Zebrafish-based identification of neuroactive and antiseizure marine natural product (MNP) extracts. MNP extracts (crude extracts and fractions of pre-fractionated extracts) underwent a primary screen for neuroactivity at 100 µg/mL (2-h incubation time, n = 1), using the embryonic zebrafish photomotor response (PMR) assay, which was followed immediately after by a toxicity evaluation. Neuroactive hits (non-toxic) underwent a secondary screen for antiseizure activity at 100 µg/mL (2-h incubation time, n = 3), using the larval zebrafish pentylenetetrazole (PTZ) seizure model, which was followed immediately after by a toxicity evaluation. Antiseizure hits (non-toxic) underwent a second round of antiseizure analysis at 100, 33, and 11 µg/mL (2-h incubation time, n = 6) using the larval zebrafish PTZ seizure model, which was followed immediately after by a toxicity evaluation.

Antiseizure hit SK0107. (A) Aspergillus insuetus IBT 28443 cultivated on czapek yeast extract agar (CYA) and yeast extract sucrose agar (YES) media for 9 days at 25 °C in the dark. Base peak chromatograms of the crude extract and bioactive fraction SK0107 in positive electrospray ionization mode. (B,C) Antiseizure activity of SK0107 in the zebrafish pentylenetetrazole (PTZ) seizure model after 2 h of incubation. PTZ-induced seizure-like behavior is expressed as mean actinteg units per 5 min (±SEM) during the 30-min recording period (B) and over consecutive time intervals (C). Means are pooled from three independent experiments with each 12 replicate wells per condition. Statistical analysis: (B) one-way ANOVA with Dunnett’s multiple comparison test, (C) two-way ANOVA with Bonferroni posttests (GraphPad Prism 5, San Diego, CA, USA). Significance levels: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. Abbreviation: vehicle, VHC.

Bioactivity-guided identification of the active compounds of antiseizure hit SK0107. (A) Aspergillus insuetus IBT 28443 cultivated on czapek yeast extract agar (CYA) media for 9 days in the dark at 25 °C. Base peak chromatogram (BPC) of the most bioactive fraction (SK1312) from first reversed-phase fractionation in positive electrospray ionization mode (ESI⁺). ESI⁺ BPC chromatograms of the two most bioactive fractions (SK1414 and SK1415) from the second reversed-phase fractionation. UV/Vis and HRMS spectra for TMC-120A (II) and TMC-120B (III). (B–D) Antiseizure activity of SK1312 (n = 23–24 replicate wells per condition) (B), SK1414 (n = 10–11 replicate wells per condition) (C), and SK1415 (n = 22 replicate wells per condition) (D) in the zebrafish pentylenetetrazole (PTZ) seizure model after 2 h of incubation at their maximum tolerated concentration (MTC), MTC/2, and MTC/4. PTZ- induced seizure-like behavior is expressed as mean actinteg units per 5 min (±SEM) during the 30-min recording period. (B,D) Data are pooled from two independent experiments with each 11–12 replicate wells per condition. (C) Data are from a single experiment with 10–11 replicate wells per condition. (B–D) Statistical analysis: one-way ANOVA with Dunnett’s multiple comparison test for comparison of sample + PTZ groups with vehicle (VHC) + PTZ control group, Kruskal–Wallis test with Dunn’s multiple comparison test (data did not pass the Shapiro–Wilk normality test) for comparison of sample + VHC groups with VHC + VHC control group (GraphPad Prism 5, San Diego, CA, USA). Significance levels: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Chemical structures of the isoquinoline alkaloids TMC-120A and TMC-120B and structural analogues TMC-120C, penicisochroman G, and ustusorane B.

Aspergillus insuetus IBT 28485 cultivated on czapek yeast extract agar (CYA) media for 9 days in the dark at 25 °C. Base peak chromatogram of the crude extract with marked peaks for TMC-120A and TMC-120B, TMC-120C, penicisochroman G, and ustusorane B in positive electrospray ionization mode.

Behavioral antiseizure analysis of TMC-120A, TMC-120B, and structural analogues in the zebrafish PTZ seizure model. Antiseizure activity of TMC-120A (A,B), TMC-120B (C,D), and their structural analogues (TMC-120C, penicisochroman G, and ustusorane B) (E,F) in the zebrafish pentylenetetrazole (PTZ) seizure model after 2 h of incubation, respectively. PTZ-induced seizure-like behavior is expressed as mean actinteg units per 5 min (±SEM) during the 30-min recording period (A,C,E) and over consecutive time intervals (B,D,F). (A–D) Means are pooled from three independent experiments with each 10–12 replicate wells per vehicle (VHC) + PTZ and compound + PTZ condition, and 6–12 replicate wells per VHC + VHC and compound + VHC condition. (E–F) Data are pooled from two single experiments. Number of replicate wells per condition: 21–22 replicate wells for VHC + PTZ and VHC + VHC conditions, and 8–11 replicate wells for compound + PTZ conditions. Statistical analysis: (A,C,E) one-way ANOVA with Dunnett’s multiple comparison test, (B,D,F) two-way ANOVA with Bonferroni posttests (GraphPad Prism 5, San Diego, CA, USA). Significance levels: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Electrophysiological antiseizure analysis of TMC-120A and TMC-120B in the zebrafish PTZ seizure model. Noninvasive local field potential recordings from the optic tectum of larvae pre-exposed to vehicle (VHC) and pentylenetetrazole (PTZ), VHC only, compound and PTZ, or compound and VHC. Larvae were incubated with 20 µg/mL TMC-120A or TMC-120B for 2 h, conform with the maximum tolerated concentrations and incubation time used in the behavioral assay. Larvae are considered to possess epileptiform brain activity when three or more epileptiform events occurred during a 10-min recording (A). Epileptiform discharges are quantified by the number (mean ±SD) (B) and cumulative duration (mean ± SD) (C) of events per 10-min recording. Number of replicate wells per condition: 19 larvae were used for VHC + PTZ controls, 16 larvae were used for VHC + VHC controls, 13–14 larvae were used for compound + PTZ conditions, and 12 larvae were used for compound + VHC conditions. Statistical analysis: (A) Fisher’s exact test with Bonferroni posttest, (B,C) Kruskal–Wallis test with Dunn’s multiple comparison test (data did not pass the Shapiro–Wilk normality test) (GraphPad Prism 5, San Diego, CA, USA). Significance levels: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Representative local field potential recordings. 10-min noninvasive local field potential recordings from the optic tectum of larvae pre-exposed to vehicle (VHC) and pentylenetetrazole (PTZ), VHC only, compound and PTZ, or compound and VHC. Larvae were incubated with 20 µg/mL TMC-120A or TMC-120B for 2 h, conform with the maximum tolerated concentrations and incubation time used in the behavioral assay.

Antiseizure activity analysis of TMC-120A and TMC-120B in the mouse 6-Hz (44 mA) psychomotor seizure model. Drug-resistant psychomotor seizures were induced by electrical stimulation through the cornea, 30 min after i.p. injection of vehicle (VHC, n = 20), positive control valproate (n = 12), TMC-120A (n = 4–8) or TMC-120B (n = 5–6). Mean seizure durations (±SD) are depicted. Statistical analysis: one-way ANOVA with Dunnett’s multiple comparison test (GraphPad Prism 5, San Diego, CA, USA). Significance levels: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.