Abstracts

Rationale: Mutations in STXBP1 are linked to epilepsy, intellectual disability, developmental delay, ataxia, tremor, and other neurological symptoms. Currently available antiseizure medications are inadequate treatment for most STXBP1 Disorder patients. CRISPR-generated zebrafish stxbp1 mutants recapitulate seizure and non-seizure phenotypes seen in this patient population (Grone et al., 2016). Recent work suggests that 4-Phenylbutyrate (4-PBA), a chemical chaperone, may reverse mutation-induced deficits like impaired locomotion behavior in a Munc18-1 (stxbp1) C.elegans model (Abramov et al., 2021; Guiberson et al., 2018). However, 4-PBA rescue in Munc18-1 (stxbp1) mutant worms often require high concentrations, potentially leading to off-target effects. In-silico structure-based screening offers a rapid strategy to identify chemically similar compounds with higher specificity and potentially fewer side effects (Abramov et al., 2021). Here we used our stxbp1a zebrafish model with profound motor deficits to test the therapeutic efficacy of 4-PBA and related analogs.


Methods: In-silico ligand-based screening was performed using the 4-PBA structure against an approved-drug library (2315 drugs) from the DrugBank database (Version 5.1.7). Flexi-LS-align was employed for flexible structural comparison, yielding 14 candidates with high similarity scores ( >0.9) and suitable water solubility (LogP values 0-5). 4-PBA treatment was optimized for zebrafish larvae by chronically treating 3 days post-fertilization (dpf) stxbp1a mutants and wild-types for 48 hrs at a variety of concentrations. Following behavioral and toxicity assessments, 25 and 50μM treatment concentrations were identified and then used for testing related analogs. Behavioral assays were conducted at 5 dpf, assessing total movement of larvae tracked using a DanioVision platform (Noldus Technology) at 25 frames per second for 20 min. Toxicity was evaluated by monitoring larval circulation every 24 hrs as well prior to and immediately after locomotion assays. All screens included the use of tetracaine, an anesthetic, as a positive control to establish baseline detection thresholds for larvae lacking locomotor function.

Results: Consistent with a movement disorder phenotype, stxbp1a mutants display a 73% reduction in total distance moved compared to WT controls (stxbp1a = 282 ± 136 mm; WT = 1029 ± 625 mm; p < 0.001 Welch’s t test). This was similar to total movement tracked for WT larvae exposed to 200 uM of the anesthetic tetracaine (301 ± 151 mm). 4-PBA failed to increase movement for stxbp1a mutants following a 48 hr exposure (p = 0.8745 Welch’s ANOVA test). 4-PBA analogs identified during in-silico screening also failed to rescue movement deficit seen in stxbp1a mutants.