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.

Figures

Figure 1

Metabolic characterization of PTZ and kcna1-MO models of epilepsy. (A) Cartoon representation of how the Seahorse bioanalyser displays mitochondria bioenergetics as modulated by pharmacological inhibitors. (B) Schematic representation of treatment paradigm for metabolic drug screen in PTZ and kcna1-MO models of epilepsy. (C) PTZ and kcna1-MO models of epilepsy exhibit significant increases in metabolic parameters. Specifically, changes in basal respiration, maximal respiratory capacity, non-mitochondria respiration, total mitochondrial respiration, proton leaks, and ATP-linked respiration were studied for both models. Non-mitochondrially mediated changes in respiration were not observed in either model. (D) Linear regression analyses showing significant correlation between basal respiration and both maximum respiratory capacity (r = 0.817; P < 0.0001) and mitochondria-mediated respiration (r = 0.895; P < 0.0001) but only a moderate correlation with non-mitochondrial respiration (r = 0.489; P < 0.001). Data in C are shown as mean ± SEM; *P < 0.05, **P < 0.001, ***P < 0.0001 (unpaired t-test); n = 6–7 fish per group.

Figure 2

ASDs are efficacious in zebrafish bioenergetics assays. (A) A double-blinded screen of 20 known ASDs and three compounds that recently failed clinical trials in epilepsy. Thirteen of 20 known ASDs restored elevated basal respiration and total mitochondria respiration to wild-type levels in PTZ-induction model. In the kcna1-MO model, 15 of the 20 known ASDs were efficacious in restoring basal respiration, maximum respiratory capacity and total mitochondrial respiration to wild-type levels. (B) List of the 20 known ASDs and three compounds that recently failed clinical trials in epilepsy used in present study. Two of the three compounds that failed were efficacious in the PTZ-induction but not in the kcna1-MO model. Data in A are shown as mean ± SEM. Statistics are shown comparing treatment group to vehicle control; wild-type and vehicle are statistically different in all graphs although not illustrated; *P < 0.05, **P < 0.001, ***P < 0.0001 (unpaired t-test); n = 6–7 fish per group. ESL = eslicarbazepine; ESM = ethosuximide; LEV = levetiracetam; PRM = primidone; RUF = rufinamide; TGB = tiagabine; TOP = topiramate; Veh = vehicle; VGB = vigabatrin; WT = wild-type.

Figure 3

A screen of 870 compounds uncovers new potential ASDs. (A) Schematic representation of the workflow for the two-step drug screen. (B) Z-score scatter plot of 870 compounds used in the primary screen. Purple dots are representative hits from behavioural screen, green squares are lead compounds from both behavioural and metabolic screens. The red triangle represents vorinostat. (C) Identification of two categories of efficacious compounds from the behavioural screen namely; compounds effective only in kcna1-MO or PTZ-induction models. (D) Representative data showing elevated basal respiration and mitochondrially-mediated respiration in both kcna1-MO and PTZ-induction model is blocked by valproic acid (a known ASD) and other compounds used in drug screen including compound 53 (vorinostat), which is a broad HDAC inhibitor. Non-mitochondrial respiration is unaffected in both kcna1-MO and PTZ-induction models. Data in D are shown as mean ± SEM; *P < 0.001 (unpaired t-test); n = 6–7 fish per group.

Figure 4

ASDs are less robust in behavioural assays. (A) Comparison between behavioural and metabolic assays indicates behavioural efficacy is not predictive of bioenergetics efficacy. Known ASDs including levetiracetam and lamotrigine that effectively reduced elevated basal and mitochondrially-mediated respiration in PTZ-induction and kcna1-MO models had no effect on altered swimming behaviour in both models. (B) List of 20 known ASDs used in the behavioural screen. Eleven of 20 ASDs were efficacious in the PTZ-induction model, 6 of 20 were effective in the kcna1-MO, and 5 of 20 effective in both models. Data shown are for drugs efficacious in the locomotion assay only. (C) Linear regression analyses indicated a very weak negative correlation between total locomotor activity and basal respiration (r = −0.228; P = 0.04) and no correlation between total locomotor activity and total mitochondrial respiration (r = −0.160; P > 0.1). Data in A are shown as mean ± SEM; statistics are shown comparing treatment group to vehicle control; wild-type and vehicle are statistically different in all graphs although not illustrated; **P < 0.001, ***P < 0.0001(unpaired t-test); n = 6–7 fish per group. LGT = lamotrigine; LEV = levetiracetam; OXC = oxcarbazepine; PHT = phenytoin; TOP = topiramate; VBT = vigabatrin; Veh = vehicle; WT = wild-type.

Figure 5

Vorinostat is efficacious for mitochondria-mediated respiration. Vorinostat effectively ameliorated the elevated metabolic parameters of basal respiration, maximum respiratory capacity, mitochondria respiration, proton leaks and ATP-linked respiration in both PTZ-induced and kcna1-MO models. Data are shown as mean ± SEM; statistics are shown comparing treatment group to vehicle control; wild-type (WT) and vehicle are statistically different in all graphs although not illustrated; *P < 0.05, **P < 0.001, ***P < 0.0001 (unpaired t-test); n = 6–7 fish per group.

Figure 6

Vorinostat blocks seizures in zebrafish and rodents. (A) Image shows agarose-embedded zebrafish and the placement of a glass microelectrode in the tectum opticum. Representative extracellular field recordings obtained from the optic tectum of 6 dpf wild-type and kcna1-MO zebrafish larvae ± vorinostat treatment (40 μM). The presence of repetitive high frequency, large amplitude spikes indicative of hyperexcitability in knockdown animals is shown, and is abolished in the presence of vorinostat. (B) Representative electrographic seizure and heat maps recorded in Kcna1-null mice. Bar graph of the mean number of seizures per day recorded from six Kcna1-null mice ± vorinostat treatment. Mice were recorded for 24 h per day for 1 day, and then received vorinostat injection (40 mg/kg body weight/day) for 6 days. Vorinostat treatment significantly reduced the number of seizures (two-way ANOVA, F = 27.7, P < 0.01). (C) Acute (30 min dosing regimen) vorinostat (100 and 300 mg/kg) administration significantly reduced corneal electrode-stimulated seizures by 50 and 75%, respectively. A 2-h dosing regimen effectively reduced seizures across all concentrations used in the 6 Hz psychomotor test but not in the maximum electroshock test.

Figure 7

Inhibition of HDAC1 and HDAC3 blocks basal respiration in zebrafish. (A) MC1568, a HDAC1 inhibitor, reduced basal respiration in kcna1-MO to wild-type levels, whereas RGFP966, a selective HDAC3 inhibitor did not. Furthermore, entinostat, SBHA, and RG2833 are HDAC1 and HDAC3 inhibitors that reduced elevated basal respiration in kcna1-MO to wild-type levels, along with the pan-HDAC inhibitors quisinostat and belinostat. (B) RG2833 (100 mg/kg body weight/day) decreases the number of seizures/day following 6 days of treatment in Kcna1-null mice. Data in B and C are shown as mean ± SEM; *P < 0.05, **P < 0.001, ***P < 0.0001 (unpaired t-test); n = 6–7 fish per group and n = 4–8 animals per group. See also Table 1.