Targeting cancer through inhibition of cathepsin B by non-peptidic small molecule thiosemicarbazones and disruption of pre-existing vasculature by colchicine-like benzosuberene analogues.

Cancer is a leading cause of death in men and women under in the United States and is characterized by uncontrolled cellular proliferation and migration (metastasis) which can impinge on surrounding organs, modify ordinary biological functions, and lead to death. This study focuses on two strategies for cancer therapy: targeting cathepsin B, an enzyme linked to tumor metastasis and progression, and the disruption of pre-existing tumor vasculature as a means to starve tumors of oxygen and nutrients. Cathepsin B is a cysteine protease involved in intra- and extracellular degradation of proteins. Increased expression of cathepsin B has been documented in a number of different cancers and is associated with a poor disease prognosis, and increased tumor vascularization, degradation of the extracellular matrix, invasion, and metastasis. Inhibition of cathepsin B has the potential to arrest cancer cell invasion and metastasis. In a collaborative project between the Trawick and Pinney laboratories at Baylor University, a focused synthetic library of non-peptidic, small molecule thiosemicarbazone derivatives was screened for their ability to inhibit cathepsin B activity as monitored by a fluorogenic enzyme assay. Five compounds were found to be effective inhibitors of cathepsin B in the low micromolar range, and the best four were characterized for their mode of inhibition. Kinetic analysis revealed that two of the active thiosemicarbazone compounds were time dependent, competitive, tight binding, slowly reversible inhibitors of cathepsin B. The other compounds analyzed were rapidly reversible, competitive inhibitors with KI values in the low micromolar range. Vascular disrupting agents (VDAs) are a promising class of anticancer drugs that selectively disrupt tumor vasculature. Tubulin-binding VDAs disrupt microtubule dynamics of endothelial cells lining tumor vasculature. A lead benzosuberene analogue exhibited extreme cytotoxicity against a panel of human cancer cell lines. The lead compound and several of its analogues were investigated for their ability to inhibit tubulin polymerization, bind to the colchicine binding site of tubulin as determined by a competitive radiometric binding assay and arrest human breast cancer cells in the G2/M phase of the cell cycle as indicated by flow cytometry. The results support the mechanism of action of the lead benzosuberene analogues as VDAs.