Advanced Kinetics of Novel, Non-peptidic Thiosemicarbazone Inhibitors of Cathepsin B

The lysosomal cysteine protease cathepsin B is considered to be an attractive target for the synthesis of anticancer drugs due to its over-expression in cancerous cells and its involvement in tumor formation. Among its many roles in the advancement of several pathological events including Arthritis and Alzheimer’s disease, cathepsin B is primarily responsible for initiating cancer metastasis as it facilitates the degradation of the extracellular matrix. In order to impede the process of angiogenesis and to prevent the spread of cancerous cells, this study aims to identify an effective, non-toxic inhibitor of cathepsin B from a library of novel, non-peptidic thiosemicarbazone (TSC) compounds which have shown to be effective inhibitors of the cysteine protease cathepsin L. To evaluate the compounds’ inhibitory effects, the concentration of the TSC inhibitors causing a 50% reduction in enzymatic activity (IC50 value) was determined. After an optimization of assay conditions, experiments were performed in a 96-well microplate using the substrate Z-arginylarginyl-aminomethylcoumarin which reacts with cathepsin B and releases the fluorescent product, aminomethylcoumarin. Advanced kinetic studies of the most potent inhibitors were performed to determine the compounds’ mechanism of action. All three of the compounds studied exhibited reversible inhibition. Two of these inhibitors, a bromo-substituted, acetylated phenolic benzophenone TSC and a bromo-substituted, di-trifluoromethyl benzophenone TSC exhibited time dependent inhibition; whereas, the monohydroxy-tetramethozy-substituted benzophenone TSC displayed classical competitive inhibition against cathepsin B activity. By developing a database consisting of TSC compounds with IC50 values in the low micromolar to nanomolar range, it is possible to examine the kinetics and structure-activity relationship of TSC inhibitors which could potentially be used medically to decrease tumor progression and cancer metastasis. These studies represent a collaborative project between the Trawick (biochemical) and Pinney (synthetic) Laboratories.