Automated in vivo compound screening with zebrafish and the discovery and validation of PD 81,723 as a novel angiogenesis inhibitor

Abstract

AbstractAngiogenesis is a critical process in tumor progression. Inhibition of angiogenesis by blocking VEGF signaling can impair existing tumor vessels and halt tumor progression. However, the benefits are transient, and most patients who initially respond to these therapies develop resistance. Accordingly, there is a need for new anti-angiogenesis therapeutics to delay the processes of resistance or eliminate the resistive effects entirely. This manuscript presents the results of a screen of the National Institutes of Health Clinical Collections Libraries I & II (NIHCCLI&II) for novel angiogenesis inhibitors. The 727 compounds of the NIHCCLI&II library were screened with a high-throughput drug discovery platform (HTP) developed previously with angiogenesis-specific protocols utilizing zebrafish. The screen resulted in 14 hit compounds that were subsequently narrowed down to one, with PD 81,723 chosen as the lead compound. PD 81,723 was validated as an inhibitor of angiogenesis in vivo in zebrafish and in vitro in human umbilical vein endothelial cells (HUVECs). Zebrafish exposed to PD 81,723 exhibited several signs of a diminished endothelial network due to the inhibition of angiogenesis. Immunochemical analysis did not reveal any significant apoptotic or mitotic activity in the zebrafish. Assays with cultured HUVECs elucidated the ability of PD 81,723 to inhibit capillary tube formation, migration, and proliferation of endothelial cells. In addition, PD 81,723 did not induce apoptosis while significantly down regulating p21, AKT, VEGFR-2, p-VEGFR-2, eNOS, and p-eNOS, with no notable change in endogenous VEGF-A in cultured HUVECs.