Synthesis of palladium complexes to inhibit papain : a cysteine protease

Enzymes are proteins that act as biological catalysts increasing the kinetics of naturally slow biochemical reactions. In the very vast diversity of enzymes, cysteine proteases contain a set of enzymes able to hydrolyze the peptide bonds of other proteins available in the environment after being recognized in their active site. They represent a subclass of the protease family by owning a cysteine containing a thiol that is catalytically active. Papain is a cysteine protease that is generally operated as an enzyme model for biochemical studies. Isolated from a fruit tree, Carica papaya L., 121 amino acids constitute it and three of them are located in the active pocket: Cys 25, His 158, Asn 175. This active site at the interface between an all-α and all-β sheet domain contains a large hydrophobic pocket providing the driven-force for the folding of the protein. Buchwald have recently reported palladium methods for the S-arylation of cysteine residues (Vinogradova et al., 2015). This metal-mediated modification of proteins is becoming an important field for the discovery of new inhibitors for its ability to combines a large diversity of transition metal catalysts and chemical biology. A new class of inhibitors has emerged which allows to go beyond the limitations brought by the irreversible and the reversible inhibitors developed so far. Targeted Covalent Inhibitors (TCI) take the features of both types to establish its mechanism of action in two steps. The enzyme firstly recognizes the inhibitor by weak interactions with the active site. Then, the formation of an irreversible covalent bond lead to the inactivation of the species. Since 2004, Joyce and Hanahan as well as Rakashanda and co-workers have shown the role of cysteine proteases in the progression and the growth of tumor and their involving in phenomena like apoptosis, cell invasion and proliferation (Joyce and Hanahan, 2004; Rakash et al., 2012). Discovery and development of irreversible and site-selective cysteine protease inhibitors is a very promising strategy to avoid tumor formation and metastasis. This master thesis proposes an organic synthesis method and biochemical evaluations of two potential targeted covalent inhibitors to inhibit papain. On one side, a chemistry part describes the way of synthesis and the characterization of these both inhibitor complexes. On the other side, a biochemistry part evaluates their activity on papain. This part encompasses the development of an enzymatic test and the set-up of two inhibition tests. A time-dependence test to quantify the dependence of the inhibition effect on the time of incubation between papain and the inhibitor characterizing an irreversible inhibitor. The establishment of a dose-response curve allows to evaluate the potency. Finally, complementary tests were performed in the presence of negative controls to discuss the specificity of inhibitors. The results constituting a preliminary work are complemented by some perspectives that can be taken in account for further studies.