15 years of zebrafish chemical screening
Introduction
Traditional methods of small molecule drug discovery relied on trial-and-error testing of chemical compounds on phenotypic outcomes in cells or animals. This approach yielded many of the drugs currently used in the clinic today. By contrast, target-driven approaches, which seek to identify novel therapeutics based on a priori knowledge of a single biological target, have received greater emphasis in recent decades but have delivered fewer first-in-class drugs [1].
There are several possible reasons (not mutually exclusive) why phenotype-driven approaches have out-performed target-driven approaches. The first is that target driven approaches depend on selection of the correct, disease-modifying target — an uncertain proposition — whereas phenotype-driven approaches can identify disease-modifying drugs even in the absence of a validated target. Second, the most efficacious drugs may benefit from activity at multiple targets. For example, complex polygenetic disorders may require a ‘magic shotgun’ drug (one exhibiting polypharmacology) rather than a ‘magic bullet’ (one exhibiting specificity for a single target) [2]. Some of the most successful drugs in use today are known to benefit from engagement of multiple targets throughout the body. Third, small molecules derived from phenotypic screens often have been further selected for positive pharmacological properties, such as low toxicity, the ability to make it to the appropriate site(s) of action, and the ability to avoid or exploit endogenous chemical metabolizing enzymes and transporters.
Whole-organism, phenotypic screening holds several advantages over other approaches to small molecule discovery. The approach is target agonistic (therefore not mechanistically biased) and holistic (all possible targets in the organism are available). This includes targets relevant not only to disease intervention but also to chemical activation, chemical transport, toxicity and other side effects.
In 2000, it was demonstrated for the first time that a chemical screen could be carried out using live zebrafish in a 96-well plate simply by adding small amounts of compounds directly to the fish water [3]. Though simpler than humans, zebrafish are also complex vertebrates and maintain similarly elaborate mechanisms for activating or mitigating the effects of exogenous chemical substances. Although differences in pharmacological effects between zebrafish and humans certainly do exist, there are now hundreds of examples of small molecules that have conserved biological activities in fish and humans. It is therefore reasonable to expect that many bioactive compounds identified in zebrafish screens will maintain their activity in humans.
In this review, we summarize the work reported in 66 zebrafish chemical screens over the past 15 years. We start by giving a bird's-eye view of the field to give readers a feel for the scope of what has been accomplished to date. Many of the design details will likely be of interest to those contemplating setting up their own zebrafish screens. We then highlight some of the more interesting examples of the phenotypic endpoints that have been examined and methods of follow-up used to uncover mechanisms of action.
Section snippets
Zebrafish screens by the numbers
In a survey of the literature, we identified 66 primary research articles each reporting results of a zebrafish chemical screen. These range from the year 2000 to the present time and form the basis for our in-depth analysis. We believe these provide a good representation of the field, but we do not claim this list is exhaustive and apologize for any studies we may have omitted. A simple plot of the number of publications per year demonstrates that zebrafish chemical screens are becoming more
Discovery of new uses for existing bioactive compounds
The majority of small molecule libraries used to date in zebrafish chemical screens have been collections of known bioactive compounds. There are advantages to using libraries of known pharmacological entities. For one, many of these compounds are already in use in humans and therefore could be fast-tracked through any potential clinical trial period, having previously been assessed for safety. Furthermore, small molecule hits with known targets provide instant hypotheses concerning mechanisms
Determination of mechanisms of action
Mechanism of action studies can be challenging, nevertheless 23 of the screens we examined (35%, Figure 3b) included follow-up work describing the discovery of a new mechanism of action (MOA) for at least one new compound. We found that these studies fall into four distinct categories based on the initial clues provided by the structure, binding, phenotype, or site of action of each compound.
Spotlight on interesting disease stories
As zebrafish chemical screening turns 15 years old, the field is entering a new developmental stage. The discoveries of the past decade and a half are now reaching clinical trial stages. Several compounds discovered from a zebrafish chemical screen have shown preclinical promise and are being developed for clinical indications.
One of these is dorsomorphin, a small molecule inhibitor of bone morphogenetic protein (BMP) signaling. As described earlier, this compound was discovered by our group in
Future directions
As zebrafish research develops further, new knowledge and tools are becoming available that will significantly impact the range and quality of in vivo chemical screening. Recent and future advances in zebrafish research sure to have an impact will include improvements in data acquisition (particularly imaging), increased automation capability, advances in genetic manipulation and development of new phenotypic endpoints.
Conclusion
We have presented here an in-depth summary of more than 60 zebrafish chemical screens conducted over the past 15 years. We conclude that there are several consistent trends across the field. The majority of screens that have taken place were done using 96-well plates, with about three fish per well. The chemical concentration used is usually in the range of 5–25 μM, and all screens (with the exception of one conducted in adults) administer chemical compounds simply by adding the small molecules
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
This work was supported by US National Institutes of Health grant R01 MH 086867 (RTP), by the Charles and Ann Sanders MGH Research Scholar Award (RTP), and NIH training grant T32 HL 007208 (AJR). We would like to acknowledge the support of our former colleague, mentor and friend the late Dr. Kenneth D. Bloch, who played important roles in the administration of the T32 grant and in the preclinical development of dorsomorphin/LDN-193189, which we have described in this review.
References (75)
- et al.
Zebrafish screen identifies novel compound with selective toxicity against leukemia
Blood
(2012) - et al.
Identification of modulators of hair cell regeneration in the zebrafish lateral line
J Neurosci
(2012) - et al.
A zebrafish compound screen reveals modulation of neutrophil reverse migration as an anti-inflammatory mechanism
Sci Trans Med
(2014) - et al.
In vivo angiogenesis screening and mechanism of action of novel tanshinone derivatives produced by one-pot combinatorial modification of natural tanshinone mixture from salvia miltiorrhiza
PloS One
(2014) - et al.
Zebrafish model of the blood–brain barrier: morphological and permeability studies
Meth Mol Biol (Clifton, NJ)
(2011) - et al.
Multidimensional in vivo hazard assessment using zebrafish
Toxicol Sci
(2014) - et al.
Chemical suppression of a genetic mutation in a zebrafish model of aortic coarctation
Nat Biotechnol
(2004) - et al.
A phenotypic screen in zebrafish identifies a novel small-molecule inducer of ectopic tail formation suggestive of alterations in non-canonical wnt/pcp signaling
PloS One
(2013) - et al.
Zebrafish chemical screening reveals an inhibitor of dusp6 that expands cardiac cell lineages
Nat Chem Biol
(2009) - et al.
Bmp type i receptor inhibition reduces heterotopic [corrected] ossification
Nat Med
(2008)
Targeted mutagenesis in zebrafish using customized zinc-finger nucleases
Nat Prot
Highly efficient generation of heritable zebrafish gene mutations using homo- and heterodimeric talens
Nucleic Acids Res
Efficient genome editing in zebrafish using a crispr-cas system
Nat Biotechnol
Chemical and metabolomic screens identify novel biomarkers and antidotes for cyanide exposure
FASEB J: Off Publ Fed Am Soc Exp Biol
How were new medicines discovered?
Nat Rev Drug Discov
Magic shotguns versus magic bullets: Selectively non-selective drugs for mood disorders and schizophrenia
Nat Rev Drug Discov
Small molecule developmental screens reveal the logic and timing of vertebrate development
Proc Natl Acad Sci U S A
A chemical screen to identify novel inhibitors of fin regeneration in zebrafish
Zebrafish
A chemical genetic screen in zebrafish for pathways interacting with cdx4 in primitive hematopoiesis
Zebrafish
Prostaglandin e2 regulates vertebrate haematopoietic stem cell homeostasis
Nature
Dhodh modulates transcriptional elongation in the neural crest and melanoma
Nature
Automated, quantitative screening assay for antiangiogenic compounds using transgenic zebrafish
Cancer Res
Diversity-oriented synthesis of biaryl-containing medium rings using a one bead/one stock solution platform
J Am Chem Soc
Split-pool synthesis of 1,3-dioxanes leading to arrayed stock solutions of single compounds sufficient for multiple phenotypic and protein-binding assays
J Am Chem Soc
A novel microtubule destabilizing entity from orthogonal synthesis of triazine library and zebrafish embryo screening
J Am Chem Soc
Drugs that induce repolarization abnormalities cause bradycardia in zebrafish
Circulation
Facilitated forward chemical genetics using a tagged triazine library and zebrafish embryo screening
J Am Chem Soc
Unraveling tissue regeneration pathways using chemical genetics
J Biol Chem
Cannabinoid receptor 2 suppresses leukocyte inflammatory migration by modulating the jnk/c-jun/alox5 pathway
J Biol Chem
Discovering chemical modifiers of oncogene-regulated hematopoietic differentiation
Nat Chem Biol
Phenothiazines induce pp2a-mediated apoptosis in t cell acute lymphoblastic leukemia
J Clin Invest
Identification of novel inhibitors of dietary lipid absorption using zebrafish
PloS One
Novel chemical suppressors of long qt syndrome identified by an in vivo functional screen
Circulation
Rapid behavior-based identification of neuroactive small molecules in the zebrafish
Nat Chem Biol
Photochemical activation of trpa1 channels in neurons and animals
Nat Chem Biol
Zebrafish behavioral profiling links drugs to biological targets and rest/wake regulation
Science
Chemical modulation of memory formation in larval zebrafish
Proc Natl Acad Sci U S A
Cited by (221)
A systematic evidence map and bibliometric analysis of the behavioural impacts of pesticide exposure on zebrafish
2024, Environmental PollutionUveal melanoma modeling in mice and zebrafish
2024, Biochimica et Biophysica Acta - Reviews on Cancer