The Role Of Caspase-1 In FS Generation And A Novel Inhibitor Targets Caspase-1 For Treatment Of Experimental Febrile Seizures

Febrile seizures (FS), the most common seizures in childhood, are induced by fever, occurring in 3 to 14 percent of infants and children six months to five years of age. FS is badly controlled and often accompanied by later complications. It is reported that about 30% of febrile seizure patients had recurrent or prolonged febrile seizures, resulting in high risks to temporal lobe epilepsy (TLE), hippocampal sclerosis or the impairment of memory. At present, the clinic treatments of FS are intermittent therapy with diazepam or continuous anticonvulsant therapies with phenobarbital and valproic acid. Although they are effective in ceasing seizures, the associated potential toxicities outweigh their therapeutic effects. In addition, they are ineffective in reducing FS recurrence and fails in prevention. And conventional antipyretics were effective in alleviating fever but not in blocking seizures. Thus, one of the main challenges is to develop alternative therapeutic approaches to prevent its occurrence.It is reported that Periodic Fever Syndrome (PFS) patients have high active Caspase-1 expression. Caspase-1 is synthesized as an inactive precursor form (pro-Caspase-1). Upon appropriate stimuli, such as fever, extracellular ATP accumulation, and potassium efflux, this inactive form is recruited by PRRs (Pattern Recognition Receptors) and ASC (Associated Speck-like Protein Containing CARD) and assembles into inflammasomes. Then pro-Caspase-1 rapidly undergoes autocatalytic processing to generate an active enzyme and mediates Caspase-1-dependent processing of cytokines such as IL-1β and IL-18. Recent data indicated that upregulated activated Caspase-1 and its containing inflammasome are observed in both epilepsy patients and animal models of TLE. Moreover, Caspase-1 can be detected in pyramidal neurons and interneurons in hippocampus and can be activated in response to AMPA receptor. Therefore, we hypothesized that Caspase-1 may participate in FS generation and be a potential therapeutic target.Using a well-established experimental FS model in mice, we sought to investigate the role of Caspase-1 in FS generation, and designed a novel inhibitor targeting Caspase-1 for treatment of FS.Pups on postnatal day 8-10 (P8-10) were used to develop FS. Mice pups were posted to different hyperthermia conditions (38℃,41℃ or 44℃). Pups displayed typical behavioral seizure with ictal activity at 41℃ and 44℃, while pups displayed no behavioral seizure or ictal activity at 38℃. The expression of cleaved Caspase-1 and its enzyme activity increased up to a significantly higher level just before seizure onset at 41℃ and 44℃ conditions, while there was no difference at 38℃ condition. Furthermore, Caspl-/- mice showed decreased neuronal excitability and displayed significantly longer latency, higher threshold and reduced incidence of FS generation when compared with littermate WT mice at 41℃ and 44℃. And intra-hippocampally injected exogenous recombinant Caspase-1 significantly shortened the latency and decreased the threshold to FS generation in WT and Caspl-/- mice. Moreover, exogenous Caspase-1 reversed the latency and threshold of Casp1-/- mice to the level of WT mice. To specifically increase or rescue the expression of Caspase-1, we in utero electroporated pCIG2-caspl constructs to the hippocampus of WT and Caspl-/- mice. Behavioral results showed that electroporation of pCIG2-caspl constructs reduced the latency and decreased the threshold to FS generation in WT mice, and reversed the latency and threshold of Caspl-/- mice to the level of WT mice. And electroporation of pCIG2-caspl constructs increased neuronal excitability.Here we aimed to develop new Caspase-1 inhibitors according to its crystal structure. Caspase-1-Cys285 is defined as the catalytic site, and Argl79, Arg341, Arg383, Gln283 and Gly238 are defined as selective aspartic acid recognition sites, which are positively charged catalytic residues. According to these sites, we designed a series of compounds by molecular docking of the Glide module in Schrodinger. Virtual screening of a large database yielded 144 compounds and were clustered for analysis. The top 50 compounds were ranked based on their docking scores, and were acquired for experimental testing. We further assessed the effects of these compounds in vitro and on LPS-pretreated PBMC (Peripheral Blood Mononuclear Cells), and four compounds (encoded as 6626080,6810755,9198978 and 7135306) demonstrated high inhibitory activities. Among the four compounds, injection (i.c.v.) of 6626080 and 9198978 significantly prolonged latency and increased the threshold to FS generation in WT mice. We further analyzed structure-activity relationship of the compounds, and examined their H-bond occupancy and binding free energy. We screened a small-molecule, brain-penetrable inhibitor 6626080, which reduced neuronal excitability and reduced the incidence of FS generation without significant side effects. Importantly, loading the small-molecule to thermo-sensitive micelles allowed the drug release after hyperthermia stimulus (39℃) and extended the effective time.Therefore, our study identified a pro-convulsant role of Caspase-1 in FS generation and further found that caspase-1 could regulate neuronal excitability. These findings indicate that Caspase-1 contributes to FS generation, and the new developed small-molecule inhibitor 6626080 may serve as a new promising treatment for FS.