TECHNOLOGIES

DRUG DISCOVERY

TODAY

Drug Discovery Today: Technologies Vol. 1, No. 1 2004

Editors-in-Chief

Kelvin Lam – Pfizer, Inc., USA

Henk Timmerman – Vrije Universiteit, The Netherlands

Target identification

Discovery of therapeutic targets byphenotype-based zebrafish screensRandall T. PetersonDevelopmental Biology Laboratory, Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, 149 13th Street, Charlestown,

MA 02129, USA

The easy identification of phenotypes in the transpar-

ent zebrafish embryo has enabled numerous genetic,

antisense morpholino oligonucleotide, and small mole-

cule screens. Can zebrafish screens also be used for

unbiased discovery of novel drug targets?

E-mail address: peterson@cvrc.mgh.harvard.eduURL: http://www.mgh.harvard.edu/cvrc/crvc/peterson.html.

1740-6749/$ � 2004 Elsevier Ltd. All rights reserved. DOI: 10.1016/j.ddtec.2004.07.002

Section Editors:Wolfgang Fischer, Rob Hooft, Michael Walker

The Zebrafish is the simplest vertebrate that is widely used for study.This model organism is the only vertebrate that can be screened in 96-

or even 384-well format for the high-throughput analysis of compoundsor genetic modifiers. Moreover, the animals are optically transparent

and easily absorb chemicals during their early phases of development.Dr. Peterson, one of the foremost experts in the area of forward

chemical genomics and genetic screens in Zebrafish, explains the prosand cons of this model organism for the discovery of small molecules

and their genetic targets.

normal biological processes and disease pathology, it remains

Introduction

Identifying novel therapeutic targets remains a bottleneck in

drug discovery. Despite a wealth of molecular data about

difficult to predict which targets will permit effective reversal

of a disease phenotype. It is also difficult to predict adverse

consequences of targeting a particular pathway. As a result,

discovery of novel, validated therapeutic targets is a slow and

uncertain process. Most new drugs are directed toward a small

number of targets, while many potential therapeutic targets

are probably going untapped.

How can novel targets be identified in the absence of

detailed information about a disease mechanism? Unbiased,

phenotype-based screens represent one promising approach.

Rather than relying upon ad hoc selection and testing of

individual target candidates, large-scale phenotype-based

screens can in principle be used to identify genetic, epigenetic,

or chemical perturbations that modify a disease phenotype.

The genes targeted by these perturbations would then be

potential targets for drug discovery. This concept has

spawned numerous phenotype-based assays performed in

cultured cells. However, although cultured cells might serve

as acceptable surrogates for some cellular processes, they are

generally inadequate models for diseases that involve multi-

ple cell types, organs, or physiological systems. The ideal

system for screening for novel therapeutic targets would

have the potential not only to identify potential targets

within a particular cell but also to identify targets through-

out the organism that are capable of modifying the disease

phenotype.

The zebrafish

The zebrafish has emerged as a powerful tool for phenotype-

based screens [1–3]. Its genome and body plan are similar to

other vertebrates, but its optical transparency and external

development make real time observation of its internal

organs simple. Numerous zebrafish disease models ranging

from congenital heart defects to cancers have been developed

as reviewed elsewhere [4–6], and the zebrafish is genetically

and pharmacologically similar to humans [7,8].

The ease with which zebrafish phenotypes can be identified

has resulted in their use in numerous genetic and chemical

screens [1,9]. And, because screening can be performed in the

whole organism, perturbation of potential therapeutic targets

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Drug Discovery Today: Technologies | Target identification Vol. 1, No. 1 2004

Figure 1. Use of zebrafish for identifying novel drug targets. Numerous

human diseases can be modeled in the zebrafish. The disease models can

then be subjected to large-scale screens to identify genetic mutations,

antisense morpholino oligonucleotides or small molecules that suppress

the disease phenotype. Once a perturbation is discovered that prevents

disease development, the perturbed gene or gene product can be

considered a target for drug discovery.

by mutations or small molecules reveals the effects of such

perturbations on the integrated physiology of the entire

organism. In this review, genetic, morpholino oligonucleo-

tide, and small molecule screens using zebrafish will be dis-

cussed in light of their potential for identifying novel

therapeutic targets (see Fig. 1).

Genetic screens

Genetic screens were the first demonstration of the facility

with which large-scale phenotype-based screens can be car-

ried out in zebrafish [10,11]. In the past decade, numerous

screens have been performed in which random chemical or

insertional mutagenesis has been followed by large-scale

phenotyping to identify thousands of distinct zebrafish

mutations. Mutations affecting virtually every organ and

observable biological process have been identified, and in

many cases the affected genes have been cloned, establishing

new links between genes and the processes they control.

Similar screens are now being carried out in mice [12], but

they have proven to be more expensive, slower, and more

laborious than zebrafish genetic screens.

How might zebrafish genetic screens contribute to the

process of identifying novel drug targets? In principle, they

make it possible to scan the entire genome for targets that,

50 www.drugdiscoverytoday.com

when disrupted, modify a disease process, and they show

what the effect of this disruption is in the context of a whole

organism. However, most zebrafish screens to date have

focused on developmental biology, not disease biology. Con-

sequently, the most well characterized mutations cause early,

lethal developmental defects, and the relationship, if any,

between these phenotypes and adult diseases is not always

obvious.

Fortuitously, some of the developmental mutations dis-

covered thus far might have significance as disease models.

For example, the heartstrings mutation, which causes heart

and pectoral fin defects in zebrafish, resides in the TBX5 gene,

mutation of which causes heart and limb defects in human

Holt-Oram syndrome [13]. Similarly, mutation of the activin

receptor-like kinase 1 gene causes arteriovenous malforma-

tions associated with the violet beauregarde phenotype in fish

and type 2 hereditary hemorrhagic telangiectasia in humans

[14]. Mutations in the titin gene cause cardiomyopathy in

zebrafish and humans [15,16]. These examples illustrate the

possibility of identifying mutations in zebrafish that contri-

bute to disease phenotypes with human disease counterparts.

As zebrafish screens for early developmental phenotypes are

complemented by screens directed at disease-related embryo-

nic and adult phenotypes, the number of zebrafish mutants

with obvious disease significance is likely to be increased.

Examples of zebrafish genetic screens directed at specific

disease-related processes, including lipid possessing and tis-

sue regeneration, have been reported [17,18].

An additional limitation of the zebrafish genetic screens

carried out thus far is that they have typically sought to

identify disruptors of normal biological processes. Although

this approach has identified novel entrance points into dis-

ease processes, the mutated gene products might not make

good drug targets, because their inhibition might be expected

to cause, rather than reverse, a disease state. Arguably, ther-

apeutic target discovery would be better served by genetic

suppressor screens in which mutations are identified that

confer resistance to the development of disease. A mutation

that suppressed or conferred resistance to disease would be an

obvious target for further drug discovery efforts. An example

of this approach has just been reported. In a forward genetic

suppressor screen, 1575 mice with genetic thrombocytopenia

were screened for genetic suppressors that ameliorate the

disease phenotype [19]. Two mutant alleles of c-Myb were

identified, suggesting that c-Myb can be an important drug

target for treatment of thrombocytopenia. Although this

suppressor screen was carried out in mice, it should be

possible to carry out similar screens with greater ease and

on a larger scale using zebrafish.

Morpholino oligonucleotide screens

Morpholino oligonucleotides are antisense oligonucleotides

that are chemically modified to increase stability. They can be

Vol. 1, No. 1 2004 Drug Discovery Today: Technologies | Target identification

designed to hybridize to the translation initiation or splicing

acceptor/donor sites of specific mRNAs, and they cause a

robust knockdown of gene function when injected into

zebrafish embryos [20]. Morpholinos have been used exten-

sively to determine the effects of knocking down individual

genes, and the idea of extending the approach to large-scale

morpholino screens has been proposed. In analogy to the

forward genetic screens that have been performed, morpho-

lino screens could seek to identify gene knockdowns that

cause interesting defects in wild-type zebrafish. This screen-

ing approach has been demonstrated on a small-scale in

Xenopus tropicalis [21] and Ciona intestinalis [22] and is appar-

ently underway using zebrafish at several companies and

academic laboratories.

From the perspective of therapeutic target discovery, mor-

pholino screens directed at preventing disease development

might be of greater value than morpholino screens using

wild-type embryos. For example, many disease states can

be induced in zebrafish by genetic mutations, pharmacolo-

gical inhibitors, or by infectious agents. A large-scale mor-

pholino screen could identify genes that, when knocked

down, prevent or slow the development of the disease state

without causing other adverse effects. Such genes could

quickly become targets for drug discovery.

One limitation of the morpholino screening approach has

been the availability of morpholino oligonucleotides. Pur-

chase of morpholinos has only been available through Gene

Tools, LLC (www.gene-tools.com), and their pricing has

made large-scale screens prohibitively expensive. However,

other synthetically stabilized antisense oligonucleotides are

being developed, including peptide nucleic acids, locked

nucleic acids, and cyclohexene nucleic acids [23]. These

competing technologies are likely to reduce the price of

antisense oligonucleotides. Gene Tools, LLC has also recently

announced the availability of a morpholino library contain-

ing antisense oligonucleotides targeting hundreds of genes.

As the size of such libraries increases and prices fall, morpho-

lino screening should become an accessible approach for

both academic and industry laboratories.

Another limitation of morpholino screens is that morpho-

linos appear to be most effective during the first two to four

days postfertilization [24]. The limited temporal utility of

morpholinos precludes their use for studying normal or dis-

ease processes that do not occur in the developing embryo. By

contrast, genetic and chemical screens can theoretically be

performed at any developmental stage, although in practice,

adult screens can be cumbersome because of increased diffi-

culty of phenotyping and demands for increased space and

small molecule quantities.

Small molecule screens

Zebrafish embryos can be generated by the thousand and

are small enough to be distributed into the wells of 96- or

384-well plates for high-throughput small molecule screens.

Most drug-like small molecules added to the water sur-

rounding zebrafish embryos are readily absorbed by the

embryo [8], and the transparency of the zebrafish makes

it possible to efficiently assess the effects of thousands of

individual compounds on the organism’s morphology and

physiology.

Two types of zebrafish small molecule screen have been

carried out. The first type is a simple developmental screen,

analogous to the genetic screens described above, in

which wild-type embryos are exposed to small molecules

from a chemical library, and small molecules that induce

developmental defects are identified. Screens of this type

have produced dozens of compounds that cause specific

defects in hematopoesis, cardiac physiology, embryonic

patterning, pigmentation, and morphogenesis of the heart,

brain, ear, and eye [25–29]. Many of the compounds dis-

covered appear to be specific, with phenotypes comparable

to those caused by specific genetic mutations, and some

of the compounds are potent, with EC50s in the low nano-

molar range [30].

A second type of zebrafish small molecule screen is the

modifier screen in which small molecules capable of mod-

ifying a disease phenotype are identified. The feasibility of

this approach was recently demonstrated by the identifica-

tion of a novel class of compounds capable of suppressing

the gridlock mutation [31]. Zebrafish gridlock mutants exhibit

a dysmorphogenesis of the aorta that prevents circulation to

the trunk and tail. Because of the location of the aortic defect

and the collateral vessels that often form to circumvent the

obstruction, gridlock mutants have been considered to be a

model of human coarctation of the aorta [32]. Gridlock

mutants were exposed to 5000 compounds from a diverse

small molecule library. Two structurally related compounds

were identified that completely restore gridlock mutants to

normal without causing additional developmental defects

[31].

Beyond their ability to suppress the gridlock phenotype in

zebrafish, the gridlock suppressor compounds promote tubu-

logenesis in cultured human endothelial cells, suggesting that

the compounds can be vasculogenic in fish and in mammals

[31]. This finding is consistent with the observation that many

drugs have similar activities in zebrafish and humans [7,8].

Therefore, it might be reasonable to imagine that some com-

pounds that suppress disease phenotypes in zebrafish might

have direct utility as lead compounds for human therapies.

More probable, however, is the possibility that small molecule

suppressor screens will reveal novel drug targets and mechan-

isms by which diseases can be modified. These targets could

then be used for conventional drug development.

To realize the hope that zebrafish small molecule screens

can be used to identify novel drug targets, it will be important

to streamline the process of determining the mechanisms of

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Drug Discovery Today: Technologies | Target identification Vol. 1, No. 1 2004

Table 1. Comparison summary table

Genetic screens Morpholino oligonucleotide screens Small molecule screens

Pros Well-established protocols Rapid and systematic Inexpensive, simple, and flexible

Specific, reproducible phenotypes Provide immediate link between gene and phenotype ’Hits’ can be useful lead compounds

Cons Require large numbers of zebrafish Expensive Small molecules can be non-specific

Labor intensive Extensive libraries do not exist Identifying small molecule targets is not yet routine

Positional cloning is time consuming Knockdown only effective during early development

action of small molecules. Thus far, it has proven easier to

identify small molecules that produce a desired phenotype in

zebrafish than to determine the mechanisms by which those

compounds function.

Presently, identifying binding partners for small molecules

whose mechanisms of action are unknown is an ad hoc pro-

cess. Many drugs in current use were originally identified

by the phenotypes they cause, and their targets were initially

unclear. These drugs include classics such as digitoxin and

coumadin, and more recent discoveries such as ezetimibe

(Zetia, Schering-Plough, Kenilworth, NJ, USA, www.schering-

plough.com) and the anti-diabetic thiazolidinediones [33–36]

(e.g. pioglitazone, Takeda Chemical Industries, Japan, www.

takeda.co.jp). The targets of these and other drugs were

eventually identified through a variety of approaches, in-

cluding affinity chromatography and expression cloning.

Although ad hoc application of these biochemical approa-

ches might continue to be successful at identifying individual

small molecule targets, methodologies that are more systema-

tic and robust need to be developed if phenotype-based

screens are to become a widespread approach for identifying

drug targets.

Efforts are underway to make small molecule target iden-

tification systematic and generalized. For example, a zebra-

fish small molecule screen has been performed using a tagged

triazine library [27]. Every member of this library possesses

the same functionalized linker that can be used to efficiently

attach the molecule to solid support. This approach allows for

facile preparation of affinity matrices from compounds that

are active in the assay. The hope is that technologies such as

this one will facilitate and systematize the process of moving

Related articles

Langheinrich, U. (2003) Zebrafish: a new model on the pharmaceutical

catwalk. Bioessays 25 (9), 904–912

MacRae, C.A. and Peterson, R.T. (2003) Zebrafish-based small molecule

discovery. Chem. Biol. 10 (10), 901–908

Pichler, F.B. et al. (2003) Chemical discovery and global gene expression

analysis in zebrafish. Nat. Biotechnol. 21 (8), 879–883

Rubenstein, A.L. (2003) Zebrafish: from disease modeling to drug

discovery. Curr. Opin. Drug Discov. Dev. 6 (2), 218–223

Stern, H.M. and Zon, L.I. (2003) Cancer genetics and drug discovery in

the zebrafish. Nat. Rev. Cancer 3 (7), 533–539

52 www.drugdiscoverytoday.com

from initial compound discovery to target identification.

An appropriately designed disease suppressor screen would

therefore identify not only potential lead compounds but

quickly lead to identification of novel therapeutic targets.

Conclusions

Tens of thousands of gene products identified by the human

genome project hold potential as therapeutic drug targets,

and yet target identification remains a bottleneck in drug

discovery. In fact, the cataloguing of the genome underscores

the complexity of the organism and the difficulty of predict-

ing the organismal impact of disrupting a specific molecular

target. In the effort to discover new drug targets, one would

ideally like to be able to modify every gene product indivi-

dually and identify those that produce the desired effect.

Such an effort requires an assay system that enables (1)

modeling of complex physiological processes, (2) systematic

disruption of every gene product, and (3) rapid assessment of

the effects of each disruption. The zebrafish meets all of these

requirements. It serves as a model of many aspects of human

development, physiology, and disease. It can be subjected to

large-scale genetic, epigenetic, and small molecule screens.

And its transparent body facilitates rapid phenotyping during

the screening process.

The three screening modalities discussed in this review

(genetic, morpholino oligonucleotide, and small molecule

screening) all have potential to identify novel therapeutic

targets, but their strengths and weaknesses differ (see Table 1).

Genetic screens are the most well-established and typically

produce specific, reproducible phenotypes, but they require

large numbers of zebrafish and are labor intensive. Positional

cloning of mutants can also be time consuming. Morpholino

screens are rapid, systematic, and provide an immediate link

between phenotype and the disrupted gene. However, mor-

pholinos are expensive, and extensive libraries do not yet

exist. Furthermore, they are only effective at knocking down

gene function during the first few days of development. Small

molecule screens are inexpensive, easy to set up and perform,

and adaptable to a wide range of disease processes. Hits

from small molecule screens might also have potential as

lead compounds for therapeutic development. However, the

effects of a small molecule might be mediated through multi-

ple targets, and the process of identifying a small molecule’s

binding partner(s) is not yet routine.

Vol. 1, No. 1 2004 Drug Discovery Today: Technologies | Target identification

Until now, zebrafish screens have primarily been per-

formed by developmental biologists interested in discovering

the key regulators of embryogenesis. However, many have

recognized that the traits that make zebrafish useful as a gene

discovery tool might make it equally useful as a drug target

discovery tool. Several small companies have been built upon

this premise, and the recent selection of a zebrafish expert as

president of the Novartis Institutes for BioMedical Research

(Cambridge, MA, USA, www.nibr.novartis.com) might herald

greater awareness of the approach within the pharmaceutical

industry [37].

At this point, the utility of zebrafish for therapeutic

target discovery is unproven. It may take years to determine

whether targets identified by zebrafish screens will lead

to novel therapies. Furthermore, each of the screening meth-

odologies described in this review continues to evolve.

Genetic screens will undoubtedly be empowered by

increases in the speed with which mutant genes can be

cloned. Morpholino screens will grow in utility to the extent

that morpholino oligonucleotide libraries become available,

and small molecule screens will benefit from technologies

that shorten the path between active compound and target.

The success of the zebrafish as a drug target discovery tool

will depend in part on how rapidly these methodologies

develop (see Outstanding issues). However, so long as it

remains difficult to predict which of the thousands of poten-

tial drug targets will be effective, methods for systematically

testing them all will remain appealing. In this regard, the

screening capabilities of the zebrafish make it worthy of a

careful look.

Acknowledgements

The author thanks Ashok Srinivasan for helpful comments on

the manuscript.

Outstanding issues

� Will genetic modifier screens be practical in zebrafish?

� Will the recently sequenced zebrafish genome and technologies such

as insertional mutagenesis continue to shorten the process of

positional cloning?

� Will larger collections of antisense oligonucleotides become available

and affordable?

� Will systematic tools for identifying small molecule binding partners

be developed?

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