A novel metabolism-based phenotypic drug discovery platform in zebrafish uncovers HDACs 1 and 3 as a potential combined anti-seizure drug target

Abstract

Despite the development of newer anti-seizure medications over the past 50 years, 30-40% of patients with epilepsy remain refractory to treatment. One explanation for this lack of progress is that the current screening process is largely biased towards transmembrane channels and receptors, and ignores intracellular proteins and enzymes that might serve as efficacious molecular targets. Here, we report the development of a novel drug screening platform that harnesses the power of zebrafish genetics and combines it with in vivo bioenergetics screening assays to uncover therapeutic agents that improve mitochondrial health in diseased animals. By screening commercially available chemical libraries of approved drugs, for which the molecular targets and pathways are well characterized, we were able to reverse-identify the proteins targeted by efficacious compounds and confirm the physiological roles that they play by utilizing other pharmacological ligands. Indeed, using an 870-compound screen in kcna1-morpholino epileptic zebrafish larvae, we uncovered vorinostat (Zolinza™; suberanilohydroxamic acid, SAHA) as a potent anti-seizure agent. We further demonstrated that vorinostat decreased average daily seizures by ∼60% in epileptic Kcna1-null mice using video-EEG recordings. Given that vorinostat is a broad histone deacetylase (HDAC) inhibitor, we then delineated a specific subset of HDACs, namely HDACs 1 and 3, as potential drug targets for future screening. In summary, we have developed a novel phenotypic, metabolism-based experimental therapeutics platform that can be used to identify new molecular targets for future drug discovery in epilepsy.

Behavioural assay

For drug screening using kcna1 morphants, 5 days post-fertilization (dpf) larval zebrafish maintained in 96-well plates were habituated for 20 min, under ambient light. This was followed by treatment with 20 μM of drugs for 20–30 min (n = 6–7 per compound), and assayed for locomotor activity, burst activity, distance moved, inactive duration and tracking in dark for 20 min in ZebraBox (ViewPoint Life Sciences). For drug screening involving PTZ induction, 5 dpf zebrafish were treated with 10 mM PTZ for 10 min, followed by 20 μM (final concentration) of drug for 20 min under ambient light, and assayed for total locomotor activity, burst activity, distance moved and inactive duration, and tracking under 100% light in ZebraBox for 20 min. All behavioural assays were repeated at least twice. Total swim activity and tracking of total distance moved as a measure of hyperactive swimming behaviour were analysed using ZebraLab V3 software (ViewPoint Life Sciences, Lyon, France).

https://academic.oup.com/brain/advance-article/doi/10.1093/brain/awx364/4823617#109328665