The synthesis, enzymatic evaluation, and molecular modeling studies of new fluorogenic tetrapeptide-based substrates selective for caspase 8, having the general structure Ac-IETD-AXX, are described. Various fluorescent reporter groups (AXX), i.e., 3- and 4-substituted coumarins and quinolin-2(1H)-ones were synthesized by von Pechmann condensation. They were subsequently coupled with the caspase-8-selective tetrapeptide Ac-IETD-OH under newly developed synthetic conditions to give the desired substrates in good yields and in high enantiomeric purity. Based on K(M) and V(max) values, the new compounds proved to be excellent substrates for recombinant human caspase 8. In contrast, the K(M) values for the same compounds as substrates for human caspase 3 were approximately 10-20-fold higher. Molecular modeling studies based on the X-ray crystal structures of both human caspases 3 and 8 revealed that there is sufficient room within both active sites to accommodate substrates with moderately bulky substituents in the 3- and 4-positions of the fluorogenic coumarins and quinolin-2(1H)-ones. Automated docking of the substrates into the active sites of both human caspases 3 and 8 with the program AutoDock 3 gave structures similar to the published crystallographic structures for the same tetrapeptide bound to caspase 8 in the form of an irreversible inhibitor. The calculated binding energies for the new substrates to either caspase 3 or 8 showed little difference between the substrates, consistent with the K(M) data. In addition, the calculated binding energies (DeltaG) to caspase 8 were considerably more negative than those to caspase 3, also consistent with the K(M) data. A possible molecular interaction that might explain the selectivity of the IETD tetrapeptide motif for caspase 8 over caspase 3 is discussed.
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.
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