In-vitro and In-vivo Exploration of Physostigmine Analogues to Understand the Mechanistic Crosstalk Between Klotho and Targets for Epilepsy

Mansi Dahalia^1,2Haya Majid³Mohd Junaid Khan⁴Akshat Rathi⁵Mohd Ashif Khan³Imran Ahmd Khan⁴Mohammed Samim⁴Sayeed ur Rehman⁵MD SALIK NOORANI⁶Divya Vohora²Nidhi ..^3*

• ¹Jamia Hamdard University, New Delhi, India
• ²School of Pharmaceutical Education and Research, Jamia Hamdard University, New Delhi, India
• ³Department of Translational and Clinical Research, School of Chemical and Life Sciences, Jamia Hamdard University, New Delhi, India
• ⁴Department of Chemistry, School of Chemical and Life Sciences, Jamia Hamdard University, New Delhi, India
• ⁵Department of Biochemistry, School of Chemical and Life Sciences, Jamia Hamdard University, New Delhi, India
• ⁶Department of Botany, School of Chemical and Life Sciences, Jamia Hamdard University, New Delhi, India

Background: Epilepsy and seizures are characterized by neuronal hyperexcitability and damage, influenced by metabolic dysregulation, neuroinflammation, and oxidative stress. Despite available treatments, many patients remain resistant to therapy, necessitating novel therapeutic strategies. Klotho, a neuroprotective, anti-inflammatory, and antioxidative protein has emerged as a potential modulator of epilepsy-related pathways.Objective: This study investigates the therapeutic potential of novel physostigmine analogues in regulating Klotho expression and its downstream targets in epilepsy.: An integrative in vitro and in vivo approach was employed in PTZ-induced kindled mice. Behavioral assessments, including the Morris Water Maze (MWM), Rota Rod, Black and White Box, and Tail Suspension tests were conducted. Biochemical analyses quantified serum glucose, lipid profiles, pro-inflammatory cytokines (TNF-α, FOXO1), and apoptotic proteins (caspase-3). Quantitative real-time PCR (qRT-PCR) was performed to assess Klotho and epilepsyassociated gene expression (STAT3, Bax, Bcl2). Results: The synthesized physostigmine analogues exhibited varying inhibitory effects on Klotho transcriptional activators, with Compound C (1,8-bis(phenylsulfonyl)-1,8-dihydropyrrolo[2,3-b] indole) showing the weakest inhibition (IC50 = 1.31 µM). In vivo, Compound C demonstrated anticonvulsant (p < 0.05), neuroprotective (5 mg/kg, p < 0.05, 10 mg/kg, p < 0.01, 20 mg/kg p < 0.0001), antidepressant (p < 0.05), and anti-inflammatory (p < 0.05) effects in PTZ-induced seizure models, improving motor function (p < 0.001), cognitive performance (p < 0.01), and reducing neuroinflammatory/metabolic markers (p < 0.05), while modulating STAT3 (p < 0.001), BAX (p < 0.001), Bcl2 (p < 0.05), and Klotho (p < 0.05) gene expression.The therapeutic potential of 1,8-bis(phenylsulfonyl)-1,8-dihydropyrrolo [2,3-b] indole in epilepsy via Klotho modulation was observed. Targeting metabolic, inflammatory, and apoptotic pathways presents a promising strategy for epilepsy management. Further research is required to optimize clinical translation and ensure long-term efficacy and safety.