Bearing this in mind, kinetic studies were performed PARP Inhibition with tideglusib and the mutant enzyme in order to compare the results obtained with the wildtype version. Progress curves in the presence of several concentrations of tideglusib were obtained, and the experimental data were processed as above. As was the case with the wild type enzyme, the kobs displayed a linear dependence on the concentration of tideglusib rendering a k3/K1 value of 1.910.15102 M1 s1 and a k4 of 8.80.8105 s1. It is noticeable that the C199A mutation led to a decrease in the k3/K1 value of 1 order of magnitude with respect to that of the wild type enzyme, suggesting that Cys 199 replacement decelerated the process of E I formation. The most relevant information was provided by the dissociation constant k4, which is 4 fold higher and, more remarkably, significantly different from zero. Still, tideglusib showed a long residence time in the mutant enzyme, but the fact that k4 was distinct from zero suggests that this GSK 3 mutation turned tideglusib from an irreversible to a longstanding reversibleinhibitor, and this might be consistent with the formation of a covalent bond between the drug and Cys 199. However, the long half life and the reduced but still significant potency suggests that the Cys residue is not totally essential for the inhibitory activity of the compound. To shed light on the mode of interaction of tideglusib with GSK 3, binding experiments with radiolabeled tideglusib were performed. The aim of these experiments was to investigate whether the compound remained bound to the enzyme after having subjected the complex to denaturing conditions. Given that the possible interaction with Cys 199 might involve the formation of a disulfide bond with the sulfur atom of the TDZD ring, the effect of a reducing agent was also explored. GSK 3 and tideglusib were mixed and incubated at room temperature before adding buffer with or without DTE. The samples then followed a double pronged strategydesigned to obtain both qualitative and quantitative information of the same approach, while a small aliquot was submitted to electrophoresis under denaturing conditions followed by fluorography, the remaining part of each sample was either treated with a chaotropic agent or left untreated before removing the unbound drug by gel filtration chromatography on Sephadex G 25 and measuring the remaining radioactivity by liquid scintillation counting. As can be observed in Fig. 7A, only the DTE untreated samples rendered a very faint band, hardly visible to the naked eye, which could be detected in the fluorography at a position corresponding to the molecular mass of GSK 3 only after very long exposure times. Although the presence of a band could suggest covalent modification, such a very weak response with research chemicals library respect to the amount of GSK 3 loaded on the gel and the specific activity of the radiolabel prevents us from obtaining clear conclusions from this result and rather points to a possible artifactual nature. Furthermore, the quantitative analysis after gel filtration shows that whereas the sample treated under non denaturing conditions presented significant levels of tideglusib binding, these levels were notably reduced when the sample was denatured wit.