Orthogonal Drug Pooling Enhances Phenotype-Based Discovery of Ocular Antiangiogenic Drugs in Zebrafish Larvae

Unbiased screening of large randomized chemical libraries is a powerful tool to find new drugs and targets. However, forward chemical screens in zebrafish can be time consuming and usually >99% of test compounds have no significant effect on the desired phenotype. Here, we sought to find bioactive drugs more efficiently and to comply with the 3R principles of replacement, reduction, and refinement of animals in research. We investigated if pooling of drugs to simultaneously test 8-10 compounds in zebrafish larvae can increase the screening efficiency of an established assay that identifies drugs inhibiting developmental angiogenesis in the eye. In a phenotype-based screen, we tested 1,760 small molecule compounds from the ChemBridge DIVERSet™ chemical library for their ability to inhibit the formation of distinct primary hyaloid vessels in the eye. Applying orthogonal pooling of the chemical library, we treated zebrafish embryos from 3 to 5 days post fertilization with pools of 8 or 10 compounds at 10 μM each. This reduced the number of tests from 1,760 to 396. In 63% of cases, treatment showed sub-threshold effects of