Epilepsy is one of chronic brain dysfunctions characterized by recurrent epileptic seizures (Calabrese V et al., 2010; Devinsky et al., 2018; Sarbassov et al., 2004). Epilepsy affects 10% of the global population and directly contributes to epilepsy in 1%–2% of the world's population (Falco et al., 2020; Wullschleger et al., 2006; Peterson et al., 2009; Lodi R et al., 2006). Numerous studies found mTOR played an essential role in seizures and treatment of epilepsy. mTOR proteins are evolutionarily conserved serine/threonine proteins in PI3K-related protein family, mTORC1 and mTORC2 are involved in the composition mTOR (Sancak et al., 2007; Calabrese V et al., 2005; Hara et al., 2002; Peterson et al., 2009). mTORC1 is composed of mTOR, AKT1 Substrate 1 (AKT1S1), LST8 Homolog (MLST8), mTOR Associated Protein and regulatory associated protein of rapamycin (Kim et al. 2002, 2003; Pearce et al., 2007; Frias et al., 2006). mTORC1 is a sensitive target protein for rapamycin and activated mTORC1 can inhibit lysosomal function by suppressing TFEB activity. In the presence of nutrient starvation or mTORC1 inhibition, TFEB is activated thereby promoting cellular autophagy (Zhou et al., 2013; Kim et al., 2002, 2011; Fang et al., 2001). In additi on mTORC1 promotes glucose uptake and glycolysisre to involve in effects on cell growth through regulation of HIF1 (Semenza et al., 2010; Hay et al., 2004; Düvel et al., 2010, Wang et al., 2011; Jacinto et al., 2004; Wang et al., 2017).

Rapamycin was originally thought to be an antifungal agent, but it was subsequently found to have immunosuppressive and modulatory effects on cellular metabolism, and rapamycin was potent mTORC1 inhibitor (Dumont et al., 1990; Singh et al., 1979; Tewari et al., 2021). Numerous studies found FKBP12 binding to rapamycin can participate in the regulation of mTORC1 (Benjamin et al., 2011; Chung et al., 1992). FKBP12 binds directly to mTOR at FRB structural domain, and the binding domain is close to the catalytic domain of mTOR (Yang et al., 2013). Studies suggested that rapamycin may inhibit mTOR activity by directly blocking substrate recruitment and further blocking exposure of protein active site. Rapamycin binding to FRB may significantly reduce the likelihood of substrates reaching the catalytic protein site. This is because the extent of mTORC1 inhibition depends on the relationship between substrate and phosphorylation site (Choo et al., 2009).Because current studies suggest that the mTORC1 pathway is also closely linked to many neurological disorders, rapamycin pharmacological inhibition of mTORC1 can provide neuroprotection in many neurodegenerative diseases (Alzheimer's disease and type 3 spinal cerebellar ataxia) (Bové et al., 2011). Muncy found that rapamycin reduced frequency of seizures in tuberous sclerosis (Muncy et al., 2009). However, the mechanism of rapamycin treating epilepsy is still unclear.

This study combined bioinformatics analysis, supercomputer simulations and cellular experiments to investigate the mechanism of rapamycin treating epilepsy through blocking phosphorylation of mTOR. Bioinformatics was used to screen and analyze targets for rapamycin and epilepsy, and epilepsy cell models were constructed to observe the effect of rapamycin on mTOR by RT-qPCR, WB and IF. Supercomputer simulations were used to calculate the binding stability of rapamycin and protein.