Protocol Development for the Discovery of Angiogenesis Inhibitors via Automated Methods Using Zebrafish and the Discovery and Validation of PD 81723 as a Novel Angiogenesis Inhibitor

Their optical clarity as larvae and embryos, small size, and high fecundity make zebrafish ideal for whole animal high-throughput screening. A high-throughput drug discovery platform (HTP) has been built to perform fully automated screens of compound libraries with zebrafish embryos. A Tg(kdrl:EGFP) line, marking endothelial cell cytoplasm, was used in this work to help develop protocols and functional algorithms for the system, with the intent of screening for angiogenesis inhibitors. Indirubin 3’ Monoxime (I3M), a known angiogenesis inhibitor, was used at various concentrations to validate the protocols. Consistent with previous studies, a dose-dependent inhibitory effect of I3M on angiogenesis was confirmed. The National Institutes of Health Clinical Collections Libraries I II (NIHCCLI) were screened for angiogenesis inhibitors with these methods as a pilot project for the HTP. The 727 compounds were screened in triplicate and 14 compounds were considered hits. The 5 compounds that corresponded to the most diminished endothelial network with a relatively normal overall morphology were selected for further analysis. A dose response experiment of each of the top 5 compounds was conducted and only PD 81723 and raclopride yielded a significantly smaller endothelial network. Evaluation of the chemical phenotypes of PD 81723 and raclopride led to the exclusion of raclopride as a possible candidate. PD 81723 appeared to be working in a dose-dependent manner and was selected as the main compound of interest. Zebrafish exposed to PD 81723 exhibited several signs of a diminished endothelial network due to the inhibition of angiogenesis. Immunochemistry experiments did not reveal any significant apoptotic or mitotic activity in the zebrafish. Assays with cultured human umbilical vein endothelial cells (HUVECs) elucidated the ability of PD 81723 to inhibit capillary tube formation, wound healing/migration, and proliferation of endothelial cells. An annexin V/PI flow cytometry assay did not reveal significant levels of apoptotic events in PD 81723 treated cells. These results indicate that PD 81723 may be driving endothelial cells into a quiescent state. In addition, immunoblotting revealed that PD 81723 significantly down regulated p21, AKT, VEGFR-2, p-VEGFR-2, eNOS, and p-eNOS, with no notable change in endogenous VEGF-A.Ph.D