TR-764 is a novel tubulin binding agent with strong antiangiogenic activity.

Tubulin binding agents (TBAs) are drugs which are commonly used in cancer therapy as antimitotic. TR-764 is a new inhibitor of tubulin polymerization, based on the 2-(alkoxycarbonyl)-3-(3′,4′,5′-trimethoxyanilino)benzo[b]thiophene molecular skeleton, with high antiproliferative activity in vitro and inhibiting in vivo the growth of a syngenic hepatocellular carcinoma in Balb/c mice. Recently it has been reported that antimitotics, like combretastatin A-4 (CA-4), present also antivascular activity. Targeting both the tumor mass and its blood vessels is a new strategy to improve the traditional chemotherapy, and to set the conditions for a more efficient anticancer therapy. To evaluate the antivascular and antiangiogenic activity, we tested TR-764 effects on human umbilical endothelial cells (HUVEC). Cell morphology was observed by optical microscopy and immunofluorescence analysis were helpful to visualize both the actin and the tubulin cytoskeleton. Cell migration was followed out at different time points by the wound assay, and cell permeability was evaluated measuring the ability of dextran-FITC to filter a HUVEC monolayer. Antiproliferative analysis were performed by MTT assay and cell cycle was analyzed by PI staining and flow cytometry. Antivascular activity was investigated plating HUVEC cells on a Matrigel matrix, which recreate capillary-like structures in vitro. TR-764 was tested on the chick embryo chorioallantoic membrane, a highly vascularised membrane normally used as an in vivo angiogenic model, and on BL16 mice allogenic tumors by administrating or a single dose (30 mg/kg) or repeated doses for 18 days of the compound. TR-764 shows high affinity to colchicines-binding site of tubulin, and it inhibits tubulin cytoskeleton polymerization. It affects HUVEC extended shape leading to a more rounded morphology, and it increases cell adhesion. In contrast, TR-764 treatment significantly impairs VEGF-stimulated cells adhesion and increases the permeability of a cell monolayer. Cell-to-cell contacts are reduced by the treatment, as demonstrated also by a downregulation of VE-cadherin phosphorylation (Tyr658). This new compound strongly reduces endothelial migration and disrupts tubule-like capillaries in vitro at non-toxic and nanomolar concentrations (1-10nM) without affecting endothelial proliferation and cell cycle regulation. These results support the hypothesis that TR-764 may act as an antivascular agent, and it is confirmed by in vivo analysis on chick chorioallantoic membrane. TR-764 (3-300nmol/egg) remarkably blocks the bFGF proangiogenic activity by significantly decreasing the number of vessels. Moreover preliminary experiments on a murine allogenic tumor model demonstrate a strong reduction of tumor mass. TR-764 is a novel antimitotic molecule with a strong potencial as antivascular disrupting agent that could improve the common anticancer therapies.