Small molecule screening platform for assessment of cardiovascular toxicity on adult zebrafish heart

<p>Abstract</p> <p>Background</p> <p>Cardiovascular toxicity is a major limiting factor in drug development and requires multiple cost-effective models to perform toxicological evaluation. Zebrafish is an excellent model for many developmental, toxicological and regenerative studies. Using approaches like morpholino knockdown and electrocardiogram, researchers have demonstrated physiological and functional similarities between zebrafish heart and human heart. The close resemblance of the genetic cascade governing heart development in zebrafish to that of humans has propelled the zebrafish system as a cost-effective model to conduct various genetic and pharmacological screens on developing embryos and larvae. The current report describes a methodology for rapid isolation of adult zebrafish heart, maintenance <it>ex vivo</it>, and a setup to perform quick small molecule throughput screening, including an in-house implemented analysis script.</p> <p>Results</p> <p>Adult zebrafish were anesthetized and after rapid decapitation the hearts were isolated. The short time required for isolation of hearts allows dissection of multiple fishes, thereby obtaining a large sample size. The simple protocol for <it>ex vivo </it>culture allowed maintaining the beating heart for several days. The in-house developed script and spectral analyses allowed the readouts to be presented either in time domain or in frequency domain. Taken together, the current report offers an efficient platform for performing cardiac drug testing and pharmacological screens.</p> <p>Conclusion</p> <p>The new methodology presents a fast, cost-effective, sensitive and reliable method for performing small molecule screening. The variety of readouts that can be obtained along with the in-house developed analyses script offers a powerful setup for performing cardiac toxicity evaluation by researchers from both academics and industry.</p

Teleost retina : A model to study neurogenesis and angiogenesis

Teleost models, zebrafish and medaka have become popular models to study various aspects of developmental biology and genetics. The rapid embryonic development, transparent embryos and the availability of many mutants for various developmental and molecular pathways contribute to the usefulness of these models. The availability of various biochemical, molecular and genetic techniques applicable on these models facilitate in dissecting developmental processes. Teleost retina shows very high similarity to that seen in mammalian retina. The arrangement of the six types of neurons and one type of glia is very similar. Zebrafish has been extensively used in gaining insight into the development and functioning of the retina. Medaka, on the other hand has not been so extensively capitalized as zebrafish. The current study characterizes expression of genes mainly from the nuclear receptor family and establishes the role of zebrafish liver x receptor in governing the size, patterning and neurogenesis of the retina in zebrafish. We also establish the time line of the retinal patterning of medaka retina. Zebrafish and medaka retina show both similarity and difference in the developmental events governing the patterning of the retina. In zebrafish, retinal neurogenesis follows a fan gradient pattern starting at the ventro-nasal region. In medaka, neurogenesis starts from the central retina. An additional, second domain of neurogenesis is seen with the patterning of photoreceptors in medaka. This observation highlights the possibility of utilizing these two species as comparative models in gaining rapid understanding of retinal development and function. This study also establishes the time line of vascular development in the zebrafish retina, an important event required for normal function. Similar to neurogenesis, vasculaturedevelops rapidly and this feature was utilized to develop a small molecule-screening assay. The screening resulted in identification of five compounds that produced phenotype ranging from decrease in the number of vessels to loss of vessels specifically in the retina. To gain insight into the mode of action, further analyses of three of the five identified compounds, using either morpholino knockdown or structural similarity search was done. This study highlights the advantage of using zebrafish model to perform medically relevant chemical screen

In Silico Analysis of Thioredoxins and Glutaredoxins

Thioredoxins (TRXs) and glutaredoxins (GRXs) are ubiquitous small redox proteins belonging to the thioredoxin (TRX) superfamily. They regulate several cellular functions via mediating a dithiol/disulphide exchange in target proteins. Thioredoxins have been classified into several subgroups based on their structural homologs. In an attempt to identify thioredoxin proteins which have not been characterized, an EST database survey of Lycopersicon esculentum, Glycine max, Helianthus annus, Secale cereale, Solanum tuberosum, Apis mellifera ligustica, Oncorhynchus mykiss, Salmo salar, and whole genome survey for Drosophila melanogaster, Rattus norvegicus and Caenorhabditis briggsae was performed. Several glutaredoxin and glutaredoxin-like proteins from Ricinus communis, Vercinia fordii, Lycopersicon esculentum, Tilia platyphyllos, Populus tremuloides, Triticum aestivum and Oryza sativa were also characterized. The deduced amino acid sequences were aligned and phylogenetic trees were constructed to determine the consensus sequences and for establishing interrelationships amongst the new and established thioredoxin and glutaredoxins. Based on the alignments, proteins were designated to their respective classes and subcellular localization predictions were used to predict their possible site of actions. In silico analysis has identified several new thioredoxins, glutaredoxins and related proteins and provided insight into their evolutionary relationships

Tetrahydroquinoline-Derived Macrocyclic Toolbox: The Discovery of Antiangiogenesis Agents in Zebrafish Assay

A novel approach to incorporate the macrocyclic rings onto the privileged substructure, i.e., tetrahydroquinoline scaffold, is developed. The presence of an amino acid-derived moiety in the macrocyclic skeleton provides an opportunity to modulate the nature of the chiral side chain. Further, evaluation in a zebrafish screen identified three active small molecules (<b>2.5b</b>, <b>3.2d</b>, and <b>4.2</b>) as antiangiogenesis agents at 2.5 μM

Macrocyclic Glycohybrid Toolbox Identifies Novel Antiangiogenesis Agents from Zebrafish Assay

A practical and modular approach to obtain a diverse set of 14-membered macrocyclic compounds from carbohydrates is developed that utilizes functional groups at C-1 and C-5. The evaluation of this toolbox in various zebrafish assays led to the identification of <b>2.7f</b> as an antiangiogenesis agent

Macrocyclic toolbox from epothilone fragment identifies a compound showing molecular interactions with actin and novel promoters of apoptosis in patient-derived brain tumor cells

A simple, practical stereoselective synthesis of the epothilone fragment is developed to obtain a diverse set of expanded 18-membered macrocyclic compounds. These macrocycles contain the C5–C8 sub-unit of epothilone and an additional amino acid moiety incorporated in the 18-membered macrocycle, which allows the synthesis of several analogs with a variation in the chiral side chain. The epothilone fragment was obtained by using an enantiopure epoxide, which was subjected to a regioselective opening, giving the key derivative. Finally, the synthesis of the 18-membered macrocyclic ring was achieved by employing two key steps: (i) acylation with an N-allylated amino acid moiety, and (ii) a ring-closing metathesis (RCM) approach. Computational studies of the macrocyclic compounds obtained from this study with actin give rise to the proposed molecular interactions with the target protein. Further, the screening of our chemical toolbox from this program (i.e., the final products and several intermediates) identified several compounds as promoters of apoptosis in patient-derived brain tumor glioma cells

14-Membered Macrocyclic Ring-Derived Toolbox: The Identification of Small Molecule Inhibitors of Angiogenesis and Early Embryo Development in Zebrafish Assay

A highly practical and modular synthesis to obtain a diverse 14-membered ring-based macrocyclic toolbox is achieved. These compounds were further tested in zebrafish assays related to early embryonic development, angiogenesis, and neurogenesis, respectively. <b>1.4c</b> was identified as an antiangiogenesis agent

Cell cycle dependent Erg1 channel expression.

<p>(A) Heat-map displaying mRNA differential expression of selected K⁺ channels in different cell cycle phases (ANOVA-test p<0.005). (B) Erg1 mRNA expression level evaluated using real-time PCR in cell cycle sorted mESCs (ANOVA-test). (C) Flow cytometry plot of live mESCs stained for an extracellular epitope of Erg1 channel. (D) Confocal images of mESCs sorted in different cell cycle stages and immunostained for Erg1 protein with cross section histograms (note that measurements across nucleoli were avoided) showing Erg1 immunostaining intensity with increased plasma membrane localization in G1.</p

Erg inhibition decreased stiffness in mESCs.

<p>(A) A representative image of the cantilever placed over a mESC during atomic force microscopy. (B) In control conditions, blebbing cells showed a trend towards higher stiffness than non-blebbing cells (n=7, p=0.17, median indicated in box) and control cells that were subjected for hypertonic medium (sucrose 20 mM) for one hour showed reduced stiffness (n=11, p<0.001). (C) After 7 h of Erg inhibition (E4031; 10 µM) treated cells (n=8) were significantly (p=0.02, t-test unequal variance) less stiff than control cells (n=18).</p