Executive Summary : | Copper is an essential bioelement for a wide range of enzymatic reactions, but the presence of high-level Cu leads to cytotoxicity, associated with several diseases such as Wilson’s disease (WD), neurological disorder, and cancer. Overloaded cellular Cu, generally, creates an abnormal Cu–protein interaction, which promotes the formation of reactive oxygen species (ROS) and aggregation of the protein. Therefore, high-level cellular Cu concentration is a primary target for the treatment of Cu-overload diseases. Several studies have reported that tetrathiomolybdate (TTM) is a promising anti-copper drug for the treatment of WD, and cancer, but unfortunately, it has limited applications in the clinic, perhaps due to its unusual binding mode and redox chemistry, and unwanted product formation with cellular components (such as glutathione, transition metals, proteins, ascorbic acid, and H₂O₂). Often these factors are neglected or overlooked, but those may lead to biased results. Therefore, the major aim of this project is to need a systematic study including experimental proof and qualitative/quantitative assay of product formation during the reaction of TTM with cellular components to understand the clinical efficacy, toxicity, and fate of TTM. This project will address a vitro model in designed conditions to mimic the cellular environment, which would be a complementary model approach to investigate the complex biochemical process. The extracellular protein, the most abundant protein, namely human serum albumin (HSA) is found in blood, raising the Cu-level cause of the protein aggregation and ROS formation. In abnormal conditions, plasma contains also free Fe. Therefore, HSA will be treated with various concentrations of Cu (or Fe) solution in the biological environment to form various intermediates. Each intermediate may have a different structural arrangement and binding site at Fe/Cu-albumin that may generate a different degree of toxicity. The toxicity of each ratio (Cu/Fe : albumin) will be measured in presence of the fixed amount of H₂O₂ and or ascorbic acid (AscH). The overload Cu/Fe-HSA will be also treated with various concentrations of TTM to screen the best condition for the formation of non-aggregated HSA. A fixed amount of Cu/Fe-TTM-HSA will be treated with a variable amount of H₂O₂ to evaluate the inhibition rate of ROS. Switch to intracellular, the intracellular redox balance and copper homeostasis are significantly controlled by glutathione (GSH). In cells, GSH can bind both Cu and Fe, yielding pro-oxidant species that may modulate the intracellular redox balance. This makes it a challenge for anti-copper metallodrug activity in cells. Therefore, the stability, redox chemistry, and mode of action of TTM with Fe/Cu in presence of GSH will be studied. Overall, the findings will help the bioinorganic/biomedical researchers and also clinical practitioners who are applying TTM to patients with WD and cancer across the world. |