Design and synthesis of cysteine protease inhibitors utilizing S-nitrosation mechanism

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)



First Committee Member

P. George Wang, Committee Chair


The natural product dephostatin and its unsubstituted analog N-methyl-N-nitroso-aniline were identified as irreversible inhibitors of cysteine protease papain. A series of substituted N -methyl-N-nitrosoanilines were then synthesized to further explore the substitution effect on inhibition potency. The inactivation process was found to be competitive and reversible. Based on this initial finding, ten peptidyl N-nitrosoanilines were designed, synthesized and evaluated as inactivators of the cysteine protease papain and the serine protease chymotrypsin. These new compounds exhibited different inhibitory activities towards the cysteine protease papain in a time- and concentration-dependent manner. No inactivation was observed for the serine protease chymotrypsin. Formation of the S-NO bond in papain is supported by several lines of evidence from both spectroscopic studies and chemical analyses. The pH profile study on the inactivation of papain was conducted over a pH range 3.2--9.2 to provide more insight into the mechanism of the inactivation process. The covalent yet reversible cysteine protease inactivation process offers mechanistic implications and endows this new family of inactivators with special properties that are suitable for the development of stable and potent cysteine protease inhibitors.The S-nitrosation inhibition mechanism was extended to the apoptosis relevant cysteine protease caspase. Quenched dynamics was used to generate a pool of accessible conformations for caspase-1 substrate Ac-YVAD-NMe, from which three representative members were studied in detail by molecular dynamics at 300 K. To predict the binding mode of a designed inhibitor and evaluate its binding contacts with a receptor, a modeling strategy was proposed in which two computational tools AUTODOCK and AMBER dynamics simulations can be used in a sequential manner. This methodology has been validated in a model study on caspase-1/Ac-YVAD-CHO complex and applied to the design of caspase-3 inhibitors. Two peptide based inhibitors were designed to utilize the S-nitrosation mechanism. They were tested against caspase-3 and showed inhibitory potency.


Chemistry, Organic; Chemistry, Pharmaceutical

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