Chemical Screening In Zebrafish Research Articles

Orthogonal Drug Pooling Enhances Phenotype-Based Discovery of Ocular Antiangiogenic Drugs in Zebrafish Larvae.

Unbiased screening of large randomized chemical libraries in vivo is a powerful tool to find new drugs and targets. However, forward chemical screens in zebrafish can be time consuming and usually >99% of test compounds have no significant effect on the desired phenotype. Here, we sought to find bioactive drugs more efficiently and to comply with the 3R principles of replacement, reduction, and refinement of animals in research. We investigated if pooling of drugs to simultaneously test 8–10 compounds in zebrafish larvae can increase the screening efficiency of an established assay that identifies drugs inhibiting developmental angiogenesis in the eye. In a phenotype-based screen, we tested 1,760 small molecule compounds from the ChemBridge DIVERSet™ chemical library for their ability to inhibit the formation of distinct primary hyaloid vessels in the eye. Applying orthogonal pooling of the chemical library, we treated zebrafish embryos from 3 to 5 days post fertilization with pools of 8 or 10 compounds at 10 μM each. This reduced the number of tests from 1,760 to 396. In 63% of cases, treatment showed sub-threshold effects of <40% reduction of primary hyaloid vessels. From 18 pool hits, we identified eight compounds that reduce hyaloid vessels in the larval zebrafish eye by at least 40%. Compound 4-[4-(1H-benzimidazol-2-yl)phenoxy]aniline ranked as the most promising candidate with reproducible and dose-dependent effects. To our knowledge, this is the first report of a self-deconvoluting matrix strategy applied to drug screening in zebrafish. We conclude that the orthogonal drug pooling strategy is a cost-effective, time-saving, and unbiased approach to discover novel inhibitors of developmental angiogenesis in the eye. Ultimately, this approach may identify new drugs or targets to mitigate disease caused by pathological angiogenesis in the eye, e.g., diabetic retinopathy or age-related macular degeneration, wherein blood vessel growth and leaky vessels lead to vision impairment or clinical blindness.

Chemical screening in zebrafish for novel biological and therapeutic discovery.

Zebrafish chemical screening allows for an in vivo assessment of small molecule modulation of biological processes. Compound toxicities, chemical alterations by metabolism, pharmacokinetic and pharmacodynamic properties, and modulation of cell niches can be studied with this method. Furthermore, zebrafish screening is straightforward and cost effective. Zebrafish provide an invaluable platform for novel therapeutic discovery through chemical screening.

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.

Phenotype-driven chemical screening in zebrafish for compounds that inhibit collective cell migration identifies multiple pathways potentially involved in metastatic invasion.

ABSTRACTIn the last decade, high-throughput chemical screening has become the dominant approach for discovering novel compounds with therapeutic properties. Automated screening using in vitro or cultured cell assays have yielded thousands of candidate drugs for a variety of biological targets, but these approaches have not resulted in an increase in drug discovery despite major increases in expenditures. In contrast, phenotype-driven screens have shown a much stronger success rate, which is why we developed an in vivo assay using transgenic zebrafish with a GFP-marked migrating posterior lateral line primordium (PLLp) to identify compounds that influence collective cell migration. We then conducted a high-throughput screen using a compound library of 2160 annotated bioactive synthetic compounds and 800 natural products to identify molecules that block normal PLLp migration. We identified 165 compounds that interfere with primordium migration without overt toxicity in vivo. Selected compounds were confirmed in their migration-blocking activity by using additional assays for cell migration. We then proved the screen to be successful in identifying anti-metastatic compounds active in vivo by performing orthotopic tumor implantation assays in mice. We demonstrated that the Src inhibitor SU6656, identified in our screen, can be used to suppress the metastatic capacity of a highly aggressive mammary tumor cell line. Finally, we used CRISPR/Cas9-targeted mutagenesis in zebrafish to genetically validate predicted targets of compounds. This approach demonstrates that the migrating PLLp in zebrafish can be used for large-scale, high-throughput screening for compounds that inhibit collective cell migration and, potentially, anti-metastatic compounds.

Discovering novel neuroactive drugs through high-throughput behavior-based chemical screening in the zebrafish.

Most neuroactive drugs were discovered through unexpected behavioral observations. Systematic behavioral screening is inefficient in most model organisms. But, automated technologies are enabling a new phase of discovery-based research in central nervous system (CNS) pharmacology. Researchers are using large-scale behavior-based chemical screens in zebrafish to discover compounds with new structures, targets, and functions. These compounds are powerful tools for understanding CNS signaling pathways. Substantial differences between human and zebrafish biology will make it difficult to translate these discoveries to clinical medicine. However, given the molecular genetic similarities between humans and zebrafish, it is likely that some of these compounds will have translational utility. We predict that the greatest new successes in CNS drug discovery will leverage many model systems, including in vitro assays, cells, rodents, and zebrafish.

Hooking the big one: the potential of zebrafish xenotransplantation to reform cancer drug screening in the genomic era.

The current preclinical pipeline for drug discovery can be cumbersome and costly, which limits the number of compounds that can effectively be transitioned to use as therapies. Chemical screens in zebrafish have uncovered new uses for existing drugs and identified promising new compounds from large libraries. Xenotransplantation of human cancer cells into zebrafish embryos builds on this work and enables direct evaluation of patient-derived tumor specimens in vivo in a rapid and cost-effective manner. The short time frame needed for xenotransplantation studies means that the zebrafish can serve as an early preclinical drug screening tool and can also help personalize cancer therapy by providing real-time data on the response of the human cells to treatment. In this Review, we summarize the use of zebrafish embryos in drug screening and highlight the potential for xenotransplantation approaches to be adopted as a preclinical tool to identify and prioritize therapies for further clinical evaluation. We also discuss some of the limitations of using zebrafish xenografts and the benefits of using them in concert with murine xenografts in drug optimization.

An In Vivo Platform for Rapid High-Throughput Antitubercular Drug Discovery

Treatment of tuberculosis, like other infectious diseases, is increasingly hindered by the emergence of drug resistance. Drug discovery efforts would be facilitated by facile screening tools that incorporate the complexities of human disease. Mycobacterium marinum-infected zebrafish larvae recapitulate key aspects of tuberculosis pathogenesis and drug treatment. Here, we develop a model for rapid in vivo drug screening using fluorescence-based methods for serial quantitative assessment of drug efficacy and toxicity. We provide proof-of-concept that both traditional bacterial-targeting antitubercular drugs and newly identified host-targeting drugs would be discovered through the use of this model. We demonstrate the model's utility for the identification of synergistic combinations of antibacterial drugs and demonstrate synergy between bacterial- and host-targeting compounds. Thus, the platform can be used to identify new antibacterial agents and entirely new classes of drugs that thwart infection by targeting host pathways. The methods developed here should be widely applicable to small-molecule screens for other infectious and noninfectious diseases.

In Situ Hybridization Assay‐Based Small‐Molecule Screening in Zebrafish

In vitro biochemical and cell-based small molecule screens have been widely used to identify compounds that target specific signaling pathways. But the identified compounds frequently fail at the animal testing stage, largely due to the in vivo absorption, metabolism and toxicity of chemicals. Zebrafish has recently emerged as a vertebrate whole organism model for small molecule screening. The in vivo bioactivity and specificity of compounds are examined from the very beginning of zebrafish screens. In addition, zebrafish is suitable for chemical screens at a large scale similar to cellular assays. This protocol describes an approach for in situ hybridization (ISH)-based chemical screening in zebrafish, which, in principle, can be used to screen any gene product. The described protocol has been used to identify small molecules affecting specific molecular pathways and biological processes. It can also be adapted to zebrafish screens with different readouts.

In VivoChemical Screening for Modulators of Hematopoiesis and Hematological Diseases

In vivo chemical screening is a broadly applicable approach not only for dissecting genetic pathways governing hematopoiesis and hematological diseases, but also for finding critical components in those pathways that may be pharmacologically modulated. Both high-throughput chemical screening and facile detection of blood-cell-related phenotypes are feasible in embryonic/larval zebrafish. Two recent studies utilizing phenotypic chemical screens in zebrafish have identified several compounds that promote hematopoietic stem cell formation and reverse the hematopoietic phenotypes of a leukemia oncogene, respectively. These studies illustrate efficient drug discovery processes in zebrafish and reveal novel biological roles of prostaglandin E2 in hematopoietic and leukemia stem cells. Furthermore, the compounds discovered in zebrafish screens have become promising therapeutic candidates against leukemia and included in a clinical trial for enhancing hematopoietic stem cells during hematopoietic cell transplantation.

Small Molecule Screening in Zebrafish

Small molecule screens have become useful tools in ongoing efforts to understand complex biologic systems and disease states. These screens can identify compounds that specifically affect signaling pathways, development, and disease processes. The zebrafish Danio rerio has increasingly been used as a whole organism model in which to perform such functional small molecule screens. Here, we review recent advances in screening approaches that focus on modulation of developmental processes and signaling pathways, along with suppressor screens of disease models. The identification of small molecule targets continues to be a challenge, and several recent approaches to this problem are discussed. The promise of chemical screens in zebrafish to identify novel biologic probes and therapeutic compounds continues to drive this rapidly growing field.

Small molecule screening in zebrafish: an in vivo approach to identifying new chemical tools and drug leads

In the past two decades, zebrafish genetic screens have identified a wealth of mutations that have been essential to the understanding of development and disease biology. More recently, chemical screens in zebrafish have identified small molecules that can modulate specific developmental and behavioural processes. Zebrafish are a unique vertebrate system in which to study chemical genetic systems, identify drug leads, and explore new applications for known drugs. Here, we discuss some of the advantages of using zebrafish in chemical biology, and describe some important and creative examples of small molecule screening, drug discovery and target identification.

The New Age of Chemical Screening in Zebrafish

ZebrafishVol. 7, No. 1 Special Issue: Chemical BiologyGuest Editors: Leonard I. Zon, M.D., and Randall Peterson, Ph.D.EditorialThe New Age of Chemical Screening in ZebrafishPublished Online:26 Apr 2010https://doi.org/10.1089/zeb.2010.9996AboutSectionsView articleView Full TextPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"The New Age of Chemical Screening in Zebrafish." , 7(1), p. 1FiguresReferencesRelatedDetailsCited ByAssessing the Morphological and Behavioral Toxicity of Catechol Using Larval Zebrafish20 July 2022 | International Journal of Molecular Sciences, Vol. 23, No. 14What Zebrafish and Nanotechnology Can Offer for Cancer Treatments in the Age of Personalized Medicine30 April 2022 | Cancers, Vol. 14, No. 9Modeling Cancer Using Zebrafish Xenografts: Drawbacks for Mimicking the Human Microenvironment27 August 2020 | Cells, Vol. 9, No. 9Reverse genetic screen reveals that Il34 facilitates yolk sac macrophage distribution and seeding of the brain8 March 2019 | Disease Models & Mechanisms, Vol. 12, No. 3A Modular Millifluidic Homeostatic Imaging Plate for Imaging of Larval Zebrafish Timo Friedrich, Alon M. Douek, Celia Vandestadt, Donald Wlodkowic, and Jan Kaslin23 April 2019 | Zebrafish, Vol. 16, No. 1Zebrafish bio-assay guided isolation of human acetylcholinesterase inhibitory trans-tephrostachin from Tephrosia purpurea (L.) PersNeuroscience Letters, Vol. 687Single-cell functional and chemosensitive profiling of combinatorial colorectal therapy in zebrafish xenografts23 August 2017 | Proceedings of the National Academy of Sciences, Vol. 114, No. 39Fish to Learn: Insights into Blood Development and Blood Disorders from Zebrafish Hematopoiesis13 April 2016 | Human Gene Therapy, Vol. 27, No. 4Endoderm specification and liver developmentA homeostatic, chip-based platform for zebrafish larvae immobilization and long-term imagingZebrafish xenotransplantation as a tool for in vivo cancer study10 April 2015 | Familial Cancer, Vol. 14, No. 3Switching to zebrafish neurobehavioral models: The obsessive–compulsive disorder paradigmEuropean Journal of Pharmacology, Vol. 759Mmp17b Is Essential for Proper Neural Crest Cell Migration In Vivo1 October 2013 | PLoS ONE, Vol. 8, No. 10Neuroprotective Effect of Zinc Chelator DEDTC in a Zebrafish (Danio rerio) Model of Hypoxic Brain Injury16 April 2013 | Zebrafish, Vol. 10, No. 1A larval zebrafish model of bipolar disorder as a screening platform for neuro-therapeuticsBehavioural Brain Research, Vol. 233, No. 2EuFishBioMed (COST Action BM0804): A European Network to Promote the Use of Small Fishes in Biomedical Research20 June 2012 | Zebrafish, Vol. 9, No. 2Distinct models of induced hyperactivity in zebrafish larvaeBrain Research, Vol. 1449Zebrafish embryos as an alternative to animal experiments—A commentary on the definition of the onset of protected life stages in animal welfare regulationsReproductive Toxicology, Vol. 33, No. 2Small molecule screening identifies targetable zebrafish pigmentation pathways22 February 2012 | Pigment Cell & Melanoma Research, Vol. 25, No. 2Mutation of zebrafish dihydrolipoamide branched-chain transacylase E2 results in motor dysfunction and models maple syrup urine diseaseDisease Models & Mechanisms, Vol. 5, No. 2Disruption of Eaat2b, a glutamate transporter, results in abnormal motor behaviors in developing zebrafishDevelopmental Biology, Vol. 362, No. 2Automated Zebrafish Chorion Removal and Single Embryo Placement: Optimizing Throughput of Zebrafish Developmental Toxicity ScreensSLAS Technology, Vol. 17, No. 1Chemical screen identifies FDA-approved drugs and target pathways that induce precocious pancreatic endocrine differentiation14 November 2011 | Proceedings of the National Academy of Sciences, Vol. 108, No. 48Zebrafish for the Study of the Biological Effects of Nicotine8 March 2011 | Nicotine & Tobacco Research, Vol. 13, No. 5Genetic inducible fate mapping in larval zebrafish reveals origins of adult insulin-producing β-cellsDevelopment, Vol. 138, No. 4Endoderm Specification, Liver Development, and Regeneration Volume 7Issue 1Mar 2010 InformationCopyright 2010, Mary Ann Liebert, Inc.To cite this article:The New Age of Chemical Screening in Zebrafish.Zebrafish.Mar 2010.1-1.http://doi.org/10.1089/zeb.2010.9996Published in Volume: 7 Issue 1: April 26, 2010PDF download

Swimming into the Future of Drug Discovery: In Vivo Chemical Screens in Zebrafish

In recent years in vivo chemical screening in zebrafish has emerged as a rapid and efficient method to identify lead compounds that modulate specific biological processes. By performing primary screening in vivo, the bioactivity, toxicity, and off-target side effects are determined from the onset of drug development. A recent study demonstrates that in vivo screening can be used successfully to perform structure-activity relationship (SAR) studies. This work validates the zebrafish as an effective model for not only drug discovery but also drug optimization.

Antipsychotics produce locomotor impairment in larval zebrafish

Zebrafish has been a favored vertebrate genetic model organism for studying developmental processes. It also holds a great potential for understanding the genetic basis of behavior and associated behavioral disorders. Despite such potential, their use in the study of behavior is greatly under-explored. It is well known that multiple classes of drugs used to treat psychiatric diseases produce extrapyramidal side (EPS) effects and consequent movement disorders in humans. The underlying molecular causes of these drug-induced movement disorders are poorly understood. Here we report that zebrafish treated with the antipsychotics fluphenazine and haloperidol (both of which can induce severe EPS in humans) develop movement defects. In contrast, another antipsychotic olanzapine, which produces mild to little EPS in humans, leads to minimal movement defects in zebrafish. These results establish a rapid assay system in which the effects of EPS-inducing agents can be assessed. Thus, future genetic screening in zebrafish shall identify genes and pathways that elucidate drug-induced movement disorder in human as well as provide insights into the brain control of locomotor activity. Future chemical screening in zebrafish may act as a preclinical test for the EPS effect of certain drugs, as well as a test used to researching drugs made to counteract the effects of EPS.