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

Expression and function of ribozymes in transgenic mice

Hammerhead and hairpin ribozymes are short RNAs that act as sequence-specific endoribonucleases by cleaving a target RNA after hybridization to complementary sequences. By changing the specific target hybridization sequence of the ribozyme, virtually any RNA can be targeted, making ribozymes a versatile tool for gene down-regulation (knockdown). This thesis addresses questions regarding expression and function of ribozymes in transgenic mice. A series of hammerhead and hairpin ribozyme transgenic mouse models were generated. The first transgenic mouse model, expressing a hammerhead ribozyme targeting beta-2-microglobulin (ß2-m), proved the principle that ribozymes can efficiently down-regulate the expression specific mRNAs in vivo in a mammalian organism. A second transgenic mouse model expressing hammerhead ribozymes targeting Wilms tumor gene 1 (WT1) generated interesting preliminary data on the involvement of WT1 in male sexual development. However, ribozyme transgene expression was gradually shut-off during breeding, correlating with a disappearance of the observed abnormalities. A third transgenic model showed the feasibility of using ribozyme transgenic mice as a model for ribozyme gene therapy against HIV-1 infection. Transgenic spleen cells expressing an anti-HIV-1 hairpin ribozyme, showed a dose-dependent resistance to infection compared with control spleen cells, when infected with HIV-1/MuLV pseudotype virus. The different trans-cleaving ribozymes targeting ß2-m, WT1 or HIV-1 in transgenic mice were expressed in an expression cassette, in which a 3' cis-cleaving ribozyme, downstream of the trans-targeting ribozyme, was used to terminate the transcript. This cis-cleavage was found to be essential for the activity of the transcleaving ribozyme. Direct proof of hammerhead ribozyme cleavage in mammalian cells in vivo was given by detection of cis-cleavage products in transgenic mouse organs. Steady state levels of cleavage products, relative to the level of the non-cleaved transcript, showed a consistent ranking order: kidney > liver > lung > spleen, which suggests a difference in hammerhead ribozyme cleavage activity in these organs. In vitro analyses of ribozyme - RNA substrate kinetics have primarily relied on indirect detection methods. We have developed a real-time analysis of interactions (i.e. association, dissociation and cleavage) between hammerhead ribozymes and substrates using the surface plasmon resonance (SPR) technology. The SPR technology permits an inclusion of proteins in in vitro analyses, which may improve the capabilities to predict ribozyme function in living cells. In summary, this work has shown that ribozymes, expressed in transgenic mice, can down-regulate the mRNA of specific target genes in a dose-dependent manner, thereby satisfying the general aim of the thesis. It has also shed light on some of the basic aspects of ribozyme expression and function in transgenic mice

Phenotypic Screen Identifies a Small Molecule Modulating ERK2 and Promoting Stem Cell Proliferation

Stem cells display a fundamentally different mechanism of proliferation control when compared to somatic cells. Uncovering these mechanisms would maximize the impact in drug discovery with a higher translational applicability. The unbiased approach used in phenotype-based drug discovery (PDD) programs can offer a unique opportunity to identify such novel biological phenomenon. Here, we describe an integrated phenotypic screening approach, employing a combination of in vitro and in vivo PDD models to identify a small molecule increasing stem cell proliferation. We demonstrate that a combination of both in vitro and in vivo screening models improves hit identification and reproducibility of effects across various PDD models. Using cell viability and colony size phenotype measurement we characterize the structure activity relationship of the lead molecule, and identify that the small molecule inhibits phosphorylation of ERK2 and promotes stem cell proliferation. This study demonstrates a PDD approach that employs combinatorial models to identify compounds promoting stem cell proliferation

(A) Flow cytometric recordings of DiBAC₄(3) loaded mESCs (n=10 000 cells, N=3) treated with 10 µM clofilium for 1 h. (B) Cell viability of mESCs treated with E4031 with and without sucrose (20 mM) or ouabain (1 µM) for 24 h. Data presented as mean ± SEM (n=4), one-way ANOVA, Tukey’ post-hoc test. (C) Images from time-lapse movies of mESCs treated with Erg inhibitor, E4031 (10 µM) and with the Na⁺,K⁺-ATPase inhibitor ouabain (1 µM).

<p>(A) Flow cytometric recordings of DiBAC₄(3) loaded mESCs (n=10 000 cells, N=3) treated with 10 µM clofilium for 1 h. (B) Cell viability of mESCs treated with E4031 with and without sucrose (20 mM) or ouabain (1 µM) for 24 h. Data presented as mean ± SEM (n=4), one-way ANOVA, Tukey’ post-hoc test. (C) Images from time-lapse movies of mESCs treated with Erg inhibitor, E4031 (10 µM) and with the Na⁺,K⁺-ATPase inhibitor ouabain (1 µM).</p