Zebrafish offer the potential for a primary screen to identify a wide variety of potential anticonvulsants

Zebrafish offer the potential for a primary screen to identify a wide variety of potential anticonvulsants

Epilepsy Research (2007) 75, 18—28 journal homepage: www.elsevier.com/locate/epilepsyres Zebrafish offer the potential for a primary screen to identi...

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Epilepsy Research (2007) 75, 18—28

journal homepage: www.elsevier.com/locate/epilepsyres

Zebrafish offer the potential for a primary screen to identify a wide variety of potential anticonvulsants Stephane Berghmans a, Julia Hunt a, Alan Roach a, Paul Goldsmith a,b,∗ a b

DanioLabs Ltd., 7330 Cambridge Research Park, Cambridge CB25 9TN, UK Department of Neurology, Addenbrooke’s Hospital, Cambridge CB2 0QQ, UK

Received 24 November 2006; received in revised form 2 March 2007; accepted 15 March 2007 Available online 7 May 2007

KEYWORDS Epilepsy; Zebrafish; Pentylene tetrazole; Screen; AEDs

Summary The search for novel anticonvulsants requires appropriate model systems in which to test hypotheses through focused compound screening or genetic manipulation, or conduct black box screening of large numbers of compounds or potential genetic modifiers. Many models are currently in existence that subserve particular roles in achieving these aims, but all have their limitations. Zebrafish have been suggested as an additional model of epilepsy, but their optimum role is unclear. They are more amenable to high throughput analysis, but are more genetically removed from humans than rodents. We therefore sought to develop assay methodology applicable to medium/high throughput screening using an automated tracking system to measure the amount of movement induced by exposure to the proconvulsant, pentylene tetrazole (PTZ). We then used this system to explore how many known anti-epileptic drugs (AEDs) would be detected when running such a screen. We were able to detect suppression of PTZ-induced excessive movements with 13 out of 14 standard AEDs. A parallel sedation and toxicity screen suggested these effects were due to direct anti-epileptic effect, although non-specific effects cannot be fully excluded. These results suggest zebrafish may be a useful high throughput primary screen to pick up potential novel AEDs. © 2007 Elsevier B.V. All rights reserved.

Introduction Background to zebrafish epilepsy models

∗ Corresponding author at: Department of Neurology, Addenbrooke’s Hospital, Cambridge CB2 0QQ, UK. Tel.: +44 1223 706460; fax: +44 1223 706461. E-mail address: [email protected] (P. Goldsmith).

A zebrafish model of epilepsy has recently been described (Baraban et al., 2005). Larval zebrafish exposed to the proconvulsant pentylene tetrazole (PTZ) progress through a distinct series of postures and movements, culminating in generalized tonic clonic seizures. The authors divided the stages of activity into three. Stage I consisted of a

0920-1211/$ — see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.eplepsyres.2007.03.015

Zebrafish compound screen dramatic increase in swimming activity, stage II, a rapid ‘‘whirlpool-like’’ behaviour, and stage III, a series of cloniclike movements leading to a loss of posture, followed by further clonic episodes. The latencies to the beginning of stages I, II, or III were concentration dependent. Tracking systems were used to determine the total amount of movement and therefore quantify the amount of seizure activity. Ictal spike and sharp wave activity, postictal depression and interictal slow wave activity were seen using tectal wholefield recordings. Upregulation of c-fos, an immediate early gene indicative of neuronal activation, was detected and pharmacological evidence of seizure suppression seen with phenytoin, sodium valproate and the benzodiazepines clonazepam and diazepam. No effects were seen with carbamazepine or ethosuximide. The drugs were tested on immobilized fish through the electrographic recordings and the fish were dosed acutely with the test AED into the fish water. Toxicity was assessed by recording heart rate. More direct neurotoxic effects are harder to exclude using this mechanism and it is unlikely steady state tissue levels would have been reached within the 1 h experimentation period. Also, the preliminary pharmacology only reported dose response curves on sodium valproate and diazepam.

Advantages of zebrafish Systems tractable for screening purposes are always desirable for drug discovery, providing they are relevant to the question in hand. A pilocarpine-induced seizure model amenable to high throughput screening has recently been described in drosophila (Stilwell et al., 2006). However, drosophila are invertebrates and relatively highly removed phylogenetically from man compared to vertebrates. Whilst very good for genetic manipulation studies, there are also questions regarding variability in compound uptake through feeding in a pharmacological assay. Zebrafish also offer the scalability and throughput of drosophila, but are vertebrates and are more tractable pharmacologically (Goldsmith, 2004). Zebrafish breed prolifically, a single pair of adults generating 200 offspring per week (Detrich et al., 1999). A modest aquarium can thus easily generate several hundred thousand offspring per year. They then develop rapidly, ex-utero, facilitating early experimentation. By 72 h, they are free swimming, can see and are beginning to feed. During the larval stages (from days 3 to 10), they can live in volumes as small as 50 ␮l, making screens in 96well plate format possible. They were originally primarily used for genetic studies, culminating in two large screens characterizing a large number of mutants created by ENU mutagenesis published in 1996 (Driever et al., 1996). They are now an established tool not only for developmental biologists, but increasingly also for disease-focused researchers (e.g. see Berghmans et al., 2005; Ninkovic and Bally-Cuif, 2006; Shafizadeh and Paw, 2004; Zon and Peterson, 2005). It is possible to inject protein, RNA, or DNA constructs into the early embryo, thus modifying gene and protein expression (Westerfield et al., 1997). A particularly powerful technique involves the use of morpholinos, modified oligonucleotides, which can be used to titrate down gene expression (Sumanas and Larson, 2002). Essentially an antisense sequence is directed towards the 5 sequence, splice

19 site or untranslated region of a gene of interest to titrate down the expression of that gene. An experienced operator can inject 500 embryos per morning with the morpholino. In terms of compound screening, the fish swallow from day 3 and do not develop scales until several weeks of age. Compounds in general appear to be readily absorbed though either their gastrointestinal tract or across their skin (although there is still only a limited understanding of pharmacokinetic issues) and a number of papers describing high throughput compound screens have been published (Goldsmith, 2004; Langheinrich, 2003; Peterson and Fishman, 2004; Stern et al., 2005). As the total volume is so small, only tiny amounts of compounds are required removing the need for scale up chemistry. Thus whilst being a non-mammalian vertebrate and thus evolutionarily more distant from humans than rodents, they compensate through cost savings in terms of throughput and ability to carry out genetic modification. When determining the exact role that zebrafish may play in a viable screening cascade, one needs to establish just how relevant the model is to the human disease in question. We have therefore further developed the assay described by Baraban (Baraban et al., 2005), optimizing a number of aspects of the assay, and have then gone on to test a wider range of anticonvulsant standards in the free-swimming fish. We show that zebrafish can detect a wide range of anticonvulsants and therefore offer the potential for an early general screen, prior to more specific assays in other systems.

Methods Animals Zebrafish of the WIK wild-type strain were reared under standard conditions (Westerfield et al., 1997). Larvae were collected from natural spawning, staged according to established criteria (Kimmel et al., 1995) and reared in embryo medium (‘E3’: 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2 , 0.33 mM Mg2 SO4 , 10−5 % methylene blue) in an incubator in the dark at 28.5 ± 0.5 ◦ C. All procedures were performed in accordance with the UK Home Office Animals Scientific Procedures Act (1986).

Toxicological evaluation The maximum tolerated concentration in the fish water (MTC) was initially determined by incubating zebrafish larvae with test compound (i.e. the AED or control) for 24 h, from 6 to 7 days post fertilization (d.p.f.) in a 96-well plate (Millipore, USA), with 3 larvae per well in 200 ␮l of 1% dimethyl sulfoxide (DMSO) in E3. The aim of this was to determine AED concentrations to use in the anti-epileptic test (‘‘Anti-epileptic experimental procedure’’). A primary assay plate was set up with four concentrations of each compound, in duplicate wells, at log intervals from the highest dissolvable concentration downwards. A second assay was set up to refine the MTC by testing four concentrations of each compound, in duplicate wells, at half log intervals downwards from around the MTC identified in the primary assay. The secondary assay was repeated with a different clutch of larvae on another day. Each assay plate included four wells of vehicle controls (1% DMSO) and assays were incubated at 28.5 ± 0.5 ◦ C. Single clutches were used per compound/control set of wells to reduce variability (i.e. offspring all from same parents and same mating). Following the 24 h incubation, the capacity of larvae to startle to taps on the plate was first evaluated and lar-

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vae that did not startle were further examined for death or signs of toxicity. The MTC was defined as the maximum concentration at which no signs of toxicity (combination of no startle response and at least one other sign of toxicity) were observed in at least 20 out of 24 larvae in treatment groups at 7 d.p.f. after 24 h exposure to the tested drugs.

Anti-epileptic experimental procedure At 6 d.p.f. zebrafish larvae were placed in a 96-well plate with one larva per well and 12 larvae per treatment group. The larvae were placed in 200 ␮l E3 containing the various treatments: (i) two groups of vehicle (1% DMSO); (ii) two groups each of three concentrations of drugs to be evaluated for their anti-epileptic properties. The highest drug concentration tested corresponded to the MTC. Larvae were exposed to the drug for between 20 and 24 h in an incubator in the dark at 28.5 ± 0.5 ◦ C. Following drug exposure, the zebrafish larvae underwent a repeat of the toxicological evaluation described above. This is because the exact MTC was observed to vary between experiments, depending perhaps on the exact clutch of fish and age of compound. Therefore the MTC during the anti-epileptic assessment was deemed most accurate. The larvae were then transferred to fresh 200 ␮l E3 containing the various treatments: (i) vehicle (1% DMSO); (ii) pentylene tetrazole (PTZ, 20 mM); (iii) three concentrations of tested drugs without PTZ; (iv) three concentrations of the test drug with PTZ (20 mM). This concentration of PTZ is slightly higher than the 15 mM concentration used in the Baraban study, but in our hands was the optimum concentration to result in clear seizures. Immediately after transfer the larvae’s behaviour was monitored in the dark for 60 min using an apparatus containing an infra-red light source and a high-resolution digital video camera (modified from Tracksys, Nottingham, UK). The assay was performed in the dark to minimize the risk of compound photodegradation. Each assay was performed in duplicate.

Data analysis The locomotor activity (LA) for each 60 min recording session was analyzed by calculating the mean ‘‘total distance moved’’ for each treatment group using EthoVision 3.1 locomotion tracking software (Noldus, Wageningen, The Netherlands). The results were statistically evaluated using an ANOVA analysis to independently establish if treatment groups statistically reduced or increased the LA in comparison to the vehicle and PTZ treated groups. The quality control criteria to accept an assay for analysis were that: (i) the total distance moved for the PTZ treated group had to significantly increase in comparison to the vehicle group; (ii) no significant increase of LA occurred for larvae treated with the tested drug alone.

Drugs The drugs used were aspirin as negative control, carbamazepine, diazepam, ethosuximide, gabapentin, lamotrigine, oxcarbazepine, phenytoin, primidone, sodium valproate, tiagabine, topiramate, zonisamide, all from Sigma—Aldrich, Gillingham, UK, and levetiracetam (LTM: Sequoia Research Products, Pangbourne, UK). The drugs were dissolved in DMSO and administered in the embryo medium at concentrations in the fish water indicated in the results section in 1% final DMSO concentration.

Results The pattern of movement induced by the PTZ was directly observed. The fish exhibited short bursts of very rapid jerking lasting up to a few seconds, propelling them through the water at high speed. This movement is unlike any other

Figure 1 This shows the total distance moved in % relative to control by a fish exposed to varying concentrations of PTZ only, with acute exposure and tracking as described above.

movement we observe in the fish, such as when fish are moved into a novel environment (where they exhibit an initial increase in activity, either exploratory or anxiety driven), in response to a range of drugs we have tested or in response to a startle. The latter results in a characteristic dart lasting less than a second. The effects of varying concentrations of PTZ on the degree of seizure activity (measured by assessment of total distance moved) was assessed to allow selection of optimum parameters to pick up anticonvulsant activity of test drugs (Fig. 1). At very high doses the fish move less, perhaps as a result of neural damage and then eventually die. A 20 mM PTZ concentration was selected for further experiments, slightly higher than the 15 mM PTZ concentration in the fish water used by Baraban et al. (2005). The MTC determined for each drug in the toxicology assay is shown in Table 1 (second column). No MTC could be established for primidone. It was therefore tested at the maximum soluble concentration of 7.5 mM (MSC). The test concentrations selected for the anti-epileptic assessment (described in ‘‘Anti-epileptic experimental procedure’’) were based around an earlier toxicity-concentration response assessment, with the actual MTCs given in Table 1 being those determined during the final anti-epileptic assessment. The final MTC given in Table 1 concurred with this initial assessment, except in the case of ethosuximide, gabapentin and lamotrigine. Ethosuximide displayed a shift in MTC between the initial toxicology and subsequent epilepsy assays. In these latter assays, performed in duplicate, 13/24 and 11/24 larvae, respectively, showed signs of toxicity (defined as a combination of absence of startle response and presence of at least one sign of toxicity) at 30 mM and 6/24 and 12/24 larvae respectively at 10 mM in the duplicate assays. This placed the MTC at 3 mM. However it may still be that the reduction in locomotor activity observed at 10 mM was caused by ethosuximide’s anti-epileptic property rather than a toxic effect because when the affected fish were taken out of the analysis a significant reduction in the ‘total distance moved’ was still observed (p < 0.01). A similar variation in toxicity results between the primary toxicity assay and the subsequent additional toxicity assessment within the epilepsy assay was observed for carbamazepine and levetiracetam. The MTC for carbamazepine was initially established at 300 ␮M. This

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Table 1 Drug

Maximum tolerated concentration

Anticonvulsant effect

Sedative effect

Carbamazepine Sodium valproate Primidone Levetiracetam Oxcarbazepine Zonisamide Phenytoin Ethosuximide Gabapentin Diazepam Lamotrigine Topiramate Tiagabine Aspirin

300 ␮M 3 mM 7.5 mMa 30 mM 250 ␮M 1 mM 1 mM 30 mM 50 mM 50 ␮M 300 ␮M 10 mM 300 ␮M 300 mM

Yes Yes No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No

?b ?b No No Yes Yes No ?b ?b Yes No No No No

a b

Maximum soluble concentration. Sedative effects seen in one assay, but not duplicate assay.

drug was then found to be toxic at 300 ␮M in both epilepsy assays, with 8/24 and 22/24 larvae showing toxicity, placing the MTC at 100 ␮M, a concentration at which all fish startled. An MTC of between 30 and 100 mM was seen for levetiracetam in the primary toxicology assays. In contrast, in the epilepsy assay, the MTC was 30 mM, with 22/24 and 17/24 larvae respectively showing toxicity at 100 mM in the duplicate assays. Finally, three other drugs (oxcarbazepine, zonisamide and gabapentin) were tested at concentrations above the initially assessed MTC, given the variability in MTC which we were observing and the fact that at least a component of the absence of startle response identified in the toxicology assay might be linked to sedation. This sedative effect was confirmed in the epilepsy assay (Fig. 3E, H and N). All larvae tested at the two highest concentrations of oxcarbazepine (790 ␮M and 2.5 mM) showed toxicity, placing the MTC at 250 ␮M where all larvae startled even though sedation was identified in PTZ-untreated larvae. Zonisamide showed toxicity for all larvae at 3 mM in both assays and at 1 mM in the second assay (16/24 larvae), providing an MTC of 1 mM and 300 ␮M respectively. Fourteen out of 24 larvae showed no startle and toxicity signs at 160 mM gabapentin in the epilepsy assays placing the MTC at 50 mM, a concentration at which all larvae startled except for a single larva in the first assay. The capacity of the investigated drugs to reduce PTZinduced convulsions in zebrafish larvae was then assessed through quantification of the total amount of distance moved. A sample movement plot is given in Fig. 2, with the results summarized in Table 1. Individual drug concentration/response data is given in Fig. 3. All drugs showed statistically significant movement suppression and therefore presumed anticonvulsant activity in zebrafish larvae except for aspirin and primidone, albeit with the caveats noted above. Aspirin was used as a negative control and the absence of activity was expected. The lack of activity of primidone could either be due to bioavailability issues as the highest concentration tested was limited by the

Figure 2 Sample movement tracking plots for experiments with (A) carbamazepine (CBZ) and (B) lamotrigine (LTG). These images show the path taken by the fish (red tracks) for a 2 min period recorded 5 min into the experiment. Each well in any given row contained the same compound. Note the lack of movement in well 2B and 8B (PTZ only, column 2 and 7) in (A) and well 2B and 7B in (B) do not represent the total 60 min locomotion tracks but only 2 min of tracking. These larvae also moved and convulsed but not within that 2 min time window. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

22 drug solubility rather than toxicity, and thus insufficient amounts reached the brain, or because zebrafish larvae do not possess appropriate enzymes to metabolize primidone to active compound and thus the overall level of active drug was less than expected. Three AEDs caused a significant decrease in locomotor activity in zebrafish larvae: oxcarbazepine, zonisamide, and diazepam, as assessed by

S. Berghmans et al. comparing the total amount of movement when exposed to AED only as compared to embryo medium only. Four further AEDs showed a significant decrease in locomotor activity in one, but not the subsequent assay (Fig. 3) at the concentration at which the putative anti-epileptic effects were seen: carbamazepine, gabapentin, sodium valproate and ethosuximide.

Figure 3 The x-axis represents the treatments including vehicle (E3), 20 mM pentylene tetrazole (PTZ), the three concentrations tested for test compound alone, and the three concentrations tested for test compound in combination with 20 mM PTZ. Each treatment group comprises 12 zebrafish larvae. The y-axis represents the average total distance moved over the 1 h of videotracking and is expressed in centimeters. Results were evaluated using an ANOVA analysis to independently establish if treatments statistically reduced or increased the LA in comparison to the PTZ treated group (level of statistical significance is indicated as * for p < 0.05 and ** for p < 0.01) and vehicle group (s for p < 0.05 and ss for p < 0.01 for hypoactivity).

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Figure 3

Discussion This study extends the earlier work described by Baraban et al. (2005), and assesses a more comprehensive range of AEDs in an acute seizure assay system amenable to higher throughput screening. Of the AEDs tested by Baraban et al., we detected activity with carbamazepine, in contrast to the earlier work which picked up activity with phenytoin, but not carbamazepine (Baraban et al., 2005). One possible explanation for the differences relates to bioavailability issues. Baraban and colleagues induced epileptic activity first with PTZ, and then subsequently applied the AED, observing for suppression of manifestations of epileptic activity for up to 1 h. In contrast we bathed the fish in

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AED for 24 h and then applied the PTZ. Whilst it is generally said that drugs are absorbed well by zebrafish, formal pK measurements have never been published (and HPLC measurements on brain tissue were beyond the scope of these studies) and because of this uncertainity, we allow a longer period of time for drugs to enter the fish. This prolonged drug loading time, plus the paradigm change of AED exposure then addition of PTZ rather than PTZ exposure then addition of AED, may explain the differences. Another possibility relates to strain difference, with Baraban using TL fish whereas we used WIK fish. However, it could just be that the nature of our assay (behavioural rather than electrophysiological) picks up more false positives.

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Figure 3

It is interesting to note that the majority of AEDs were positively identified in this screen. PTZ induces convulsions in mice, rats, cats and primates (Fisher, 1989). The mechanism of PTZ is assumed to be principally through an inhibition of GABA activity at the GABA-A receptor, although many non-GABAergic drugs are also capable of preventing PTZinduced seizures (Olsen, 1981). This makes the zebrafish PTZ screen relatively non-discriminatory, in contrast to those in the mouse and rat where PTZ seizures can be used to identify those anticonvulsants acting through GABA. In this respect, the zebrafish resembles the audiogenic mouse model which also detects a wide range of anticonvulsants (Loscher, 1984). This is not without precedent, since for example the maximum electroshock (MES) assay also picks

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up some antidepressants and neuroleptics, as well as standard AEDs such as phenytoin and carbamazepine, both of which are very potent anticonvulsants in the MES assay (Mody and Schwartzkroin, 1998). The other interpretation is that the assay is relatively discriminatory but that we picked up false positives, as indicated above. In this study we ran parallel assays in an attempt to identify compounds with non-specific (neurotoxic or sedative) effects, or ones with general effects on locomotor activity. The fish exhibit spontaneous movement in the absence of any drug, consisting of repeated short swims (as shown with the no AED, no PTZ baseline in Fig. 1). Suppression of this activity was seen with three compounds (oxcarbazepine, zonisamide and diazepam) and possibly another

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Figure 3

four compounds (carbamazepine, sodium valproate, ethosuximide and gabapentin) and it is possible that the decrease in ‘seizure activity’ seen was just an inhibitory ‘braking’ effect of the sedation on the overall amount of movement, although the relative magnitude of the changes and the potent and absolute level of movement seen with PTZ makes this less likely. Nevertheless these remain potential confounding factors in a screen based around locomotor activity. It remains to be addressed exactly what the false positive rate is for the identification of a new anticonvulsant, although this in part depends on the parameters set for the identification of a positive compound to take forward. The present study shows the MTC not to be absolute as we observed variation in the MTC of up to a log interval between assays. It is therefore advised to monitor toxicity in

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all assays performed, as was done in this study. In zebrafish, the therapeutic effect of some anti-epileptic drugs seems to be present at concentrations close to those causing toxicity, most notably in this study with ethosuximide and oxcarbazepine. As discussed above, a further potential confounding effect to be considered relates to bioavailability. The fish has a functional gastrointestinal tract from day 3, with the gut continuing to mature over subsequent days, so the oral route of absorption is possible (Wallace and Pack, 2003). Zebrafish also do not develop scales until several weeks of age, so until this time transdermal absorption is also possible, and thereafter absorption across the gills. However, different drugs will be absorbed to differing extents. There are also active transport mechanisms present (e.g. pgp, unpublished obser-

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Figure 3

vations). Ultimately one can infer a compound is absorbed if activity is seen, but the converse is not necessarily true if no activity is seen. This is one of the rationales as to why we employed the MTC as the upper limit for the screen on the assumption that the compound is likely to have been absorbed at that concentration to manifest its toxicity. It is possible to measure actual drug concentrations with HPLC and this extra data would be beneficial, especially to rule out false negatives. This is under active development. The first anti-epileptic drugs to be developed were identified using non-hypothesis driven random screening (Merritt and Putnam, 1984; Swinyard et al., 1952; Swinyard and Kupferberg, 1985). Various animal models continue to be used for initial screens. These models utilize both chemically and electrically induced seizures, as well as several genetic and reflex seizure models. None is capable of identifying all of the anti-epileptics currently in use and all are of relatively low throughput. One therefore needs to prioritize the compounds to be applied to the model. It seems that zebrafish offer an opportunity to have a pre-mammalian in vivo anticonvulsant screening assay (Fig. 4). The majority of known anticonvulsants were picked up by this assay suggesting it can function as a useful primary screen, prior to dissecting out more specific mechanistic considerations with rodent assays. One could therefore envisage using drosophila-based epilepsy assays (Stilwell et al., 2006) for early genetic and possibly also pharmacological screens and zebrafish for early pharmacological and possibly also genetic screens,

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before testing hypotheses thrown up by these systems in conventional assay systems such as those utilized in the NINDS Anticonvulsant Screening Program (details listed at http://www.ninds.nih.gov/funding/research/asp/index.htm) (Stables et al., 2002). In terms of what such an assay might be used for, possibilities are indicated in Fig. 4. So called black box screening, where a random compound is screened in a whole organism, has been and continues to be a very useful method of drug discovery, particularly in the field of AED discovery. However one critique of this approach is that if the target of the compound is not known it is much harder to chemically optimize the hit. A converse and popular drug discovery process involves the identification of a target, in vitro screening using the particular target to find chemicals that bind to or interact with it, functional assays to see if the interaction has a desired effect, alteration of the structure of the lead compound to improve its bioavailability and minimize its toxic tendency without losing its beneficial effects, then testing through standard safety assays. This lead optimisation is more difficult if the target is not known, and also it may be harder to get FDA approval if the mechanism of action is not known. It is not that it is not possible to get a drug to market without this approach (e.g. the mechanism of action of levetericatem was unknown at launch), but it is more difficult. This zebrafish assay may therefore complement existing approaches, enabling aspects of black box and rational design screening to be combined by using pharmacological

Zebrafish compound screen

Figure 4

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This shows a current screening cascade (A) and how zebrafish might be incorporated into this cascade (B).

probes as the test set. It is much easier to reach the correct conclusion with such a set, with the throughput of the assay making the screening of a large set of probes feasible. We also undertake the screening of known or abandoned drugs, partly because these are often useful probes, but also because regardless of this, the fact that they have already been into man de-risks their development compared to an NCE (new chemical entity). Whilst large numbers of compounds have been screened in rodent assays, a zebrafish screen in 96-well plate format still offers the potential for a much greater throughput for equivalent costs. The small volume of fluid into which the test compound is added also means only tiny amounts of compound are needed. This both makes larger screens of novel compounds possible and also makes in vivo SAR (structure activity relationship) assessment feasible and the iteration in drug discovery between optimizing functional activity and minimizing toxicity easier without requiring scale up chemistry, although a key factor is then how well conserved the target is between zebrafish and humans. The zebrafish genome has been sequenced and so homology analysis is possible. The automated ensemble annotation is error prone on account of the way the zebrafish genome sequence was originally derived and therefore when looking for a zebrafish gene it is best to begin by looking at the Vega database (http://vega.sanger.ac.uk/Danio rerio/index.html). This contains high quality, manually annotated gene sequence, although is still incomplete. Gene profiling experiments are being undertaken in various labs to look for genes which may protect or predispose to epilepsy. Such analyses often have considerable noise, so systems which might be able to prioritize candidate genes would be useful. On account of their genetic tractability zebrafish offer one such system. Morpholinos are a very powerful technique in zebrafish research to knockdown a particular gene. However currently the morpholinos are injected at the one cell stage, so a developmental phenotype may mask a later phenotype. Furthermore, as the embryo grows the morpholinos become diluted out so an effect at day 7 will be missed. To pursue such a strategy one would really need to see if the assay could be run at earlier time points, something we have not yet done. Injecting

DNA or RNA constructs is also possible to look for overexpression effects, although similar caveats to the morpholino approach apply. Finally, whilst monotherapy is the goal in epilepsy therapy, combination effects are an important consideration, whether these be between two concurrently prescribed drugs, or between a drug and a genetic alteration. Looking at combination effects is very difficult using conventional systems on account of the number of data points necessary. This is a role that zebrafish may fulfil. In conclusion, we show that a zebrafish PTZ seizure assay is relevant to human epilepsy and is capable of picking up a range of anticonvulsant drugs. It should therefore be considered as part of anti-epileptic screening cascades.

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