Selectivity of new caspase 3 and 8 tetrapeptide substrates can be explained by automated docking analysis

Abstract

Caspases are cysteine proteases and are considered keymediators in apoptotic cell death. Selective quantification of the various caspase activities in cancer cells is important for detecting cell death caused by cancer therapy. We have designed and synthesized a series of novel fluorogenic tetrapeptide substrates for caspase 8 and investigated the substrates for selective cleavage by either caspases 3 or 8 in enzyme assays. At the same time we have used the automated docking program AutoDock (ver 3, [1,2]) to dock the new substrates into the active sites of X-ray crystal structures of human caspases 3 and 8, respectively. Auto-Dock was confirmed to be an appropriate tool for substrate binding prediction because substrate docking results are comparable with documented X-ray crystal structure of caspase 3 and 8 bounded with analogous tetrapeptide inhibitors [3,4]. Enzyme-substrate conformations with changes in free energy of binding (ΔG) were calculated with AutoDock and compared to the experimental determined Michaelis-Menten constant Km. A significant correlation between the experimental Km and theoretical ΔG was found. Enzyme kinetics showed the substrates to have 100-fold lower Km-values for caspase 8 compared to caspase 3. This selectivity was reflected in the significantly larger negative ΔG-values between the substrates docked to caspase 8 as opposed to caspase 3. These results will help in the design of even more selective caspase substrates.