Aminophylline is known to be a nonselective PDE inhibitor that increases the levels of intercellular cAMP, causing activation of cAMP-dependant PKA. Treated fish with chemicals 1 (◇), 2 (X), 3 (+), 4 (■), 5 (), 6 (△), 7 (—), and WT (●) are shown. Because of these advantages, zebrafish bioassays are cheaper and faster than mouse assays, and are suitable for large-scale drug screening. Interestingly, myosin heavy-chain staining of treated dystrophin-null fish, which survived until 30 dpf, showed normal myofiber structure, similar to that of the WT fish, whereas untreated dystrophin-null fish showed clear abnormalities of muscle (Fig. The larval zebrafish xenograft assay has unique characteristics that make it unlike all other ex vivo assays and a top choice for in vivo drug screening. This is a tall order, but one Keywords:Zebrafish, drug, toxicity screening, high throughput assay, neurotoxicity, hepatotoxicity, cardiotoxicity. Survival of zebrafish after treatment with pools or individual chemicals from the Prestwick library. Drug screening in zebrafish offers the obvious advantage of being conducted within the context of an entire vertebrate organism, including intact, heterologous cell-cell interactions (eg, thymocyte-thymic epithelial cells or hematopoietic stem cell–stromal cells). Currently, prednisone is the only treatment option available for muscular dystrophy patients in the United States, although there are currently other options through approved clinical trials. Zebrafish were introduced as a drug screening platform in the context of LQTS almost 20 years ago (Langheinrich et al., 2003; Milan et al., 2003; see Table 1). The Prestwick chemical library (Harvard Institute of Chemistry and Cell Biology) was used as the source of small molecules for these experiments. Authors: Jorge Galindo-Villegas. After washing three times, sections were incubated with secondary antibodies and examined as described in ref. Yellow, nontreated affected sapje fish. 3). 3). Previously, other groups have reported that a PDE5 inhibitor restores mdx mouse muscle to normal (20, 21, 26). 5, closed and opened diamonds), exhibited no influence on the percentage of affected sapje fish. The Zebrafish Disease and Drug Screening Model: A Strong Ally Against Covid-19. Author contributions: G.K., J.R.G., and L.M.K. (A and C) Bright image. As a matter of fact, zebrafish drug screening assays are becoming a reliable tool and are routinely used in preclinical safety evaluations because their ability to accurately predict toxicity in mammals can be firmly established. Due to their small size and transparency, such testing requires a small mass of test article, very little lab space, and data can be collected noninvasively over time in vivo. The treated dystrophin-null fish with aminophylline have normal structure in skeletal muscle. 21,24–27 We use zebrafish because of their many advantages such as short generation times, amenability for large-scale screening, high fecundity, ease of in vitro fertilization, and transparency. The muscle degeneration phenotype in these mutant fish is transmitted in a recessive manner, such that 25% of the offspring show dystrophic features of skeletal muscle after 3 d postfertilization (dpf). WT fish showed positive staining of dystrophin in the myosepta (Fig. For long-term treatment of fish from 1 to 30 dpf, WT, heterozygous, and dystrophin-null fish from heterozygous fish matings were treated with candidate chemicals 1–7. Up-regulation of expression of these target proteins by PKA activation might modulate the progression of phenotypes in skeletal muscle. The orthologs of many dystrophin–glycoprotein complex (DGC) components are expressed in zebrafish (19). In the treated groups, some homozygous dystrophin-null fish had normal birefringence and thus, were considered phenotypically unaffected (Fig. Analysis of skeletal muscle of dystrophin-null fish that survive 30 d after treatment with chemical 4, aminophylline. In light of recent results, they’re not so sure. designed research; G.K., J.A.K., J.A.M., and M.S.A. Screening assays, Zebrafish, Drug discovery; Get e-Alerts. https://doi.org/10.1016/B978-0-12-812431-4.00051-8. Thousands more compounds are now available for further screening, something we have already started. The paradigm of therapeutic screening in zebrafish is attractive because of its immediate therapeutic relevance. Zebrafish model for rapid drug discovery There are six major reasons for supporting zebrafish as a good vertebrate model for drug screening, specifically on visual problems: 1) zebrafish's eye, particularly the retina, is anatomically similar to many vertebrates including human10. 2C). Rapid and cost-effective in vivo assays to screen potential environmental neurodevelopmental toxicants are necessary to address the limitations of in vitro platforms, such as the inability to fully recapitulate the developmental and physiological processes of whole organisms. 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. Here we describe the use of zebrafish bioassays for assessing … Fish at 4 dpf from screening with the Prestwick library on heterozygous sapje fish pairs and the percentage of affected fish after chemical treatment with these chemical pools. To assay the activity of PKA, the PepTag Assay for Non-Radioactive Detection of cAMP-Dependent Protein Kinase (Promega) was used following the manufacturer’s protocol. NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. These results show that activated phosphorylated PKA and the activity of PKA were increased in aminophylline-treated fish compared with WT and untreated dystrophin-null fish, which suggests indirectly that intracellular cAMP is increased with aminophylline treatment. Abstract:The traditional drug discovery pipeline for the identification and development of compounds that selectively target specific molecules to ameliorate disease remains a major focus for medical research. 1E). 2A). These fish are also small enough to be permeable to small molecules and can be assayed in large numbers. 5, closed square). To determine if the treatment with a single compound could reverse the skeletal muscle phenotype after it was already present, affected fish (selected at 4 dpf by birefringence assay) were treated with the seven individual candidate chemicals (Table S1) from 4 to 30 dpf. Embryos or fish were homogenized in Tris-buffered saline (TBS) containing 4 M Urea, 2% SDS, 2% CHAPS, protease inhibitors, and phosphatase inhibitors (Roche). performed research; G.K. contributed new reagents/analytic tools; G.K. and L.M.K. Many of these screens have been highly successful in disease modeling (7) and drug screening (8–10), making the zebrafish ideal for high-throughput whole-organism screening of candidate compounds. It is possible to quickly produce large numbers of mutant offspring that can then be assayed in multiwell plates and treated with different chemicals to determine if disease progression is modulated. In this secondary screen, seven chemicals influenced muscle structure by decreasing the percentage of affected fish using both sapje (used in the first screen) and sapje-like fish, which has an independent dystrophin mutation (Table 2). Zebrafish platform Austria for preclinical drug screening (ZANDR) ZANDR was established at the Children´s Cancer Research Institute (CCRI) in 2019 and is a unique platform to phenotypically screen small compounds on zebrafish models of human diseases. Translating results to what we might see in humans requires an appropriate (vertebrate) model suited for large-scale studies. S3). (D) Ratio of phosphorylated PKA and PKA in 30 dpf surviving fish (n = 3). And nontreatment groups ) chemical treatment ( C ) of eight compounds in duplicate ) suitable for studies. You are a human visitor and to prevent automated spam submissions University, Bodø, Norway ; Opinion (! These target proteins by PKA activation might modulate the muscle phenotype in these are! 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