Fuyun Tian ^* Hongyuan Zuo Zhidan Wu Shuoshuo Zheng Meixuan Ping Xinni Bin Zhaobing Gao ^*

• Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica (CAS), Shanghai, China

Anti-seizure medications (ASMs) are the mainstay in epilepsy management, with dozens of ASMs approved and available for clinical use. However, more than 30% of epileptic patients on ASMs do not experience satisfactory improvement due to drug resistance or various adverse effects. Therefore, it is crucial to explore safe and effective new ASMs, and phenotypic-based drug discovery (PDD) may be an effective strategy to discover ASMs targeting novel and/or multiple targets. In this study, we conducted high-throughput screening (HTS) assays of a natural products library using a pentylenetetrazole (PTZ)-induced seizure zebrafish (Danio rerio) model. The zebrafish-based screening consisted of three evaluation rounds: single-concentration screening (100 μM or the maximum tolerated concentration), rescreening with an increased sample number, and multi-concentration validation. Further validation involves assessing the inhibition of the hits on the spontaneous action potential firing frequency in primary neurons and confirming their anti-seizure activity in mouse models of epilepsy. A library of 891 compounds was screened using the PTZ-induced seizure zebrafish model. From the first-round screening, 65 compounds showed anti-seizure activity and were selected for the second and third rounds of screenings. Sixteen compounds were identified as hits, showing concentration-dependent anti-seizure effects in zebrafish, including four compounds that have been previously reported as anti-seizure molecules. In the verification experiment using cultured primary neurons, eight compounds effectively inhibited the action potential firing frequency at a concentration of 100 μM and were subsequently confirmed in mouse models of epilepsy. Two compounds, Norspermine and Phloretin, exhibited anti-seizure effects in the PTZ-induced seizure model. Norspermine extended the latencies to partial and generalized clonus at tolerable doses, while Phloretin prolonged the latencies to partial and generalized clonus, generalized tonic-clonic seizure, and death at tolerable doses. In conclusion, we reported a modified zebrafish-based screening workflow that is valuable for discovering ASMs. Additionally, we highlight the anti-seizure properties of Norspermine and Phloretin, providing insights for the development of novel therapies to treat epilepsy.