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

Caspases are cysteine proteases and are considered key-mediators 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. AutoDock 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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Correspondence to R Schulz.

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Open Access This article is distributed under the terms of the Creative Commons Attribution 2.0 International License (https://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Keywords

  • Apoptotic Cell Death
  • Dock
  • Cysteine Protease
  • Docking Result
  • Detect Cell Death