Thiosemicarbazones Regulate ROS EPR Signaling By Inhibiting SOD1 Activity

Reactive oxygen species (ROS), produced in the process of the aerobic metabolism, collect terms of chemically reactive molecules containing oxygen. ROS can not only affect the structure and function of proteins through a variety of approaches, but also act as cellular signaling molecules.As signaling molecules, ROS transmit signals from extracellular to cells, and then interact with intracellular target proteins to stimulate the signaling cascade reaction through cytomembrane. Mitogen-activated protein kinases (MAPKs) are important protein kinases that could transmit signals from cell surfaces to cell nucleus. Therefore, activation of MAPKs is the Centre of various signaling pathways, which plays a key role in cell proliferation signaling pathway and upregulates the expression of many essential genes. And so, activation of ERK pathway, whose symbol is the phosphorylation of ERK, is closely related with differentiation, cancerization and deterioration.ROS palys an important role in the activation of MAPK. Signaling regulation highly depends on the level of intracellular ROS. In detail, locally high concentrations of H2O2 can oxidize the cysteine residues in active sites to induce the inactivation of PTP, and thus enhances the phosphorylation of tyrosine residues of ERK to active the signal transduction in MAPK signaling pathway. SOD 1,which is the most important antioxidant enzyme to maintain the homeostasis of O2- and H2O2 via the dismutation, is one of the key proteins that regulate redox signal transduction in cells like MAPK. Inhibition of SOD1 can decrease the level of H2O2, which will effect the oxidation of PTP, the dephosphorylation of ERK1/2, and the redox signaling networks.Specific chelators were used to control the activity of SOD1, the concentration of H2O2 and O2-, and the ERK signaling pathway. Understanding the activation mechanism of kinases in MAPK signaling pathway was significant for disease pathogenesis and physiological process.We mainly carried out the work in following areas:1. Series of chelators were synthesized, and structural optimization of chelators had obvious advantages in coordination, trans-membrane, and inhibiting ability. Reasonable combination of chelate groups and active groups provided a basis for inhibitor design and structural optimization.2. RT-PCR and Western Bolt were used to research the expression of SOD 1, which confirmed the approach of SOD1's activity inhibition.3. Potentiometric titration, EPR, CD spectroscopy, SAXS and AutoDock were used to research the structure, the property, the structure activity relationship, and the mechanism of inhibitors. Based on the structure and catalytic mechanism of SOD1, synergies and inhibitory mechanisms of the chelate groups and active groups were discussed.4. Based on theoretical and experimental results, the relationship between the dosages of inhibitors, the IC50, and the concentration of intracellular H2O2 and O2·- was analyzed. Considering the effects on the level of intracellular ERK1/2 and its phosphorylation by inhibitors, the relationship between SOD1 activity and MARK signaling pathway was determined, which was significant for the research of ERK and NF-?B signaling pathways.5. By detection the activities of cytochrome c oxidase and tyrosinase, the effects on the transmission of intracellular copper ions and the selectivities for other copper-containing proteins were analyzed, which was important to study inhibitors targeting.6. The mechanisms of regulating apoptosis and death by intracellular redox signal transduction were discussed via controlling the intracellular relative levels of H2O2 and O2- in different cell lines.