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Phenotype-based screening
We are currently witnessing a resurgence of interest in As a direct result, compounds advancing from phenotypic
A screen in which the assay phenotype-based screening in drug discovery. Phenotypic screens are often of higher quality than hits from invitro
output is a complex cellular or effects of small molecules were historically the basis of target-based screens.
organismal phenotype that all drug discovery, but over the past 30years this strategy
integrates multiple biochemical
has been largely replaced by target-based approaches. Advantages of screening in zebrafish
pathways and often captures
much of the native biological There is now a growing appreciation that firstinclass Small-molecule screens in zebrafish represent a small
context. drug discovery successes are emerging disproportion- but growing fraction of phenotype-based screens
ately from the remaining phenotype-based efforts. (TABLE1). Zebrafish screens provide the advantages of
For example, a recent analysis of firstinclass drugs phenotype-based screens outlined above, but they also
approved between 1999 and 2008 revealed that 62% were offer unique advantages that come from screening in an
1
Cardiovascular Medicine discovered by phenotype-based screens, despite the fact intactanimal.
and Network Medicine that such screens represented only a small subset of the
Divisions, Brigham and overalltotal1. Broad range of accessible biology. Most phenotypic
Womens Hospital, Boston,
Massachusetts 02115, USA.
Several factors may explain the apparent superiority screens are carried out in cultured cells, which limits
2
Harvard Stem Cell Institute, of phenotype-based approaches over target-based the screens to cell-autonomous phenotypes or those end
Cambridge, Massachusetts screening efforts. First, phenotypic screens can discover points that can be observed in relatively simple culture
02138, USA. efficacious drugs in the absence of a validated target. systems. Zebrafish screens are typically carried out in
3
Department of Medicine,
For example, ezetimibe was discovered based on its living zebrafish embryos or larvae, which exhibit a
Harvard Medical School,
Boston, Massachusetts cholesterol-lowering activity years before NiemannPick diverse repertoire of biological processes and possess
02115, USA. C1like protein 1 (NPC1L1) was validated as a thera- fully integrated vertebrate organ systems. As such, a
4
Broad Institute of MIT peutic target 2,3. Second, phenotypic screens can identify much broader range of phenotypes can be assayed in
and Harvard, Cambridge, compounds that produce a therapeutic effect through zebrafish than in cultured cells. Pain, sedation, tumour
Massachusetts 02142, USA.
5
Cardiovascular Research
simultaneous activity at multiple targets. Thus, amioda metastasis, vascular tone, and gut motility are examples
Center, Massachusetts rone, which remains the definitive antiarrhythmic agent of disease-relevant phenotypes that are observable in
General Hospital, Charlestown, decades after its serendipitous discovery, exhibits activity zebrafish yet simply inaccessible to modelling in cultured
Massachusetts 02129, USA. on multiple ion channels, adrenergic receptors, and cells. The advantages of the intact animal as a focus for
6
Department of Systems
possibly even nuclear hormone pathways4. In addition, screening are particularly evident for neuroscience drug
Biology, Harvard
Medical School, Boston, there is evidence that the acute effects of the intravenous discovery, in which the complexities of cellcell interac-
Massachusetts 02115, USA. form may be attributable in part to the solvent Tween80 tions and endocrine signalling defy even modelling with
e-mails: camacrae@bics.bwh. (REF.5). Third, phenotypic screens often combine screen- patient-derived induced pluripotent stem cells (iPSCs).
harvard.edu; peterson@cvrc. ing and counter-screening (for example, against relevant By contrast, with emerging automated technologies and
mgh.harvard.edu
doi:10.1038/nrd4627
organ-specific toxicity reporter lines) in the same assay, zebrafish screening, it is possible to imagine relatively
Published online discovering compounds that produce a desired effect unbiased capture of a substantial proportion of the
11 September 2015 while parsing out compounds with undesirable qualities. complete phenotypic repertoire.
Early insight into toxicity. Whereas cell-based assays at active sites in the enzymes, channels and receptors that
provide limited information about the absorption, distri- are frequently the targets of drugs because these sites have
bution, metabolism, excretion and toxicity (ADME-Tox) retained the ability, through evolution, to bind to the same
of screening compounds, zebrafish screens often reveal metabolites, ions and other biomolecules. For example,
insights about these pharmacological characteristics. the zebrafish glucocorticoid receptor, which is only about
Zebrafish larvae possess functional livers, kidneys and 50% identical to the human receptor overall, is 74% iden-
bloodbrain barriers68. To produce invivo phenotypes tical in the carboxyterminal half of the protein, which
in zebrafish assays, compounds must exhibit the ability contains the ligand-bindingdomain.
to be absorbed, reach the target tissue, and avoid rapid
metabolism and excretion. This fact may explain the Physiology. Among model systems that are amenable
observation that several compounds that were discov- to screening, zebrafish stand out for their highly con-
ered in zebrafish screens have been rapidly translated to served integrative physiology. Unlike yeast, worms or
invivo mammalian models with minimal optimization flies, zebrafish possess recognizable organ systems
of pharmacological properties. livers, hearts, kidneys, pancreases and so on. Although
there are some major differences resulting from adapta-
Are zebrafish relevant for human drug discovery? tion to aquatic life, most zebrafish organs perform the
Being able to perform high-throughput phenotypic same functions as their human counterparts and exhibit
screens in an invivo context is theoretically very attrac- well-conserved physiology. For example, the zebrafish
tive, but how relevant is the output from zebrafish screens pancreas contains islets comprising , , , and cells
for human biology? Recent studies have cast doubt on the that regulate glucose homeostasis by secreting glucagon,
validity of some well-established rodent disease models, insulin, somatostatin and ghrelin, just as in humans11.
reminding us again of the potential to be misled by models Drugs that modulate glucose homeostasis in humans
of any form9. If it is important to understand the capa- have been shown to have the same effects in zebrafish12.
bilities and limitations of mammalian models, the need The zebrafish haematopoietic system is highly similar
is even greater for zebrafish, which are phylogenetically to the human system and consists of the same cell types
further removed from humans. Over the past few years, erythrocytes, neutrophils, eosinophils, lymphocytes,
we have begun to understand how zebrafish compare to macrophages and so on13. Haematopoietic processes are
humans in terms of targets, physiology, drug metabolism conserved, as are globin switching and iron homeostasis
and pharmacology, in particular during the first few days through the hepcidinferroportin pathway 14,15. In fact,
of life when it is possible to house them in multi-well key components of these processes were first discov-
plates suited for screening (FIG.1). Nevertheless, questions ered in zebrafish16. Drugs affecting haematopoiesis and
about relevance to humans remain of central importance anaemia in humans have similar effects in zebrafish17.
to the use of zebrafish in drug discovery. Cardiovascular physiology is also highly conserved
between humans and zebrafish at anatomical, cellular
Targets. With a high-quality zebrafish genome now avail- and membrane-biology levels. Many human cardiovas-
able, it appears that 71% of human proteins (and 82% of cular drugs have been shown to have identical effects
disease-causing human proteins) have an obvious ortho- on zebrafish physiology, and numerous human cardio-
logue in zebrafish10. Orthologous zebrafish proteins are vascular disorders have been recapitulated in zebrafish
reasonably similar to their human counterparts, particu- genetic models18. Interestingly, the cardiac electrophysi-
larly within functional domains. For example, the protein ology of humans is more similar to that of zebrafish than
targets of the ten most-prescribed drugs have zebrafish it is to that of rodents, adding to a list of cases in which
orthologues with sequence identity ranging from 54% zebrafish physiology may be more relevant than the
(glucocorticoid receptor) to 91% (thyroid receptor). Given rodent counterpart 1921. Many other examples of orthol-
the sequence divergence between zebrafish and human ogy between humans and zebrafish exist; however, per-
proteins, one might expect a modest rate of conservation haps the ultimate consequence of the throughput feasible
of pharmacological effect. In reality, however, the rates of in this organism will be a much more global understand-
conservation are relatively high (see below). One potential ing of how representative zebrafish are of human biology
explanation for this is that the target similarity is greater compared to most other preclinicalmodels.
detail elsewhere39. Here, we focus on select examples of replicate the dorsalized phenotype in zebrafish embryos,
disease-relevant compounds that have been discovered suggesting that AMPK was not the dorsalizing target.
by zebrafish screens in the hope that they will illustrate Instead, dorsomorphin-treated animals were indistin-
the diverse methodologies accessible for drug discovery guishable from a previously identified genetic mutant
in this organism. called lostafin, which harboured a mutation in Alk8
(REF.44). This phenotypic similarity suggested that dor-
Prohema. Prohema is a stabilized derivative of prosta- somorphin may inhibit Alk8 in zebrafish and ALK2 in
glandin E2 (PGE2) that is currently in PhaseII trials in humans a hypothesis that has since been confirmed.
patients undergoing umbilical cord blood (UCB) trans- As the first small-molecule inhibitor of the BMP path-
plantation for leukaemia or lymphoma. Prohema was way, dorsomorphin and derivatives such as LDN193189
discovered in a zebrafish screen for compounds that have become widely used probes for manipulating BMP
increased or decreased the numbers of haematopoietic signalling 45.
stem cells (HSCs)40. Automated insitu hybridization was Hyperactive BMP signalling causes a variety of dis-
used to stain HSCs in the aortagonadmesonephros ease states, including FOP and anaemia of inflammation.
region of the embryo, and compounds that enhanced The dorsomorphin derivative LDN193189 has proven
PGE2 synthesis were found to increase HSC numbers. to be effective in treating rodent models of FOP and
The ability of PGE2 to enhance production of HSCs was anaemia of inflammation15,25,46. In addition, it has been
later tied to its interaction with WNT signalling 41. shown to be effective in treating mammalian models of
The therapeutic potential of Prohema was first dem- other BMP-linked conditions, including arterial calci-
onstrated in adult zebrafish, in which it enhanced the fication and inflammatory bowel disorder 4749. Because
rate of marrow recovery after sub-lethal irradiation. of the apparent promise of BMP inhibitors for treating
In mice, whole bone marrow exposed to Prohema these conditions, the US National Institutes of Health
exvivo formed more spleen colony-forming units when has selected dorsomorphin and its derivatives for pre-
transplanted into irradiated mice than did untreated clinical development through its TRND (Therapeutics
marrow 40. Similarly, Prohema-treated whole bone mar- for Rare and Neglected Diseases) and BrIDGs (Bridging
row more effectively reconstituted the haematopoietic Interventional Development Gaps) programmes, which
system and contributed more HSCs to short-term will support preclinical development through early
and long-term repopulation than did untreated whole clinical trials in FOP and anaemia of inflammation,
bonemarrow. respectively.
For humans undergoing HSC transplantation as
therapy for leukaemia or lymphoma, UCB has become PROTO1. PROTO1 and its derivatives are benzothio-
an important source of transplantable HSCs. However, phene carboxamides that are currently in development
the number of HSCs present in a typical UCB sample for preventing antibiotic-induced hearing loss. Because
is often suboptimal for treating adults. Therefore, the aminoglycoside antibiotics can cause hearing loss by
apparent ability of Prohema to boost HSC numbers and killing hair cells in the human ear, a zebrafish screen
its repopulating ability may enhance the effectiveness of of 10,960 compounds was conducted to identify those
UCB transplantation. On the basis of this idea, Prohema compounds that could protect zebrafish hair cells from
is currently in a PhaseII clinical trial for the exvivo con- aminoglycoside-induced death50. PROTO1 and a struc-
ditioning of UCB before transplantation into patients turally related benzothiophene carboxamide were found
with leukaemia or lymphoma42,43. to be potent otoprotectants in zebrafish, and to protect
hair cells from aminoglycoside death in cultured murine
Dorsomorphin. Dorsomorphin and its derivatives are utricles.
Fibrodysplasia ossificans inhibitors of the bone morphogenetic protein (BMP) PROTO1 has been licensed to Oricula Therapeutics,
progressiva receptor: in humans this protein is activin receptor-like a company dedicated to developing the benzothio-
(FOP). A rare autosomal
kinase 2 (ALK2, also known as ACVR1) and in zebrafish phene carboxamides as otoprotectants. Oricula reports
dominant condition caused by
gain-of-function mutations in it is Alk8 (also known as Acvr1l). These compounds are that lead optimization has yielded analogues with 100
the gene encoding activin currently in development as therapies for fibrodysplasia times higher potency than PROTO1, a wide safety
receptor-like kinase 2 (ALK2). ossificans progressiva (FOP) and for anaemia of inflammation margin and efficacy in rat models of aminoglycoside-
These mutations result in (FIG.2). Dorsomorphin was discovered in a zebrafish induced hearing loss. Investigational new drug (IND)-
chronic activation of the bone
morphogenetic protein (BMP)
screen that sought to identify compounds that perturb enabling studies are ongoing (Oricula Therapeutics and
pathway with resultant the establishment of the basic body organization during M.Gleser, personal communication).
formation of ectopic bone in early embryogenesis44. In screening a library of a few
muscle tissue. Restriction of thousand compounds, a pyrazolopyrimidine was iden- Repurposing existing drugs
the thoracic skeleton then
tified that causes dorsalization the expansion of dorsal In addition to discovering novel compounds with thera-
leads to respiratory failure,
usually in childhood. tissues at the expense of ventral tissues. Treated embryos peutic potential, zebrafish screens have proven useful for
developed with reduced or missing tails, and the pyra- identifying novel uses for existing drugs. As the follow-
Anaemia of inflammation zolopyrimidine was named dorsomorphin to reflect its ing examples illustrate, repurposing screens can provide
A form of anaemia that is dorsalizing activity. an abbreviated path to clinical investigation because the
characterized by a block in iron
availability for haematopoiesis
The dorsalizing pyrazolopyrimidine had previously compounds they identify have already been approved
and is observed in many been described as an inhibitor of AMP-activated protein for human use or at least have been subjected to prior
chronic inflammatory diseases. kinase (AMPK), but other AMPK inhibitors failed to pharmacokinetic analysis and safety testing.
N
N N
Gene X
N
Figure 3 | Zebrafish phenotypes reveal drug targets. Drug-induced phenotypes in embryonic or larval zebrafish can
be compared with databases containing thousands of mutation-associated zebrafish phenotypes. On several occasions,
the discovery of phenotypic similarity between drug- and mutation-induced phenotypesNature
has revealed the| mechanism
Reviews of
Drug Discovery
action of a poorly understood compound.
led to the hypothesis that kalihinol F chelates copper, toxicology may be the most prevalent use of zebrafish
which was confirmed biochemically. In fact, the addi- in industry, with the majority of large pharmaceutical
tion of exogenous copper was able to rescue the kali- companies reporting some use of zebrafish for toxicology.
hinolFinduced developmental defects in zebrafish, It should be noted that the role of zebrafish in toxicol-
and treatment with kalihinol F was able to rescue ogy is often different from that of mammalian species.
copper overload toxicity in zebrafish embryos and Owing to the cost and effort associated with mamma-
mammaliancells. lian toxicology, it is often performed relatively late in
It should be noted that this type of multidimensional preclinical development, with the goals of determining
phenotype matching, or phenoclustering, in zebrafish the safety of advanced preclinical lead compounds and
can also be used to determine MOAs for compounds obtaining regulatory approval for a clinical trial. By con-
that were not originally discovered using zebrafish trast, zebrafish toxicology is much less expensive and can
screens. Fumagillin, an anti-angiogenic natural product, be performed rapidly on large numbers of compounds
was discovered more than 60years ago, and its binding in parallel. As such, zebrafish can be deployed much
target, methionine aminopeptidase 2 (MetAP2), was earlier in preclinical development. For example, toxicol-
discovered more than 15years ago. However, pheno- ogy testing can be performed on hundreds or thousands
type matching in zebrafish revealed a key insight into of hits from a primary high-throughput screen to eliminate
its MOA. Zebrafish embryos treated with fumagillin toxic compounds at an early stage and to prioritize hits
exhibited a distinctive gastrulation phenotype previously for further development. Indeed, this implicit counter-
observed in zebrafish with mutations in the non-canonical screen for toxicity is an integral part of disease sup-
Wnt5, revealing that MetAP2 inhibition by fumagil- pressor screens in the organism. Therefore, zebrafish
lin ultimately disrupts non-canonical WNT signalling can perform an invaluable function in enabling toxic
downstream of the Frizzled receptor 59. compounds to fail fast, before substantial resources
Because the number and variety of phenotypes have been wasted on their preclinical advancement.
that can be distinguished in a whole organism greatly As suggested earlier, zebrafish screens may generate
exceed the number distinguishable in cultured cells, lead compounds from primary screens that are already
phenotype matching is a powerful way of determin- pre-evaluated for several major forms of toxicity.
ing small-molecule MOAs. As the number of described Zebrafish have been shown to be a good model for
genephenotype pairs continues to rapidly increase, the predicting several types of drug toxicities (FIG.4). Cardio
utility of this approach should continue to grow. We toxicity, for example, appears to be highly conserved
anticipate that phenoclustering will be an important between zebrafish and humans. Systematic studies of
tool for MOA determination, not only for compounds drugs that cause QT prolongation in humans show a
discovered in zebrafish screens but also for drugs discov- >95% conservation of effect in zebrafish19. In the identifi-
ered by any other means. These observations also sug- cation of clinically significant repolarization toxicity, this
gest the potential benefits of more systematic approaches single assay performed as well as the combination of an
to compound annotation in the zebrafish. assay for human ERG (hERG) inhibition and toxicological
experiments on rabbit whole heart and canine Purkinje
Other uses for zebrafish in drug development fibres. Similarly close correlations have been observed
Although much of the academic effort in zebrafish chem- for hepatotoxicity, nephrotoxicity and reproductive tox-
ical biology has been focused on small-molecule discov- icity, in which all of the known toxicants in preclinical
ery, it is by no means the only way in which zebrafish are mammalian models or humans have similar effects in
contributing to drug discovery and development. In fact, zebrafish60,61. Nevertheless, it will require considerable
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Organ-specic toxicity Behavioural toxicity Systemic phenotyping Metabolomics Toxicity reporter lines
Figure 4 | Use of zebrafish in toxicology. Zebrafish have been validated for high-throughput screening focused on
specific organ toxicity (liver, kidney and heart) or behavioural toxicity. Alternatively, they have been used to profile
Nature Reviews
compounds systematically for toxicity across multiple systems simultaneously. New technologies, | Drug
including Discovery
metabolomic
profiling and toxicity reporter lines, promise to extend the utility of zebrafish further. m/z, mass to charge ratio.
additional effort, and much higher-resolution assessment Behavioural screens for novel neuroactive drugs. Diseases
of the mechanisms of toxicity in each system, to establish of the nervous system are arguably the area of greatest
the full extent of the homology between zebrafish and challenge for drug discovery. Despite the prevalence of
human toxicology. nervous system disorders, there are generally few effec-
tive pharmacological therapies. Developing new central
Future opportunities for zebrafish nervous system (CNS) drugs is slow, expensive, and
Technologies for working with zebrafish are evolving fraught with failures. Challenges include the fact that
rapidly and opening new opportunities to contribute there are few well-validated targets for most nervous sys-
to improved drug development. Zebrafish are poised to tem disorders. Furthermore, it is difficult to create and
make a difference in a number of areas in the near future, assess faithful models of many CNS disorders. Psychiatric
as outlinedbelow. end points such as depression and psychosis are difficult
to assess in animals, and neurodegenerative end points
Genome engineering to generate sensitized models for are often slow to develop and laborious to quantify.
screening. The advent of transcription activator-like Zebrafish behavioural screens offer an intriguing
effector nucleases (TALENs) and CRISPRCas (clustered alternative approach to CNS drug discovery. Zebrafish
regularly interspaced short palindromic repeatCRISPR- possess a rich repertoire of behaviours, including
associated) nucleases has dramatically changed the simple stimulusresponse behaviours and more com-
landscape for zebrafish-based research. Withinthe past plex behaviours such as learning and sleep. Many of
2years, CRISPRCas technology in particular has made these behaviours have been shown to be amenable to
it possible to rapidly generate targeted lossoffunction assessment in a 96well format, and high-throughput
mutants and knockin lines62,63. For the first time, it is now screens have discovered compounds that alter these
possible to recreate human disease alleles in zebrafish6467. behaviours in specific, reproducible ways7274.
The potential impact of this advance on drug discovery is Although zebrafish and human behaviours differ, the
immense. With thousands of human disease-associated underlying circuits, cells and receptors are frequently
mutations being discovered by human geneticists, the conserved. Therefore, it may be possible to identify
challenge now is to figure out the functions of these muta- compounds that modulate disease-relevant processes by
tions and determine how to translate that knowledge into their ability to modulate zebrafish behaviours controlled
new therapies. Because targeted mutants can be generated by homologous pathways. For example, it may be diffi-
and phenotyped so much more efficiently in zebrafish cult to model schizophrenia in a zebrafish or to assess
than in rodents, zebrafish are likely to be a popular choice schizophrenia-like symptoms directly. However, most of
for testing the relevance of candidate diseasegenes. the proteins and cell types associated with human schizo
As genome editing technologies continue to improve, phrenia are conserved in zebrafish75,76. Antipsychotic
it will be possible to reconstruct tens or hundreds of drugs would be expected to modulate such proteins and
human mutations in zebrafish, followed by rapid assess- cell types, causing specific and distinctive behavioural
ment of their phenotypic effects. Perhaps more impor- changes in zebrafish. With a sufficiently granular under-
tantly, lines that are found to be disease-relevant can standing of the correlations between human disease and
quickly form the basis of large-scale chemical suppressor zebrafish behaviour, it would theoretically be possible to
screens. It has already been demonstrated that zebrafish identify neuroactive drugs simply by screening for com-
screens can identify compounds that reverse the effects pounds that produce the desired behavioural profile in
of genetic mutations30,52,55,6871. Thus, any line that pro- zebrafish.
duces a disease-relevant phenotype could be quickly For such an approach to work, the challenge will be
expanded and used to screen for chemical suppressors to determine what behavioural phenotypes are indica-
of the disease-associated phenotype. tive of a therapeutic candidate. Two approaches seem
Cl
NH
Compound X
Figure 5 | Shared behavioural phenotypes reveal shared mechanisms and utilities. Neuroactive compounds often
produce distinctive behavioural changes in zebrafish that can be quantified in high-throughput behavioural assays.
As databases of compound-induced behavioural profiles grow, it is becoming possible Nature
to identify mechanistic
Reviews | Drug Discovery
relationships between compounds and search large chemical libraries for desirable profiles that indicate therapeutic
potential for nervous system disorders.
promising in this regard. The first is to use existing drugs false negatives are not problematic for drug discovery
to establish what the behavioural profile is for a particu- screens, in which the occasional false negative is well
lar therapeutic class. It has been shown that some classes tolerated. In toxicology screening, however, false nega-
of known neuroactive drugs produce distinctive and tives can be more troubling, as they could potentially
reproducible behavioural signatures in zebrafish behav- result in a failure to identify a compounds significant
ioural assays72,73. Establishment of a behavioural signa- toxicity. Improved methods for predicting or quantify-
ture for a therapeutic class would enable subsequent ing drug uptake could dramatically improve the utility
high-throughput screening for novel compounds that of zebrafish for drug discovery.
produce a similar behavioural effect in zebrafish (FIG.5). Of course, several other challenges remain. Natural
A second potential approach is to use genome engineer- variation exists in many invivo phenotypes, especially
ing to recreate human disease-associated mutations in complex phenotypes such as animal behaviours. Creating
zebrafish, then phenotype these animals carefully for robust, high-throughput assays to measure such pheno-
any resulting behavioural deficits. Mutant lines could types is challenging, but has been overcome in a variety
then be subjected to behavioural screening to identify of creative ways by numerous different research groups
compounds that suppress the abnormal behaviour and (TABLE 1) . Another challenge is producing enough
return the behavioural profile tonormal. zebrafish to enable truly large-scale, high-throughput
screens. This can be particularly difficult when work-
Unresolved questions and remaining challenges ing with fragile or inbred transgenic lines. To date,
The zebrafish has filled the niche for a screenable verte- most screens have involved production and screen-
brate in biology and drug discovery, but the emergence ing of a few hundred to tens of thousands of zebrafish
of the model and its rational appraisal as a tool for drug per day 39. Improved methods for mass production and
discovery has also served to highlight several challenges handling of zebrafish embryos would be highly beneficial
that remain. One of the most substantial issues is the to thefield.
challenge of controlling and quantifying drug exposures. Moving forward, substantial innovation and invest-
Although it is easy to control the concentration of a drug ment will be required to generate phenotypes that are
in the water bathing the zebrafish, it is more difficult to both relevant to human disease traits and scalable
predict how much drug will be absorbed. Some drugs for high throughput. Even as genome editing enables
appear to be absorbed poorly, whereas others are taken truly mechanistic modelling in different organisms
up readily. Drug levels are often measured directly in (overcoming some of the limitations inherent to target
serum or tissues of mammalian models, but such meas- choice), the development of adequately characterized
urements are more challenging to make in microscopic phenotypes will be a rate-limiting step in zebrafish
zebrafish, especially for high-throughput applications. chemical screens. It will also be important to ensure
Although some physicochemical attributes of com- that the precise details of each screen are well docu-
pounds, especially logP, have been shown to be predic- mented, as numerous variables can influence screen
tive of drug uptake in zebrafish, predictions are still output. These include the well volume, timing and
imperfect. As a consequence, false negatives are possible mode of exposure, temperature, lighting, pH, assay sen-
in zebrafish screens, whereby a compound that could sitivity and specificity, and linearity of assay response
be a good drug candidate fails to produce a biological characteristics. As experience accumulates, assays
effect owing to poor uptake. Generally speaking, such will hopefully become standardized, and more formal
relationships with other preclinical animal models and integration of the zebrafish with traditional approaches
with quantitative metrics of human efficacy or toxicity to drug development will help to realize this vision of
will be established. medicines that empirically balance efficacy and toxicity,
A new scale and efficiency of drug discovery is nec- facilitating the discovery of new classes of therapeutics.
essary to move towards the individualized preventive
therapeutics that medicine will demand in the future. Note added in proof
To achieve these ends, diseases must be stratified and While this review was going to press, several important
specific drugs must be tailored for each new disease zebrafish chemical screens were published, including
entity. As we intervene earlier in disease, we will also screens for modifiers of hedgehog signalling102, cell migra-
require nuanced drug discovery with the goal of pathway tion103, liver tumorigenesis104, marrow engraftment105, and
normalization rather than target inhibition. Successful pancreatic -cell mass106.
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discovered? Nat. Rev. Drug Discov. 10, 507519 the vertebrate cardiac conduction system. PLoS Biol. Chan,E.C. An investigation of the bioactivation
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& Bache,R.J. Effects of amiodarone with and without tool for analysing the shape and performance of the 24, 5870 (2014).
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