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Zygogen Uses Zebrafish to Find Angiogenesis Inhibitor; Findings Translate to Human CBAs

BOSTON — A prospective study by researchers at Zygogen have found what they believe to be the first anti-angiogenic compound initially identified in live zebrafish whose activity they were able to translate to human cell-based assays.
Eric Sandberg, a Zygogen scientist, told CBA News this week that up until his firm’s research, all studies looking at the effect of anti-angiogenic compounds in zebrafish were done retrospectively.
The anti-angiogenic compound, called 619, is now being tested in mouse angiogenesis assays in vivo, said Sandberg. The investigators hope to have those results in the next two weeks or so, and plan to submit a publication within a few weeks after that, said Sandberg, who presented his team’s work at the Assay Development and HTS conference held here last week.
Sandberg said that he and his colleagues have every reason to believe that 619 will show anti-angiogenic activity in the mouse in vivo assays because it has already shown activity in zebrafish and endothelial cell-based assay systems.
One-Hit Wonder
Zygogen began its research by collaborating with Emory University, part of the NIH Molecular Library Screening Center Network, to screen the LOPAC1280 compound library. Emory and Zygogen each screened 640 compounds.
To demonstrate reproducibility of the assay, Emory screened 80 of the compounds originally screened by Zygogen, while Zygogen screened 80 of the compounds originally screened by Emory. Each was able to verify the hits found by the other, Sandberg said.
The zebrafish were arrayed into multi-well plates and treated with 30 μM of each test compound at day one post fertilization, Sandberg said. On day two, the researchers imaged the embryos using the Discovery-1 high-content imaging system made by Molecular Devices, which is now owned by MDS.
Sandberg said the researchers assessed angiogenic growth in the embryos using Zygogen’s automated quantitative algorithms. He said that hits were identified as compounds that were found to inhibit angiogenesis greater than three standard deviations from the mean.
The investigators identified two known anti-angiogenic compounds in addition to compound 619. Each compound inhibited angiogenesis in a dose-dependent manner.
According to Sandberg, compound 619 was the most potent of the three hits pulled out of the assays. He reported that 619 did not inhibit endothelial cell migration, but did inhibit endothelial tube formation and cellular proliferation during angiogenesis.
These findings demonstrate some specificity of 619 for certain cellular processes during angiogenesis, said Sandberg. They also indicate that the inhibition of endothelial tube formation and cell proliferation are not due to global cell cytotoxicity, he said.
User-Friendly Assay
In a follow-up interview with CBA News this week, Sandberg said that that the company believes the live zebrafish assay is “appropriate” for a secondary screen, and pointed out that it could be used for the primary screening of a small library as well.
Sandberg said that, ideally, he and his colleagues see the assay being used to confirm cell-based assay results and determine which compounds to carry forward into mammalian assays.
Sandberg explained that using the company’s Z-Tag assays, the investigators generated transgenic zebrafish expressing green fluorescent reporter proteins under the control of the vascular endothelial growth factor receptor 2 promoter for blood-vessel restricted expression. They used these fish to screen the LOPAC1280 compound library. 
Zygogen has developed several lines of zebrafish in-house that express different green fluorescent proteins under the control of tissue-specific promoters, including a neuron line under the control of the GATA-2 promoter; a heart line under the control of the cMLC-2 promoter, which is used for evaluating cardiotoxicity; a thrombocyte/platelet line under the control of the GpIIB promoter; as well as the vasculature line that was the focus of Sandberg’s presentation at the Assay Development and HTS conference.

“We have every reason to believe that [619] will show anti-angiogenic activity in the mouse in vivo assays, because it has already shown activity in zebrafish and endothelial CBA systems.”

Sandberg said that zebrafish have several advantages as an animal model. The embryos develop very rapidly, and 100 to 200 embryos are produced per adult mating pair. This allows for rapid turnaround of the data, he said. In addition, rapid organ development means that assays can be run at an earlier stage.
During embryonic development, zebrafish are transparent. This is particularly useful when combined with Z-Tag technology that uses fluorescent reporter proteins.
Zygogen has taken steps to automate Z-Tag assays, Sandberg said. “We’ve demo’ed Union Biometrica’s COPAS embryo-sorter twice now in-house. It can automatically distribute embryos into multi-well plates with greater than 95-percent accuracy.”
Sandberg said that the COPAS can also distinguish live embryos from dead embryos and fluorescent embryos from non-fluorescent embryos.
The company plans to integrate the sorter into its automation scheme, he said, though he declined to specify a timeline for doing this because the timetable is “ever-changing.”
All liquid handling at this point is robotic, which in this research was done by Caliper’s Sciclone ALH 3000.
Others share Sandberg’s enthusiasm for the zebrafish model. It is a lot cheaper to work with zebrafish compared to other animals, said Phylonix President Pat McGrath.
Phylonix won the solicitation for the zebrafish component of the EPA’s ToxCast program, whose animal component was limited to zebrafish.
“We see growth and increased interest in doing zebrafish studies year-over-year,” said McGrath. “We are living through a period where it is still something new.”
Users have to get comfortable with the data, she said, and become familiar with the differences between zebrafish and other animal models.
“We do not argue that it is going to replace CBAs or rodent studies,” McGrath said. “But it is a bridge to saving money on moving to rodent studies if you do not need to, or to go faster if you do need to.”

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