Rapid antiepileptic activity identification of isopimpinellin using a multi-model of epilepsy based on behavior-biomarker-BBB screening pipeline.

Abstract

BACKGROUND: Epilepsy is a common neurological disorder that manifests as sudden abnormal electrical discharges and convulsions, affecting more than 70 million people worldwide. Current antiepileptic drugs (AEDs) can inhibit seizures, while long-term use has side effects. In addition, one-third of patients do not respond to the available AEDs. In recent years, traditional Chinese medicines (TCMs), which have multi-target and multi-pathway actions with low side effects, have offered a promising alternative for treating epilepsy. PURPOSE: This study aims to develop a multi-tiered screening strategy combining behavioral, biomarker, blood-brain barrier (BBB) permeability, and computational approaches to identify optimal antiepileptic compounds from TCMs. METHODS: A TCMs library was initially screened based on pentylenetetrazol (PTZ)-induced seizures and biomarkers (c-fos and c-jun), which was followed by further screening using parallel artificial membrane permeation assay (PAMPA). Then, a multi-indicator in silico approach, including cluster analysis, Lipinski's Rule of 5, and clinical drug similarity assessment, was employed to discover hit compounds. Mouse epilepsy models confirmed the antiepileptic candidates. Finally, the underlying mechanism was investigated through proteomics and further verified by analyzing the key targets CSF1/CSF1R, microglial activation, and target intervention studies. RESULTS: Overall, 11 antiepileptic candidates were identified from a total of 730 TCMs, yielding a hit rate of 1.5%. ISOP exhibited the desirable BBB permeability and the optimal antiepileptic activity via multi-indicator in silico analysis. Accordingly, ISOP mitigated seizures in zebrafish and mice, including delayed seizures by 2-3 times, inhibited neuronal discharges by 60-70%, and reduced Mouse Racine's score by 3 points. A total of 27 differentially expressed proteins (DEPs) were revealed, which were enriched in the inflammatory response, among other functions. By suppressing CSF1, ISOP led to the downregulation of CSF1R, thus inhibiting microglial activation, ultimately exerting antiepileptic effects. CONCLUSION: A rapid antiepileptic drug screening strategy was generated for the first time, integrating BBB permeability early in the screening process. By using this screening pipeline, ISOP was identified, which represents a novel CSF1R-targeting candidate for the treatment of epilepsy.