15 years of zebrafish chemical screening

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Highlights

  • More than 60 zebrafish chemical screens have been reported since 2000.

  • We summarize the current practices in design of high-throughput zebrafish screens.

  • We summarize the types of phenotypic endpoints that have been used.

  • We describe the impact of zebrafish on chemical biology.

  • We highlight interesting examples of how mechanisms of action have been discovered.

In 2000, the first chemical screen using living zebrafish in a multi-well plate was reported. Since then, more than 60 additional screens have been published describing whole-organism drug and pathway discovery projects in zebrafish. To investigate the scope of the work reported in the last 14 years and to identify trends in the field, we analyzed the discovery strategies of 64 primary research articles from the literature. We found that zebrafish screens have expanded beyond the use of developmental phenotypes to include behavioral, cardiac, metabolic, proliferative and regenerative endpoints. Additionally, many creative strategies have been used to uncover the mechanisms of action of new small molecules including chemical phenocopy, genetic phenocopy, mutant rescue, and spatial localization strategies.

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.

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