Aminoacyl tRNA synthetases (ARSs) link specific amino acids with their cognate transfer RNAs in a critical early step of protein translation. Mutations in ARSs have emerged as a cause of recessive, often complex neurological disease traits. Here we report an allelic series consisting of seven novel and two previously reported biallelic variants in valyl-tRNA synthetase (VARS) in ten patients with a developmental encephalopathy with microcephaly, often associated with early-onset epilepsy. In silico, in vitro, and yeast complementation assays demonstrate that the underlying pathomechanism of these mutations is most likely a loss of protein function. Zebrafish modeling accurately recapitulated some of the key neurological disease traits. These results provide both genetic and biological insights into neurodevelopmental disease and pave the way for further in-depth research on ARS related recessive disorders and precision therapies.

There is a high need for the development of new and improved antiseizure drugs (ASDs) to treat epilepsy. Despite the potential of marine natural products (MNPs), the EU marine biodiscovery consortium PharmaSea has made the only effort to date to perform ASD discovery based on large-scale screening of MNPs. To this end, the embryonic zebrafish photomotor response assay and the larval zebrafish pentylenetetrazole (PTZ) model were used to screen MNP extracts for neuroactivity and antiseizure activity, respectively. Here we report the identification of the two known isoquinoline alkaloids TMC-120A and TMC-120B as novel antiseizure compounds, which were isolated by bioactivity-guided purification from the marine-derived fungus Aspergillus insuetus. TMC-120A and TMC-120B were observed to significantly lower PTZ-induced seizures and epileptiform brain activity in the larval zebrafish PTZ seizure model. In addition, their structural analogues TMC-120C, penicisochroman G, and ustusorane B were isolated and also significantly lowered PTZ-induced seizures. Finally, TMC-120A and TMC-120B were investigated in a mouse model of drug-resistant focal seizures. Compound treatment significantly shortened the seizure duration, thereby confirming their antiseizure activity. These data underscore the possibility to translate findings in zebrafish to mice in the field of epilepsy and the potential of the marine environment for ASD discovery.

With the aim to discover interesting lead compounds that could be further developed into compounds active against pharmacoresistant epilepsies, we first collected 14 medicinal plants used in traditional Chinese medicine (TCM) against epilepsy. Of the six extracts that tested positive in a pentylenetetrazole (PTZ) behavioral zebrafish model, only the ethanol and acetone extracts from Magnolia off icinalis (M. off icinalis) also showed effective antiseizure activity in the ethylketopentenoate (EKP) zebrafish model. The EKP model is regarded as an interesting discovery platform to find mechanistically novel antiseizure drugs, as it responds poorly to a large number of marketed anti-epileptics. We then demonstrated that magnolol and honokiol, two major constituents of M. off icinalis, displayed an effective behavioral and electrophysiological antiseizure activity in both the PTZ and the EKP models. Out of six structural analogues tested, only 4-O-methylhonokiol was active and to a lesser extent tetrahydromagnolol, whereas the other analogues (3,3′-dimethylbiphenyl, 2,2′-biphenol, 2-phenylphenol, and 3,3′,5,5′-tetra-tert-butyl-[1,1′-biphenyl]-2,2′-diol) were not consistently active in the aforementioned assays. Finally, magnolol was also active in the 6 Hz psychomotor mouse model, an acute therapy-resistant rodent model, thereby confirming the translation of the findings from zebrafish larvae to mice in the field of epilepsy. We also developed a fast and automated power spectral density (PSD) analysis of local field potential (LFP) recordings. The PSD results are in agreement with the visual analysis of LFP recordings using Clampfit software and manually counting the epileptiform events. Taken together, screening extracts of single plants employed in TCM, using a combination of zebrafish-and mouse-based assays, allowed us to identify allyl biphenol as a chemical scaffold for the future development of compounds with potential activity against therapy-resistant epilepsies.

SummaryDravet syndrome (DS) is a severe genetic epileptic encephalopathy with onset during the first year of life. Zebrafish models recapitulating human diseases are often used as drug discovery platforms, but also for drug repurposing testing. It was recently shown that pharmacological modulation of three serotonergic (5‐HT) receptors (5‐HT 1D, 5‐HT 2C, 5‐HT 2A) exerts antiseizure effects in a zebrafish scn1Lab −/− mutant model of DS. Using the zebrafish DS model, our aim was to examine the possibility of repurposing efavirenz (EFA), lisuride (LIS), and rizatriptan (RIZA), marketed medicines with a 5‐HT on‐ or off‐target profile, as antiepileptic drugs for DS. To examine whether these compounds have a broader antiseizure profile, they were tested in pentylenetetrazol and ethyl ketopentenoate (EKP) zebrafish models. Pharmacological effects were assessed by locomotor behavior, local field potential brain recordings, and bioluminescence. EFA was active in all models, whereas LIS was selectively active in the zebrafish DS model. Mainly, a poor response was observed to RIZA. Taken together, our preclinical results show that LIS could be a potential candidate for DS treatment. EFA was also active in the EKP model, characterized by a high level of treatment resistance, and hence these data are potentially important for future treatment of drug‐resistant epilepsy.

PharmaSea performed large-scale in vivo screening of marine natural product (MNP) extracts, using zebrafish embryos and larvae, to identify compounds with the potential to treat epilepsy. In this study, we report the discovery of two new antiseizure compounds, the 2,5-diketopiperazine halimide and its semi-synthetic analogue, plinabulin. Interestingly, these are both known microtubule destabilizing agents, and plinabulin could have the potential for drug repurposing, as it is already in clinical trials for the prevention of chemotherapy-induced neutropenia and treatment of non-small cell lung cancer. Both halimide and plinabulin were found to have antiseizure activity in the larval zebrafish pentylenetetrazole (PTZ) seizure model via automated locomotor analysis and non-invasive local field potential recordings. The efficacy of plinabulin was further characterized in animal models of drug-resistant seizures, i.e., the larval zebrafish ethyl ketopentenoate (EKP) seizure model and the mouse 6 Hz psychomotor seizure model. Plinabulin was observed to be highly effective against EKP-induced seizures, on the behavioral and electrophysiological level, and showed activity in the mouse model. These data suggest that plinabulin could be of interest for the treatment of drug-resistant seizures. Finally, the investigation of two functional analogues, colchicine and indibulin, which were observed to be inactive against EKP-induced seizures, suggests that microtubule depolymerization does not underpin plinabulin’s antiseizure action.