RESUMEN
Numerous drug treatments that have recently entered the clinic or clinical trials have their genesis in zebrafish. Zebrafish are well established for their contribution to developmental biology and have now emerged as a powerful preclinical model for human disease, as their disease characteristics, aetiology and progression, and molecular mechanisms are clinically relevant and highly conserved. Zebrafish respond to small molecules and drug treatments at physiologically relevant dose ranges and, when combined with cell-specific or tissue-specific reporters and gene editing technologies, drug activity can be studied at single-cell resolution within the complexity of a whole animal, across tissues and over an extended timescale. These features enable high-throughput and high-content phenotypic drug screening, repurposing of available drugs for personalized and compassionate use, and even the development of new drug classes. Often, drugs and drug leads explored in zebrafish have an inter-organ mechanism of action and would otherwise not be identified through targeted screening approaches. Here, we discuss how zebrafish is an important model for drug discovery, the process of how these discoveries emerge and future opportunities for maximizing zebrafish potential in medical discoveries.
Asunto(s)
Ensayos Clínicos como Asunto/estadística & datos numéricos , Modelos Animales de Enfermedad , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos/métodos , Animales , Ensayos Analíticos de Alto Rendimiento , Humanos , Pez CebraRESUMEN
The availability of nucleotides has a direct impact on transcription. The inhibition of dihydroorotate dehydrogenase (DHODH) with leflunomide impacts nucleotide pools by reducing pyrimidine levels. Leflunomide abrogates the effective transcription elongation of genes required for neural crest development and melanoma growth in vivo1. To define the mechanism of action, we undertook an in vivo chemical suppressor screen for restoration of neural crest after leflunomide treatment. Surprisingly, we found that alterations in progesterone and progesterone receptor (Pgr) signalling strongly suppressed leflunomide-mediated neural crest effects in zebrafish. In addition, progesterone bypasses the transcriptional elongation block resulting from Paf complex deficiency, rescuing neural crest defects in ctr9 morphant and paf1(alnz24) mutant embryos. Using proteomics, we found that Pgr binds the RNA helicase protein Ddx21. ddx21-deficient zebrafish show resistance to leflunomide-induced stress. At a molecular level, nucleotide depletion reduced the chromatin occupancy of DDX21 in human A375 melanoma cells. Nucleotide supplementation reversed the gene expression signature and DDX21 occupancy changes prompted by leflunomide. Together, our results show that DDX21 acts as a sensor and mediator of transcription during nucleotide stress.
Asunto(s)
ARN Helicasas DEAD-box/genética , Melanocitos/metabolismo , Cresta Neural/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Receptores de Progesterona/genética , Proteínas de Pez Cebra/genética , Animales , Línea Celular Tumoral , ARN Helicasas DEAD-box/metabolismo , Dihidroorotato Deshidrogenasa , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Humanos , Leflunamida/farmacología , Melanocitos/efectos de los fármacos , Melanocitos/patología , Cresta Neural/efectos de los fármacos , Cresta Neural/crecimiento & desarrollo , Nucleótidos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Progesterona/metabolismo , Unión Proteica , Receptores de Progesterona/metabolismo , Transducción de Señal , Estrés Fisiológico/genética , Elongación de la Transcripción Genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
Zebrafish are a major model for chemical genetics, and most studies use embryos when investigating small molecules that cause interesting phenotypes or that can rescue disease models. Limited studies have dosed adults with small molecules by means of water-borne exposure or injection techniques. Challenges in the form of drug delivery-related trauma and anesthesia-related toxicity have excluded the adult zebrafish from long-term drug efficacy studies. Here, we introduce a novel anesthetic combination of MS-222 and isoflurane to an oral gavage technique for a non-toxic, non-invasive and long-term drug administration platform. As a proof of principle, we established drug efficacy of the FDA-approved BRAF(V600E) inhibitor, Vemurafenib, in adult zebrafish harboring BRAF(V600E) melanoma tumors. In the model, adult casper zebrafish intraperitoneally transplanted with a zebrafish melanoma cell line (ZMEL1) and exposed to daily sub-lethal dosing at 100â mg/kg of Vemurafenib for 2â weeks via oral gavage resulted in an average 65% decrease in tumor burden and a 15% mortality rate. In contrast, Vemurafenib-resistant ZMEL1 cell lines, generated in culture from low-dose drug exposure for 4â months, did not respond to the oral gavage treatment regimen. Similarly, this drug treatment regimen can be applied for treatment of primary melanoma tumors in the zebrafish. Taken together, we developed an effective long-term drug treatment system that will allow the adult zebrafish to be used to identify more effective anti-melanoma combination therapies and opens up possibilities for treating adult models of other diseases.
Asunto(s)
Antineoplásicos/administración & dosificación , Antineoplásicos/uso terapéutico , Evaluación Preclínica de Medicamentos , Neoplasias/tratamiento farmacológico , Pez Cebra/metabolismo , Administración Oral , Animales , Animales Modificados Genéticamente , Antineoplásicos/farmacología , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Indoles/farmacología , Indoles/uso terapéutico , Melanoma/tratamiento farmacológico , Melanoma/patología , Trasplante de Neoplasias , Neoplasias/patología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/metabolismo , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , VemurafenibRESUMEN
Ex vivo expansion of satellite cells and directed differentiation of pluripotent cells to mature skeletal muscle have proved difficult challenges for regenerative biology. Using a zebrafish embryo culture system with reporters of early and late skeletal muscle differentiation, we examined the influence of 2,400 chemicals on myogenesis and identified six that expanded muscle progenitors, including three GSK3ß inhibitors, two calpain inhibitors, and one adenylyl cyclase activator, forskolin. Forskolin also enhanced proliferation of mouse satellite cells in culture and maintained their ability to engraft muscle in vivo. A combination of bFGF, forskolin, and the GSK3ß inhibitor BIO induced skeletal muscle differentiation in human induced pluripotent stem cells (iPSCs) and produced engraftable myogenic progenitors that contributed to muscle repair in vivo. In summary, these studies reveal functionally conserved pathways regulating myogenesis across species and identify chemical compounds that expand mouse satellite cells and differentiate human iPSCs into engraftable muscle.
Asunto(s)
Evaluación Preclínica de Medicamentos , Desarrollo de Músculos/efectos de los fármacos , Animales , Colforsina/farmacología , Técnicas de Cultivo , AMP Cíclico/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Músculo Esquelético/citología , Músculo Esquelético/fisiología , Distrofias Musculares/terapia , Células Satélite del Músculo Esquelético/metabolismo , Trasplante de Células Madre , Pez Cebra/embriología , Pez Cebra/metabolismoRESUMEN
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.
Asunto(s)
Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos/métodos , Embrión no Mamífero/fisiología , Ensayos Analíticos de Alto Rendimiento , Terapia Molecular Dirigida/métodos , Bibliotecas de Moléculas Pequeñas/farmacología , Pez Cebra/fisiología , Animales , Automatización de Laboratorios , Conducta Animal/efectos de los fármacos , Conducta Animal/fisiología , Bases de Datos Factuales , Embrión no Mamífero/efectos de los fármacos , Humanos , Inmunohistoquímica , Fenotipo , Proyectos de Investigación , Pez Cebra/embriologíaRESUMEN
cdx4, a caudal-related homeodomain-containing transcription factor, functions as a regulator of hox genes, thereby playing a critical role in anterior-posterior (A-P) patterning during embryogenesis. In zebrafish, homozygous deletion of the cdx4 gene results in a mutant phenotype known as kugelig, with aberrant A-P patterning and severe anemia characterized by decreased gata1 expression in the posterior lateral mesoderm. To identify pathways that interact with cdx4 during primitive hematopoiesis, we conducted a chemical genetic screen in the cdx4 mutant background for compounds that increase gata1 expression in cdx4 mutants. Among 2640 compounds that were tested, we discovered two compounds that rescued gata1 expression in the cdx4-mutant embryos. The strongest rescue was observed with bergapten, a psoralen compound found in bergamont oil. Another member of the psoralen family, 8-methoxypsoralen, was also found to rescue gata1 expression in cdx4-mutant embryos. The psoralen compounds also disrupted normal A-P patterning of embryos. These compounds modify the cdx4-mutant phenotype and will help elucidate signaling pathways that act downstream or parallel to the cdx4-hox pathway.
Asunto(s)
Hematopoyesis/efectos de los fármacos , Proteínas de Homeodominio/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Tipificación del Cuerpo/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Pruebas Genéticas , Proteínas de Homeodominio/genética , Masculino , Fenotipo , Factores de Transcripción , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
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.
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Descubrimiento de Drogas/métodos , Bibliotecas de Moléculas Pequeñas , Pez Cebra/metabolismo , Animales , Evaluación Preclínica de Medicamentos/métodosRESUMEN
Chemical genetic screening is an effective strategy to identify compounds that alter a specific biological phenotype. As a complement to cell line screens, multicellular organism screens may reveal additional compounds. The zebrafish embryo is ideal for small molecule studies because of its small size and the ease of waterborne treatment. We first examined a broad range of known cell cycle compounds in embryos using the mitotic marker phospho-histone H3. The majority of the known compounds exhibited the predicted cell cycle effect in embryos. To determine whether we could identify novel compounds, we screened a 16 320-compound library for alterations of pH3. This screen revealed 14 compounds that had not been previously identified as having cell cycle activity despite numerous mitotic screens of the same library with mammalian cell lines. With six of the novel compounds, sensitivity was greater in embryos than cell lines, but activity was still detected in cell lines at higher doses. One compound had activity in zebrafish embryos and cell lines but not in mammalian cell lines. The remaining compounds exhibited activity only in embryos. These findings demonstrate that small molecule screens in zebrafish can identify compounds with novel activity and thus may be useful tools for chemical genetics and drug discovery.
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Ciclo Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Embrión no Mamífero/efectos de los fármacos , Preparaciones Farmacéuticas/administración & dosificación , Pez Cebra/embriología , Animales , Afidicolina/administración & dosificación , Ciclo Celular/genética , Ciclo Celular/fisiología , Línea Celular , ADN/metabolismo , Dimetilsulfóxido/administración & dosificación , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Citometría de Flujo , Histonas/metabolismo , Inmunohistoquímica , Ratones , Mitosis/efectos de los fármacos , Mitosis/genética , Mitosis/fisiología , Células 3T3 NIH , Nocodazol/administración & dosificación , Preparaciones Farmacéuticas/clasificación , Fenotipo , Fosforilación/efectos de los fármacos , Tubulina (Proteína)/metabolismo , Pez Cebra/genéticaRESUMEN
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.
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Diseño de Fármacos , Modelos Animales , Pez Cebra/fisiología , Animales , Evaluación Preclínica de Medicamentos/métodos , Humanos , Pruebas de Toxicidad/métodos , Pez Cebra/embriologíaRESUMEN
For decades immunologists have relied heavily on the mouse model for their experimental designs. With the realization of the important role innate immunity plays in orchestrating immune responses, invertebrates such as worms and flies have been added to the repertoire. Here, we discuss the advent of the zebrafish as a powerful vertebrate model organism that promises to positively impact immunologic research.