Zebrafish Chemical Screening Research Articles

Chemical-Induced Cleft Palate Is Caused and Rescued by Pharmacological Modulation of the Canonical Wnt Signaling Pathway in a Zebrafish Model.

Cleft palate is one of the most frequent birth defects worldwide. It causes severe problems regarding eating and speaking and requires long-term treatment. Effective prenatal treatment would contribute to reducing the risk of cleft palate. The canonical Wnt signaling pathway is critically involved in palatogenesis, and genetic or chemical disturbance of this signaling pathway leads to cleft palate. Presently, preventative treatment for cleft palate during prenatal development has limited efficacy, but we expect that zebrafish will provide a useful high-throughput chemical screening model for effective prevention. To achieve this, the zebrafish model should recapitulate cleft palate development and its rescue by chemical modulation of the Wnt pathway. Here, we provide proof of concept for a zebrafish chemical screening model. Zebrafish embryos were treated with 12 chemical reagents known to induce cleft palate in mammals, and all 12 chemicals induced cleft palate characterized by decreased proliferation and increased apoptosis of palatal cells. The cleft phenotype was enhanced by combinatorial treatment with Wnt inhibitor and teratogens. Furthermore, the expression of tcf7 and lef1 as a readout of the pathway was decreased. Conversely, cleft palate was prevented by Wnt agonist and the cellular defects were also prevented. In conclusion, we provide evidence that chemical-induced cleft palate is caused by inhibition of the canonical Wnt pathway. Our results indicate that this zebrafish model is promising for chemical screening for prevention of cleft palate as well as modulation of the Wnt pathway as a therapeutic target.

Systematic Assessment of Exposure Variations on Observed Bioactivity in Zebrafish Chemical Screening.

The embryonic zebrafish is a powerful tool for high-throughput screening of chemicals. While this model has significant potential for use in safety assessments and chemical prioritization, a lack of exposure protocol harmonized across laboratories has limited full model adoption. To assess the potential that exposure protocols alter chemical bioactivity, we screened a set of eight chemicals and one 2D nanomaterial across four different regimens: (1) the current Tanguay laboratory’s standard protocol of dechorionated embryos and static exposure in darkness; (2) exposure with chorion intact; (3) exposure under a 14 h light: 10 h dark cycle; and (4) exposure with daily chemical renewal. The latter three regimens altered the concentrations, resulting in bioactivity of the test agents compared to that observed with the Tanguay laboratory’s standard regimen, though not directionally the same for each chemical. The results of this study indicate that with the exception for the 2D nanomaterial, the screening design did not change the conclusion regarding chemical bioactivity, just the nominal concentrations producing the observed activity. Since the goal of tier one chemical screening often is to differentiate active from non-active chemicals, researchers could consider the trade-offs regarding cost, labor, and sensitivity in their study design without altering hit rates. Taken further, these results suggest that it is reasonably feasible to reach agreement on a standardized exposure regiment, which will promote data sharing without sacrificing data content.

A high throughput zebrafish chemical screen reveals ALK5 and non-canonical androgen signalling as modulators of the pkd2\u2212/\u2212 phenotype

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic cause of end-stage renal failure in humans and results from germline mutations in PKD1 or PKD2. Despite the recent approval of tolvaptan, safer and more effective alternative drugs are clearly needed to slow disease progression. As a first step in drug discovery, we conducted an unbiased chemical screen on zebrafish pkd2 mutant embryos using two publicly available compound libraries (Spectrum, PKIS) totalling 2,367 compounds to identify novel treatments for ADPKD. Using dorsal tail curvature as the assay readout, three major chemical classes (steroids, coumarins, flavonoids) were identified from the Spectrum library as the most promising candidates to be tested on human PKD1 cystic cells. Amongst these were an androgen, 5α−androstane 3,17-dione, detected as the strongest enhancer of the pkd2 phenotype but whose effect was found to be independent of the canonical androgen receptor pathway. From the PKIS library, we identified several ALK5 kinase inhibitors as strong suppressors of the pkd2 tail phenotype and in vitro cyst expansion. In summary, our results identify ALK5 and non-canonical androgen receptors as potential therapeutic targets for further evaluation in drug development for ADPKD.

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.

Abstract 745: A non-redox reactive allosteric inhibitor of MAPK phosphatases with selective toxicity to human cancer cells

Abstract Mitogen-activated protein kinase phosphatases (MKPs) are members of a family of dual specificity phosphatases (DUSPs) that regulate cellular signaling homeostasis by dephosphorylating mitogen-activated kinases, key mediators of cell death and survival. The archetype MKP, DUSP1 (also termed MKP-1/CL100), is overexpressed in ovarian, breast, and prostate cancers and found elevated in the “oncogenic signature” of RAS mutant tumor cells. DUSP1 mediates cancer cell migration and invasion, and protects cells from apoptosis by clinically used antineoplastic agents and gamma-irradiation. Despite the evidence that DUSP1 is a promising anticancer target, efforts to discover small molecule inhibitors have been largely unsuccessful. The MKP active site is shallow and contains a catalytic cysteine that is sensitive to oxidation. Hence, prior discovery screens have mostly identified redox-reactive structures with promiscuous biological activities. Using a zebrafish chemical screen, we recently discovered a new DUSP inhibitory chemotype that has no redox-reactive structural features and that is thought to bind to an allosteric site adjacent to the catalytic cavity. Structure-activity relationship studies in transgenic zebrafish identified an analog (VNK-215) that retained DUSP1 and DUSP6 inhibitory activity but was well tolerated by the developing embryo. Here we hypothesized that VNK-215 could be selectively cytotoxic to cancer cells due to its ability to inhibit DUSP1 and lack of redox-reactive features. We found that in MDA-MB-231 human breast cancer cells, VNK-215 had apoptotic and antimigratory activity at concentrations that inhibited DUSP1 in mammalian cells, and induced phosphorylation of the DUSP1 substrates ERK, p38, and JNK. Studies in primary rat hepatocytes and live zebrafish embryos revealed that VNK-215, unlike previously described DUSP inhibitors, did not induce reactive oxygen species, mitochondrial damage, or toxicity. Our data suggest that the promiscuous activities of existing DUSP inhibitors are the cause of redox reactivity rather than target inhibition, and posit that DUSP1 is a druggable anticancer target for small molecule allosteric inhibitors. Citation Format: Laura Vollmer, Lawrence Vernetti, Ahmet Bakan, Vasiliy Korotchenko, Ivet Bahar, Billy Day, Michael Tsang, Andreas Vogt. A non-redox reactive allosteric inhibitor of MAPK phosphatases with selective toxicity to human cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 745. doi:10.1158/1538-7445.AM2014-745

Estrogens Act As a Dorsal-Ventral Limiting Factor To Pattern The Hematopoietic Stem Cell Niche

While the genetic control of hematopoietic stem cell (HSC) function is increasingly understood, less is known about factors that pattern the embryonic hematopoietic niche and specify the location of HSC emergence. 17beta-estradiol (E2) and related estrogenic compounds were identified in a zebrafish chemical genetic screen as modifiers of the number of runx1+ HSCs in the Aorta-Gonad-Mesonephros region. Exposure to exogenous E2 during hematopoietic specification significantly decreased production of runx1+ HSCs by in situ hybridization (ISH) and qPCR (p<0.01); this finding was confirmed by CD41 FACS (p<0.001). Morpholino knockdown of the estrogen receptors indicated E2-mediated loss of HSCs was due to Esr2a signaling. Zebrafish that express GFP under the control of the estrogen response element (ERE:GFP) showed endogenous estrogen activity and a 1.5 fold enhancement of GFP expression by FACS upon E2 treatment (p<0.01) confirming that zebrafish embryos contain endogenous estrogen and the transcriptional machinery to respond to exogenous E2; estradiol immunoassay revealed that endogenous estrogen is preferentially located in the yolk of developing embryos. E2 treatment from 12-24 hours post fertilization (hpf), the time in which hemogenic endothelium is specified, disrupted vessel formation as shown by flk1 (kdrl) and altered the assignment of hemogenic arterial identity as assessed by the arterial markers ephrinB2a and tbx20. As arterial specification is mediated by a cascade of sonic hedgehog, VEGF, and Notch, we examined if these pathways were altered. Consistent with a defect in the specification of hemogenic endothelium, E2 decreased expression of VEGFAa and arterial Notch pathway components deltaC and notch3 by ISH and qPCR (p<0.05). Induction of VEGFAa or Notch activity via heat-shock inducible lines rescued E2-mediated hematopoietic defects, suggesting that alterations in these networks underlie observed hematopoietic phenotypes. Further, repression of endogenous estrogen by the aromatase inhibitor anastrozole or the estrogen receptor antagonist fulvestrant increased levels of runx1, the arterial markers ephrinB2a and tbx20, and VEGFAa expression. Expression of venous flt4 was correspondingly decreased, demonstrating that endogenous estrogen signaling regulates arterial/venous identity. These results suggest that maternally deposited estrogens act as a novel morphogen organizer in patterning the HSC niche during early development. It further indicates that endogenous estrogen signaling acts to limit the ventral boundary of hemogenic endothelium by antagonizing somitic VEGFAa, thereby implicating estrogen as a critical regulator of the spatially restricted induction of HSCs. To determine if E2 could impact HSCs independently of its role in specification of hemogenic endothelium, we exposed embryos to E2 from 27-34 hpf, after arterial establishment and initiation of blood flow. Here, E2 enhanced HSCs by ISH and qPCR (runx1; p<0.05). qPCR levels of the cell cycle markers cyclinD1 (p<0.01) and c-myc (p<0.001) and AGM phospho-histone H3 immunoreactivity (p<0.01) increased, suggesting E2 enhances cell cycling. To determine if the ability of E2 to enhance proliferation was restricted to HSCs or was apparent on any blood progenitor population, we assessed the impact of E2 on primitive hematopoiesis. E2 treatment from 12-36 hpf enhanced expression of gata1 by ISH, FACS, and qPCR (p<0.05) while repressing the expression of globin (p<0.05), suggesting that while E2 enhances the self-renewal of progenitors, it impairs their differentiation into mature lineages. To analyze conservation of effect, we examined hematopoietic development in murine embryos with 5-alpha reductase deficiency. These embryos fail to metabolize testosterone into dihydroxytestosterone and as a result have more testosterone available for conversion by aromatase into estradiol, leading to increased estrogen levels. Homozygote pups show fewer phenotypic HSCs by FACS; heterozygotes display increased erythrocyte progenitors but no increase in mature erythrocytes, suggesting that the impact of estrogen on HSC induction and erythropoiesis is conserved in a mammalian system and is sensitive to dose. Together, these data indicate a novel role for estrogen in the regionalization of the hematopoietic niche and identify estrogen as an enhancer of HSC proliferation and maturation. Disclosures: No relevant conflicts of interest to declare.

17beta-estradiol has a biphasic effect on the formation of hematopoietic stem cells

While the mechanisms regulating hematopoietic stem cells (HSCs) are increasingly understood, less is known about the impact of environmental factors on this process. Estrogenic compounds were isolated as modifiers of runx1 expression in a zebrafish chemical screen. Exposure to 17beta-estradiol (E2) significantly decreased runx1+ HSCs in the AGM compared to controls by in situ hybridization and qPCR (p<0.01) which was confirmed by CD41 FACS (p<0.0001). E2 treatment from 12-24 hours post-fertilization disrupted vessel formation and angiogenic sprouting, as indicated by flk1 (kdrl) and altered the assignment of ephrinb2+ arterial identity.

Advances In Zebrafish Chemical Screening Technologies

Due to several inherent advantages, zebrafish are being utilized in increasingly sophisticated screens to assess the physiological effects of chemical compounds directly in living vertebrate organisms. Diverse screening platforms showcase these advantages. Morphological assays encompassing basic qualitative observations to automated imaging, manipulation, and data-processing systems provide whole organism to subcellular levels of detail. Behavioral screens extend chemical screening to the level of complex systems. In addition, zebrafish-based disease models provide a means of identifying new potential therapeutic strategies. Automated systems for handling/sorting, high-resolution imaging and quantitative data collection have significantly increased throughput in recent years. These advances will make it easier to capture multiple streams of information from a given sample and facilitate integration of zebrafish at the earliest stages of the drug-discovery process, providing potential solutions to current drug-development bottlenecks. Here we outline advances that have been made within the growing field of zebrafish chemical screening.

Zebrafish Chemical Screening Reveals the Impairment of Dopaminergic Neuronal Survival by Cardiac Glycosides

Parkinson's disease is a neurodegenerative disorder characterized by the prominent degeneration of dopaminergic (DA) neurons among other cell types. Here we report a first chemical screen of over 5,000 compounds in zebrafish, aimed at identifying small molecule modulators of DA neuron development or survival. We find that Neriifolin, a member of the cardiac glycoside family of compounds, impairs survival but not differentiation of both zebrafish and mammalian DA neurons. Cardiac glycosides are inhibitors of Na+/K+ ATPase activity and widely used for treating heart disorders. Our data suggest that Neriifolin impairs DA neuronal survival by targeting the neuronal enriched Na+/K+ ATPase α3 subunit (ATP1A3). Modulation of ionic homeostasis, knockdown of p53, or treatment with antioxidants protects DA neurons from Neriifolin-induced death. These results reveal a previously unknown effect of cardiac glycosides on DA neuronal survival and suggest that it is mediated through ATP1A3 inhibition, oxidative stress, and p53. They also elucidate potential approaches for counteracting the neurotoxicity of this valuable class of medications.

121 Identification, validation and mechanisms of action of novel therapeutic compounds modulating angiogenesis

Abnormal angiogenesis is a signature feature in over 70 health conditions and is critical for tumour growth and metastasis. Therefore, discovering novel therapeutic compounds, that stimulate or abrogate this biological process would be, potentially, clinically invaluable. The zebrafish has emerged as an important model for studying vascular development and angiogenesis. Advantages over other models include rapid embryonic development and optical clarity of the embryos. The small body size of the zebrafish embryo and accessibility of embryonic developmental structures within water make zebrafish embryos suitable for high through-put drug screens. We performed an angiogenesis chemical genetic screen with two compound libraries (LOPAC and Spectrum Collection) using Flk1:EGFP (fluorescently labelled endothelial cells) transgenic zebrafish embryos. The compound bioactivity was measured by numbers of the zebrafish trunk intersegmental vessel (ISV) number. The screens were performed in 96 well plates and a total of ten antiangiogenic compounds and three proangiogenic compounds were identified. A CFI-funded high throughput screening facility will be use for identification of more lead compounds. The goal of the current study was to validate the potential therapeutic efficacy of selected compounds and investigate their molecular mechanisms. Retinoic acid p-hydroxyanilide (4HPR; Fenretinide) was identified as a pro-angiogenic compound. At 4 μM, 4HPR promoted ISV development with increased ISV numbers (ISV = 32.82 ± 0.98; control ISV = 27.58 ± 0.76; P < 0.05). In HUVEC cell proliferation assay, 4HPR stimulated human endothelial cell proliferation at low molar concentrations (0.06-0.12 μM). The effects of this compound on physiological angiogenesis are currently being tested on larvae and adult zebrafish fin regeneration models. Indirubin-3-oxime (IO) was identified as a very potent antiangiogenic compound, which inhibited zebrafish ISV development in a dose-dependent manner (Fig. 1). At 16 μM, IO almost completely inhibited ISV development. In tissue regeneration models, IO inhibited larval and adult zebrafish fin regeneration. The efficacy of IO in cancer therapy will be further studied in a zebrafish cancer metastasis model. Possible molecular mechanisms responsible for IO and 4HPR action such as VEGF and notch signaling will be studied. Using the zebrafish chemical genetic screen, we demonstrated that indirubin-3-oxime is a potent angiogenic inhibitor which inhibits zebrafish angiogenesis and fin regeneration. IO may prove to be effective as an anti-cancer drug. Retinoic acid p-hydroxyanilide was identified as a pro-angiogenic effector, which may be clinically effective in promoting tissue regeneration and in treatment of myocardial infarction.

Rosuvastatin, Identified From a Zebrafish Chemical Genetic Screen for Antiangiogenic Compounds, Suppresses the Growth of Prostate Cancer

BackgroundProstate cancer (PCa) is the most common malignancy in males in Western countries. Despite improvements in standard treatments such as surgery, radiotherapy, and chemotherapy, many patients still progress to advanced stages. Recent clinical trials have shown encouraging results regarding the application of angiogenic inhibitors in the treatment of angiogenesis-dependent diseases, paving the way for novel PCa therapies. ObjectiveTo identify new antiangiogenic compounds and examine their therapeutic potential in models of PCa. Design, setting, and participantsWe performed a chemical genetic screen in developing zebrafish embryos to identify small molecules inhibiting zebrafish angiogenesis. Transgenic Tg(flk1:EGFP) zebrafish embryos were used in the screening of the Spectrum Collection compound library. Subsequently, the antiangiogenic mechanism of an identified lead compound, rosuvastatin, was studied by conducting endothelial cell function assays and examining antitumor efficacy in a PCa xenograft mouse model. Measurements, results and limitationsSeven lead compounds, including isorotenone, dihydromunduletone, aristolochic acid, simvastatin, mevastatin, lovastatin, and rosuvastatin, were identified to inhibit the growth of the zebrafish intersegmental vessels. Of these seven leads, rosuvastatin was further evaluated for its antiangiogenic mechanism and anticancer efficacy. Rosuvastatin decreased the viability of the human umbilical endothelial cells (HUVECs) (one-half inhibitory concentration: 5.87μM) by inducing G1 phase arrest and promoting apoptosis. Moreover, rosuvastatin remarkably inhibited the migration of HUVECs and dose-dependently inhibited the HUVEC capillary-like tube formation in vitro. Furthermore, we demonstrated that rosuvastatin suppressed xenografted PPC-1 prostate tumors in nonobese diabetic severe combined immunodeficiency (NOD-SCID) mice associated with decreased microvessel density (MVD) and tumor cell apoptosis. ConclusionsCollectively, our data suggest that rosuvastatin possesses antiangiogenic and antitumor activities and has therapeutic potential for the treatment of PCa. This study represents the first zebrafish antiangiogenic chemical genetic screen to identify a lead compound that targets cancer angiogenesis.

Zebrafish chemical screening reveals an inhibitor of Dusp6 that expands cardiac cell lineages

The dual specificity phosphatase 6 (Dusp6) functions as a feedback regulator of fibroblast growth factor (FGF) signaling to limit the activity of extracellular signal regulated kinase (ERK) 1 and 2. We have identified a small molecule inhibitor of Dusp6, (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI), using a transgenic zebrafish chemical screen. BCI treatment blocked Dusp6 activity and enhanced FGF target gene expression in zebrafish embryos. Docking simulations predicted an allosteric binding site for BCI within the phosphatase domain. In vitro studies supported a model that BCI inhibits Dusp6 catalytic activation by ERK2 substrate binding. A temporal role for Dusp6 in restricting cardiac progenitors and controlling heart organ size was uncovered with BCI treatment at varying developmental stages. This study highlights the power of in vivo zebrafish chemical screens to identify novel compounds targeting Dusp6, a component of the FGF signaling pathway that has eluded traditional high-throughput in vitro screens.