80) = 14.41, ***p < 0.0001 or **p < 0.0003) (Figure 3a).

188 Representative 10 min recording epochs for each compound, and an age-matched scn1lab mutant

189 larvae control are shown in Figure 3B. Note the presence of robust interictal- and ictal-like

190 events in scn1lab controls and following exposure to compound #900799 but the near complete

191 absence of events with exposure to five different SCs.

192

193 Discussion

194 Here we describe the first large-scale phenotype-based screen of a synthetic cannabinoid library

195 using a zebrafish model of Dravet syndrome. Our approach to screening compounds for anti-

196 seizure activity in scn1lab mutant zebrafish builds on earlier model validation12, acute drug

197 exposure protocols17, and a database now exceeding 3200 compounds13, 17, 23, 24. Interest in

198 cannabinoid-based treatment options for DS began with anecdotal reports and ultimately led to

199 FDA approval of cannabidiol (Epidiolex®) for the treatment of seizures associated with this

200 disorder. Using a stringent two-stage screening strategy, we identified five SCs that exert

201 significant anti-seizure activity in scn1lab mutant zebrafish. SCs add to a growing list of DS

202 relevant drugs successfully identified in scn1 zebrafish that include “standard-of-care” AEDs

203 (e.g., valproate, stiripentol, benzodiazepines, bromides) and experimental drugs (e.g.,

204 fenfluramine, lorcaserin, trazodone, clemizole).

205

206 Synthetic cannabinoids (SCs), first developed in the 1940s, represent a variety of compounds

207 engineered to bind cannabinoid receptors. SCs bind G-protein coupled CB1 receptors located at bioRxiv preprint doi: https://doi.org/10.1101/815811; this version posted October 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

208 the presynaptic terminal and are also thought to interact with voltage-gated potassium channels,

209 voltage-gate sodium channels, N-and P/Q-type-calcium channels, and an orphan G-protein

210 coupled receptor (GPR55). Whether one, or several, of these potential sites of action are

211 responsible for the anti-seizure effects noted with SCs (or CBD) remains a controversial area of

212 investigation. In vivo studies using intracerebroventricular administration of a CB1-receptor

213 agonist, arachidonyl-2-chloroethylamide (ACEA), significantly decreased the frequency of

214 penicillin-induced epileptiform activity in rats25. CMYL-4CN-BINACA, another CB1 receptor

215 agonist, elicited pro-convulsant effects in mice26 and a synthetic cannabinoid (AM2201) induced

216 epileptiform activity and convulsive behaviors in mice that could be blocked by the selective

217 CB1 receptor antagonist AM25127. Investigating alternatives to a CB1/2 receptor mechanism,

218 Kaplan et al.7 showed that antagonism of GPR55 receptors mimic the effects of CBD in vitro and

219 could explain anti-seizure effects of high concentrations of CBD in Scn1+/- mice. However,

220 Huizenga et al.6 reported that nonselective CB1/2 and selective CB1 agonists suppress activity in

221 neonatal seizure models (though these findings were not replicated with GPR55 agonists). In

222 patch-clamp recordings from isolated mouse cortical neurons, CBD decreased neuronal firing

223 activity and significantly reduced peak whole-cell voltage-activated sodium currents28. In

224 contrast, in a recent study using rodent and human induced pluripotent stem cell (iPSC)-derived

225 excitatory or inhibitory neurons, CBD reduced neuronal firing activity but did not alter voltage-

226 gated sodium channel conductance29.

227

228 Zebrafish possess orthologues for 82% of human disease-associated genes30. Orthologous

229 zebrafish proteins are often similar to human within their functional domains. For example,

230 between 85% and 95% of the amino acids in a ligand-binding domain of the zebrafish bioRxiv preprint doi: https://doi.org/10.1101/815811; this version posted October 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

231 glucocorticoid receptor are similar to those in human. These properties combined with the ease

232 of large-scale pharmacological screening make larval zebrafish a good model system for

233 investigating drug targets. A recent study31, limited only to locomotion assay read-outs in larval

234 zebrafish, reported a synergistic effect of Δ-9-tetrahydrocannabinol (THC) and cannabidiol

235 (CBD) on hyperactive behaviors. Unfortunately, these studies failed to include

236 electrophysiology measures of efficacy and were limited to only these two compounds. Here we

237 screened 370 different synthetic cannabinoids in a locomotion assay at two different drug

238 concentrations, initially identifying 20 compounds as exerting potential therapeutic benefit.

239 With repeated biological replicates, newly sourced compound re-tests and sensitive secondary

240 electrophysiologay assays, we narrowed this initial list to five SCs that were classified as

241 effective in suppressing spontaneous seizures (behavioral and electrographic) in scn1lab

242 mutants. This scientifically stringent two-stage approach further validates the selectivity of our

243 screening strategy as only 1.3% of all SCs tested were identified as positive hits. A caveat of this

244 strategy is that pharmacokinetic data for how these SCs are absorbed, distributed or metabolized

245 in larval zebrafish is not available which would suggest an error in false negative designations.

246 Interestingly, as several of these SCs are classified as indole-derivatives these data also suggest a

247 potential target not recognized in previous preclinical studies. Indole-derived cannabinoids have

32 248 strong binding affinity for the 5HT2B receptor . This serotonin receptor subtype was recently

249 identified by our group as potentially mediating the anti-seizure effects of clemizole33. Taken

250 together, a convergence of evidence now exists to suggest that activation of a serotonin 2B

251 receptor is a potential target for DS therapy.

252 bioRxiv preprint doi: https://doi.org/10.1101/815811; this version posted October 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

253 Although further preclinical studies investigating SCs identified here are warranted, a note of

254 caution needs to be extended to clinical translation of these data. Substantial evidence of serious

255 adverse effects accompany these compounds. These include, and may not be limited to, panic

256 attacks, memory distortions, paranoia, psychotic reactions, disorganized behavior, and suicidal

257 thoughts34, 35. Reports of acute ischemic stroke36, 37, seizures38, 39, and sudden death40-42 with SCs

258 are not uncommon. In particular, indole- and indazole-based SCs are associated with clinical

259 signs of reduced consciousness, paranoia and seizures in humans43. Acknowledging these

260 limitations, the current studies provide tools for further preclinical research investigating the

261 anti-seizure potential of the endocannabinoid signaling system. This information is valuable for

262 investigators and clinician-scientists interested in the potential benefits of cannabinoid receptor

263 agonists for intractable seizure disorders.

264

265 Acknowledgements

266 This work was supported from NINDS R01 grants no. NS079214 and NS103139 (S.C.B).

267 We would like to thank members of the Baraban laboratory for their useful discussions during

268 the course of these studies; Matthew Dinday, Chinwendu Ononuju and Harrison Ramsay in

269 particular for their support in zebrafish facility maintenance.

270

271 Author contributions

272 Locomotion-based screening assays were performed by M.A., K.H. and A.G. Electrophysiology

273 experiments were performed by S.C.B. The design, conceptualization, interpretation of data,

274 data analysis and writing of the manuscript was done by A.G. and S.C.B. All authors

275 contributed to the editing process. bioRxiv preprint doi: https://doi.org/10.1101/815811; this version posted October 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

276

277 Conflict of Interest

278 S.C.B. is a co-Founder and Scientific Advisor for EpyGenix Therapeutics. S.C.B is on the

279 Scientific Advisory Board of ZeClinics.

280 Data availability

281 The datasets generated and analyzed in the current study are available from the corresponding

282 author on reasonable request.

283

284

285

286 bioRxiv preprint doi: https://doi.org/10.1101/815811; this version posted October 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

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374 35. Tait, R.J., Caldicott, D., Mountain, D., Hill, S.L. & Lenton, S. A systematic review of 375 adverse events arising from the use of synthetic cannabinoids and their associated 376 treatment. Clin Toxicol 54, 1-13 (2016). 377 36. Takematsu, M. et al. A case of acute cerebral ischemia following inhalation of a 378 synthetic cannabinoid. Clin Toxicol 52, 973-975 (2014). 379 37. Freeman, M.J. et al. Ischemic stroke after use of the synthetic marijuana “spice”. 380 Neurology 81, 2090 (2013). 381 38. Schwartz, M.D. et al. A Common Source Outbreak of Severe Delirium Associated with 382 Exposure to the Novel Synthetic Cannabinoid ADB-PINACA. J Emerg Med 48, 573-580 383 (2015). 384 39. Schneir, A.B. & Baumbacher, T. Convulsions Associated with the Use of a Synthetic 385 Cannabinoid Product. J Med Toxicol 8, 62-64 (2012). 386 40. Shanks, K.G., Clark, W. & Behonick, G. Death Associated With the Use of the Synthetic 387 Cannabinoid ADB-FUBINACA. J Anal Toxicol 40, 236-239 (2016). 388 41. Shanks, K.G. & Behonick, G.S. Death after use of the synthetic cannabinoid 5F-AMB. 389 Forensic Sci Int 262, e21-e24 (2016). 390 42. Behonick, G. et al. Four Postmortem Case Reports with Quantitative Detection of the 391 Synthetic Cannabinoid, 5F-PB-22. J Anal Toxicol 38, 559-562 (2014). 392 43. Hill, S.L. et al. Human Toxicity Caused by Indole and Indazole Carboxylate Synthetic 393 Cannabinoid Receptor Agonists: From Horizon Scanning to Notification. Clin Chem 64, 394 346 (2018). 395 bioRxiv preprint doi: https://doi.org/10.1101/815811; this version posted October 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

396 FIGURE LEGANDS AND TABLES

397

398 Figure 1. A library of synthetic cannabinoids was screened for their ability to reduce the high

399 velocity seizure-like swim behavior of 5 day old zebrafish larvae. Compounds were screened at

400 (a) 250 µM, and (b) 10 µM. Each data point represents the mean velocity change in swim

401 behavior of six fish treated with an individual compound. The red data points represent

402 compounds that failed to go into solution or identified as toxic after 90-min exposure. (c)

403 Summary of compound effects after screening 370 synthetic cannabinoids at 250 µM and 10

404 µM. The threshold for inhibition of seizure activity was determined as a reduction in mean swim

405 velocity of ≥40%. (d) The total number of compounds identitied as positive from the 250 µM

406 and 10 µM library screen. (e) Heat map representing the 20 compounds which successfully

407 reduced the mean swim velocity by >40% in the 250 µM and 10 µM screening.

408

409 Figure 2. Behavioral screening of 20 compounds which were identified as positive from the

410 library screens. (a) Heat map representing the change of mean swim velocity of the 20 hit

411 compounds. The 20 synthetic cannabinoids which were identified from the blind screens were

412 retested at 1µM, 10µM and 100µM to confirm a dose response effect. Compounds marked with a

413 cross were identified as toxic. (b) Representative swim behavior traces obtained during a 10 min

414 recording epoch for the top two compounds, #9001523 and #147666.

415

416 Figure 3. Electrophysiological assay for compounds identified in the locomotion-based

417 screening assay. (a) Local field potential (LFP) recordings were obtained with an glass micro-

418 electrode placed under visual guidance in the midbrain of agar-immobilized scn1lab larvae that bioRxiv preprint doi: https://doi.org/10.1101/815811; this version posted October 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

419 had previously showed reduced seizure-like behavior in the locomotion assay. Representative

420 examples of events classified as interictal- or ictal-like are shown. Scale bars: 1 mV, 0.5 sec. (b)

421 Bar graphs show the frequency of epileptiform events in a 10 min recording epoch for scn1lab

422 larvae exposed to DMSO vehicle (scn1lab mutants; n = 17), JWH 018 N-(5-chloropentyl) analog

423 (compound #10521; n = 10), JWH 018 N-(2-methylbutyl) isomer (compound #10690; n = 12), 5-

424 fluoro PB-22 5-hydroxyisoquinoline isomer (compound #14550; n = 11), 5-fluoro ADBICA

425 (compound #14766; n = 13), JWH 018 adamantyl analog (compound #9000799; n = 13), and

426 AB-FUBINACA 3-fluorobenzyl isomer (compound #90001523; n = 11). Mean ± SEM and

427 individual data points are shown. One-way analysis of variance with Dunnet’s multiple

428 comparisons was used to test for significance. **p < 0.001; *** p < 0.0001. (c) Representative

429 electrophysiology traces (10 min) are shown for SC compouns 10521, 10690, 14550, 14766,

430 9000799 and 900015323 compared to an scn1lab mutant zebrafish (red). Scale bars: 1 mV, 10

431 sec.

432

433 Figure 4. Structural comparison of SC identified to reduce spontaneous seizures in the scn1lab

434 Dravet syndrome zebrafish mutant. (a) 14550; 5-fluoro PB-22 5-hydroxyisoquinoline isomer, (b)

435 10521; JWH 018 N-(5-chloropentyl) analog, (c) 14766; 5-fluoro ADBICA, (d) 10690; JWH 018 N-(2-

436 methylbutyl) isomer, and (e) 9001523; AB-FUBINACA 3-fluorobenzyl isomer. (f) These indol derived

437 cannabinoids share structural similarity with serotonin and clemizole (currently in clinical trial for Dravet

438 syndrome). They are structurally unrelated to cannabidiol. bioRxiv preprint doi: https://doi.org/10.1101/815811; this version posted October 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

439 Table 1

Compound No. Compound Name Description 10521 JWH 018 N-(5-chloropentyl) analog JWH 018 is a synthetic cannabinoid that potently activates the central cannabinoid (CB1) and peripheral cannabinoid (CB2) receptors (Ki = 9.0 and 2.94 nM, respectively). JWH 018 N-(5-chloropentyl) analog differs structurally from JWH 018 by having chlorine added to the 5 position of the pentyl chain. 10690 JWH 018 N-(2-methylbutyl) isomer JWH 018 2-methylbutyl homolog is an analog of JWH 018, a mildly selective agonist of the central cannabinoid receptor (Ki = 8.9 nM) derived from the aminoalkylindole WIN 55,212-2. 14550 5-fluoro PB-22 5-hydroxyisoquinoline 5-fluoro PB-22 is an analog of 5-fluoropentyl JWH- isomer type cannabimimetics. 5-fluoro PB-22 5- hydroxyisoquinoline isomer differs from 5-fluoro PB- 22 by having the quinoline group replaced with an isoquinoline group attached at its 5 position. 14766 5-fluoro ADBICA This compound is a derivative of ADBICA featuring a fluorine atom added to the terminal carbon of the pentyl group. 9000799 JWH 018 adamantyl analog JWH 018 adamantyl analog is a mildly selective agonist of the peripheral cannabinoid receptor, where the naphthalene ring is substituted with an adamantyl group. 9001523 AB-FUBINACA 3-fluorobenzyl isomer AB-FUBINACA is an indazole-based synthetic cannabinoid that potently binds the central CB1 receptor (Ki = 0.9 nM). 440 Table 1: Synthetic cannabinoid compounds identified in a two-stage screening process bioRxiv preprint doi: https://doi.org/10.1101/815811; this version posted October 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/815811; this version posted October 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/815811; this version posted October 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/815811; this version posted October 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.