Abstract
This study was designed to examine the structure activity relationships (SARs) between thiol peroxidase (TPx) activity, cytotoxicity and anti-oxidant versus pro-oxidant properties for homologous series of phenyl alkyl selenides and tellurides PhMCnH2n+1 (M=Se, Te; n=4-7 i.e. Se4-7 and Te4-7). Theoretically, these compounds can act as anti-oxidant glutathione peroxidase (GPx) mimics and protect cells from OS by catalysing the reduction of reactive oxygen species (ROS) at the expense of the cellular thiol glutathione (GSH), which would be oxidised to the disulphide (GSSG). However, they could also act as pro-oxidants by catalysing the oxidation of alternative thiol substrates within the cell. Currently the SARs which determine the choice of thiol substrate, and therefore the overall pro- or anti-oxidant properties of TPx mimics, remain unknown. This thesis examines the relationship between the pharmacological properties of the phenyl alkyl selenides and tellurides and their lipophilicity and ability to catalyse the oxidation of the cellular thiols GSH (GPx activity) and dihydrolipoic acid (DHLA) (TPx activity) by H2O2.
Relative lipophilicity of the compounds was determined based on their retention in reversed phase HPLC. GPx activity (as GSH kobs rate constants) and TPx activity (as DHLA kobs rate constants) were determined using both an indirect GSSG reductase (GDR) coupled assay and a direct electrochemical assay (ECA). Cytotoxicity was determined toward A549 lung cancer cells. Anti-oxidant versus pro-oxidant activity was evaluated by exposing A549 cells to non-toxic concentrations of the TPx mimics in the presence of increasing concentrations of H2O2.
The results of both the ECA and GDR coupled assays were consistent in showing that tellurium compounds are stronger GPx mimics than their selenium analogues. However, the H2O2 removal rate in the GDR coupled assay was much bigger than in the ECA resulting from an artefactual increase in the apparent GPx activity of compounds in the presence of GDR and NADPH. The results of the study show that GPx activity was not related to lipophilicity and only Se7 and Te4 were active. In contrast, with the exception of Se4, all compounds displayed TPx activity using DHLA as substrate. Individual SAR trends were detected within series with DHLA kobs increasing with chain length for Se compounds and decreasing with chain length for Te compounds. All Se compounds displayed low cytotoxicity to A549 cells (IC50 ⩾100 µM) whereas all Te analogues were more cytotoxic with IC50 values in the range 10-30 µM. There was no relationship between cytotoxicity and GSH kobs but a correlation was identified between cytotoxicity and DHLA kobs, where the resulting SAR data could be fitted to the exponential function IC50=10+280e-5(DHLA kobs).
Investigation of the anti-oxidant or pro-oxidant activity of the compounds revealed neither Se nor Te based TPx mimics provided protection against H2O2 cytotoxicity. However, Te4 and Te5, the compounds with the highest DHLA kobs values, augmented the cytotoxic effects of H2O2 and therefore acted as pro-oxidants. The relationship between DHLA kobs and cytotoxicity could be fitted to the initial stages of a sigmoidal concentration-response curve indicating that there may be a relationship between TPx activity with DHLA and pro-oxidant action. Interestingly, however, simultaneous exposure of A549 cells to extracellular DHLA and Te4 provided some protection against H2O2 toxicity suggesting that Te4 may switch in mechanism from pro-oxidant to anti-oxidant under conditions of external DHLA administration. In conclusion, the cytotoxicity and pro-oxidant activity of these phenyl alkyl selenides and tellurides correlates with their ability to oxidise DHLA, but not their lipophilicity or ability to oxidise GSH.