Screening Platforms for Genetic Epilepsies-Zebrafish, iPSC-Derived Neurons, and Organoids.

Recent advances in molecular and cellular engineering, such as human cell reprogramming, genome editing, and patient-specific organoids, have provided unprecedented opportunities for investigating human disorders in both animals and human-based models at an improved pace and precision. This progress will inevitably lead to the development of innovative drug-screening platforms and new patient-specific therapeutics. In this review, we discuss recent advances that have been made using zebrafish and human-induced pluripotent stem cell (iPSC)-derived neurons and organoids for modeling genetic epilepsies. We also provide our prospective on how these models can potentially be combined to build new screening platforms for antiseizure and antiepileptogenic drug discovery that harness the robustness and tractability of zebrafish models as well as the patient-specific genetics and biology of iPSC-derived neurons and organoids.

Developing zebrafish disease models for in vivo small molecule screens.

The zebrafish is a model organism that allows in vivo studies to be performed at a scale usually restricted to in vitro studies. As such, the zebrafish is well suited to in vivo screens, in which thousands of small molecules are tested for their ability to modify disease phenotypes in zebrafish disease models. Numerous approaches have been developed for modeling human diseases in zebrafish, including mutagenesis, transgenesis, pharmacological approaches, wounding, and exposure to infectious or cancerous agents. We review the various strategies for modeling human diseases in zebrafish and discuss important considerations when developing zebrafish models for use in in vivo small molecule screens.

High-throughput screening for selective appetite modulators: A multibehavioral and translational drug discovery strategy.

How appetite is modulated by physiological, contextual, or pharmacological influence is still unclear. Specifically, the discovery of appetite modulators is compromised by the abundance of side effects that usually limit in vivo drug action. We set out to identify neuroactive drugs that trigger only their intended single behavioral change, which would provide great therapeutic advantages. To identify these ideal bioactive small molecules, we quantified the impact of more than 10,000 compounds on an extended series of different larval zebrafish behaviors using an in vivo imaging strategy. Known appetite-modulating drugs altered feeding and a pleiotropy of behaviors. Using this multibehavioral strategy as an active filter for behavioral side effects, we identified previously unidentified compounds that selectively increased or reduced food intake by more than 50%. The general applicability of this strategy is shown by validation in mice. Mechanistically, most candidate compounds were independent of the main neurotransmitter systems. In addition, we identified compounds with multibehavioral impact, and correlational comparison of these profiles with those of known drugs allowed for the prediction of their mechanism of action. Our results illustrate an unbiased and translational drug discovery strategy for ideal psychoactive compounds and identified selective appetite modulators in two vertebrate species.

Chemical Screening in Zebrafish.

Phenotypic small molecule screens in zebrafish have gained popularity as an unbiased approach to probe biological processes. In this chapter we outline basic methods for performing chemical screens with larval zebrafish including breeding large numbers of embryos, plating larval fish into multi-well dishes, and adding small molecules to these wells. We also highlight important considerations when designing and interpreting the results of a phenotypic screen and possible follow-up approaches, including popular methods used to identify the mechanism of action of a chemical compound.

Zebrafish as tools for drug discovery.

The zebrafish has become a prominent vertebrate model for disease and has already contributed to several examples of successful phenotype-based drug discovery. For the zebrafish to become useful in drug development more broadly, key hurdles must be overcome, including a more comprehensive elucidation of the similarities and differences between human and zebrafish biology. Recent studies have begun to establish the capabilities and limitations of zebrafish for disease modelling, drug screening, target identification, pharmacology, and toxicology. As our understanding increases and as the technologies for manipulating zebrafish improve, it is hoped that the zebrafish will have a key role in accelerating the emergence of precision medicine.

Pharmacological HIF2α inhibition improves VHL disease-associated phenotypes in zebrafish model.

Patients with a germline mutation in von Hippel-Lindau (VHL) develop renal cell cancers and hypervascular tumors of the brain, adrenal glands, and pancreas as well as erythrocytosis. These phenotypes are driven by aberrant expression of HIF2α, which induces expression of genes involved in cell proliferation, angiogenesis, and red blood cell production. Currently, there are no effective treatments available for VHL disease. Here, using an animal model of VHL, we report a marked improvement of VHL-associated phenotypes following treatment with HIF2α inhibitors. Inactivation of vhl in zebrafish led to constitutive activation of HIF2α orthologs and modeled several aspects of the human disease, including erythrocytosis, pathologic angiogenesis in the brain and retina, and aberrant kidney and liver proliferation. Treatment of vhl(-/-) mutant embryos with HIF2α-specific inhibitors downregulated Hif target gene expression in a dose-dependent manner, improved abnormal hematopoiesis, and substantially suppressed erythrocytosis and angiogenic sprouting. Moreover, pharmacologic inhibition of HIF2α reversed the compromised cardiac contractility of vhl(-/-) embryos and partially rescued early lethality. This study demonstrates that small-molecule targeting of HIF2α improves VHL-related phenotypes in a vertebrate animal model and supports further exploration of this strategy for treating VHL disease.

15 years of zebrafish chemical screening.

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.

Chemical and metabolomic screens identify novel biomarkers and antidotes for cyanide exposure.

Exposure to cyanide causes a spectrum of cardiac, neurological, and metabolic dysfunctions that can be fatal. Improved cyanide antidotes are needed, but the ideal biological pathways to target are not known. To understand better the metabolic effects of cyanide and to discover novel cyanide antidotes, we developed a zebrafish model of cyanide exposure and scaled it for high-throughput chemical screening. In a screen of 3120 small molecules, we discovered 4 novel antidotes that block cyanide toxicity. The most potent antidote was riboflavin. Metabolomic profiling of cyanide-treated zebrafish revealed changes in bile acid and purine metabolism, most notably by an increase in inosine levels. Riboflavin normalizes many of the cyanide-induced neurological and metabolic perturbations in zebrafish. The metabolic effects of cyanide observed in zebrafish were conserved in a rabbit model of cyanide toxicity. Further, humans treated with nitroprusside, a drug that releases nitric oxide and cyanide ions, display increased circulating bile acids and inosine. In summary, riboflavin may be a novel treatment for cyanide toxicity and prophylactic measure during nitroprusside treatment, inosine may serve as a biomarker of cyanide exposure, and metabolites in the bile acid and purine metabolism pathways may shed light on the pathways critical to reversing cyanide toxicity.

An in vivo zebrafish screen identifies organophosphate antidotes with diverse mechanisms of action.

Organophosphates are a class of highly toxic chemicals that includes many pesticides and chemical weapons. Exposure to organophosphates, either through accidents or acts of terrorism, poses a significant risk to human health and safety. Existing antidotes, in use for over 50 years, have modest efficacy and undesirable toxicities. Therefore, discovering new organophosphate antidotes is a high priority. Early life stage zebrafish exposed to organophosphates exhibit several phenotypes that parallel the human response to organophosphates, including behavioral deficits, paralysis, and eventual death. Here, we have developed a high-throughput zebrafish screen in a 96-well plate format to find new antidotes that counteract organophosphate-induced lethality. In a pilot screen of 1200 known drugs, we identified 16 compounds that suppress organophosphate toxicity in zebrafish. Several in vitro assays coupled with liquid chromatography/tandem mass spectrometry-based metabolite profiling enabled determination of mechanisms of action for several of the antidotes, including reversible acetylcholinesterase inhibition, cholinergic receptor antagonism, and inhibition of bioactivation. Therefore, the in vivo screen is capable of discovering organophosphate antidotes that intervene in distinct pathways. These findings suggest that zebrafish screens might be a broadly applicable approach for discovering compounds that counteract the toxic effects of accidental or malicious poisonous exposures.

Systematic approaches to toxicology in the zebrafish.

As the current paradigms of drug discovery evolve, it has become clear that a more comprehensive understanding of the interactions between small molecules and organismal biology will be vital. The zebrafish is emerging as a complement to existing in vitro technologies and established preclinical in vivo models that can be scaled for high-throughput. In this review, we highlight the current status of zebrafish toxicology studies, identify potential future niches for the model in the drug development pipeline, and define the hurdles that must be overcome as zebrafish technologies are refined for systematic toxicology.

Chemical informatics and target identification in a zebrafish phenotypic screen.

Target identification is a core challenge in chemical genetics. Here we use chemical similarity to computationally predict the targets of 586 compounds that were active in a zebrafish behavioral assay. Among 20 predictions tested, 11 compounds had activities ranging from 1 nM to 10,000 nM on the predicted targets. The roles of two of these targets were tested in the original zebrafish phenotype. Prediction of targets from chemotype is rapid and may be generally applicable.

Designing zebrafish chemical screens.

The zebrafish is proving to be highly amenable to in vivo small molecule screening. With a growing number of screens successfully completed, a rich interface is being created between disciplines that have historically used zebrafish (e.g., embryology and genetics) and disciplines focused on small molecules (e.g., chemistry and pharmacology). Navigating this interface requires consideration of the unique demands of conducting high-throughput screening in vivo. In this chapter, we discuss design elements of successful zebrafish screens, established screening methods, and approaches for mechanism of action studies following discovery of novel small molecules. These methods are enabling the zebrafish to have an increasingly positive impact on biomedical research and drug development.

Using the zebrafish photomotor response for psychotropic drug screening.

Because psychotropic drugs affect behavior, we can use changes in behavior to discover psychotropic drugs. The original prototypes of most neuroactive medicines were discovered in humans, rodents and other model organisms. Most of these discoveries were made by chance, but the process of behavior based drug discovery can be made more systematic and efficient. Fully automated platforms for analyzing the behavior of embryonic zebrafish capture digital video recordings of animals in each individual well of a 96-well plate before, during, and after a series of stimuli. To analyze systematically the thousands of behavioral recordings obtained from a large-scale chemical screen, we transform these behavioral recordings into numerical barcodes, providing a concise and interpretable summary of the observed phenotypes in each well. Systems-level analysis of these behavioral phenotypes generate testable hypotheses about the molecular mechanisms of poorly understood drugs and behaviors. By combining the in vivo relevance of behavior-based phenotyping with the scale and automation of modern drug screening technologies, systematic behavioral barcoding represents a means of discovering psychotropic drugs and provides a powerful, systematic approach for unraveling the complexities of vertebrate behavior.

Dehydro-alpha-lapachone, a plant product with antivascular activity.

Antivascular agents have become a standard of treatment for many malignancies. However, most of them target the VEGF pathway and lead to refractoriness. To improve the diversity of options for antivascular therapy, we applied a high-throughput screen for small molecules targeting cell adhesion. We then assayed the resulting antiadhesion hits in a transgenic zebrafish line with endothelial expression of EGFP (Tg(fli1:EGFP)(y1)) to identify nontoxic molecules with antivascular activity selective to neovasculature. This screen identified dehydro-α-lapachone (DAL), a natural plant product. We found that DAL inhibits vessel regeneration, interferes with vessel anastomosis, and limits plexus formation in zebrafish. Furthermore, DAL induces vascular pruning and growth delay in orthotopic mammary tumors in mice. We show that DAL targets cell adhesion by promoting ubiquitination of the Rho-GTPase Rac1, which is frequently up-regulated in many different cancers.

A forward chemical screen in zebrafish identifies a retinoic acid derivative with receptor specificity.

BACKGROUND: Retinoids regulate key developmental pathways throughout life, and have potential uses for differentiation therapy. It should be possible to identify novel retinoids by coupling new chemical reactions with screens using the zebrafish embryonic model. PRINCIPAL FINDINGS: We synthesized novel retinoid analogues and derivatives by amide coupling, obtaining 80-92% yields. A small library of these compounds was screened for bioactivity in living zebrafish embryos. We found that several structurally related compounds significantly affect development. Distinct phenotypes are generated depending on time of exposure, and we characterize one compound (BT10) that produces specific cardiovascular defects when added 1 day post fertilization. When compared to retinoic acid (ATRA), BT10 shows similar but not identical changes in the expression pattern of embryonic genes that are known targets of the retinoid pathway. Reporter assays determined that BT10 interacts with all three RAR receptor sub-types, but has no activity for RXR receptors, at all concentrations tested. CONCLUSIONS: Our screen has identified a novel retinoid with specificity for retinoid receptors. This lead compound may be useful for manipulating components of retinoid signaling networks, and may be further derivatized for enhanced activity.

Fishing for new antimicrobials.

The discovery of antibiotics and other antimicrobial agents in the 1930s is arguably the most significant therapeutic advance in medical history. Penicillin and the sulfa drugs touched off the search for and discovery of countless derivative compounds and several new antibiotic classes. However, the pace of discovery has slowed down, and there is growing appreciation that much of the low-lying fruit accessible to traditional methods of antimicrobial discovery has been harvested. Combating emerging drug-resistant strains of infectious agents may require the adoption of fresh approaches to drug target validation, small-molecule discovery and safety assessment. The recent development of several infectious disease models in zebrafish raises the possibility of a new paradigm in antimicrobial discovery.

In vivo drug discovery in the zebrafish.

The zebrafish has become a widely used model organism because of its fecundity, its morphological and physiological similarity to mammals, the existence of many genomic tools and the ease with which large, phenotype-based screens can be performed. Because of these attributes, the zebrafish might also provide opportunities to accelerate the process of drug discovery. By combining the scale and throughput of in vitro screens with the physiological complexity of animal studies, the zebrafish promises to contribute to several aspects of the drug development process, including target identification, disease modelling, lead discovery and toxicology.

Induction of reversible hemolytic anemia in living zebrafish using a novel small molecule.

We used zebrafish to screen and identify small molecules that affect the process of vertebrate hematopoietic development. Zebrafish embryos were exposed to a library of 5000 synthetic compounds and screened for defects in primitive erythropoiesis. Here, we present the characterization of hemolytic anemia induced in zebrafish by the small molecule 5115318 (3-[5-methyl-furan 2-yl]-propionic acid N'-phenyl-hydrazide). This compound is capable of generating hemoglobin aggregates and Heinz bodies in red cells in vivo only. The induced anemia is reversible and treated fish recover in about 4 days. This study shows the feasibility of using zebrafish to screen for small molecules that can modulate the specific process of erythropoiesis.