Mechanistic Investigations On The Protein Reactions Of Platinum-and Arsenic-Based Antitumor Drugs

Platinum-and arsenic-based drugs are successfully used in clinic for the cancer chemotherapies. The mechanisms of platinum and arsenite drugs are closely related to their interactions with the sulfur-containing of proteins. The sulfur-containing copper transport proteins are proposed to be important for the cellular uptake, transfer, and efflux of platinum drugs. Zinc finger proteins (ZFPs) are proved to be one of the important and direct targets of arsenite compounds. In this dissertation, the interactions of platinum compounds with the copper chaperone Cox17are investigated, and the interaction of arsenite with zinc finger proteins are studied. The influences of celluar small molecules on these interactions were also studied.In the first chapter, the mechanism of platinum drugs and arsenic compounds are reviewed, including the DNA targets of platinum drugs and mechanisms of DNA damage induced apoptosis; the multi-targets of platinum drugs; the relationship of platinum drugs and the copper transport proteins. The dual function of arsenic in both the carcinogenicity and the cancer chemotherapy are discussed.In chapter2, the interaction of cisplatin and human copper chaperone Cox17was investigated. The protein was overexpressed in E. coli and purified using a Ni-NTA column and gel filtration chromatography. UV-visible spectroscopy, High Performance Liquid Chromatography (HPLC), ICP-MS and ESI-MS method were used to study the interaction of Cox172s-s with cisplatin. In order to verify the coordination of copper binding site in the cisplatin reactions, experiments are also performed on the oxidized state Cox173s.s as the control. Results show the copper binding site of Cox17is also the major binding site of cisplatin. The formation of Pt-S bond causes the release of ammine ligands of cisplatin and the nacked platinum binding is observed with long time reaction. In addition, cellular uptake assay on cancer cells demonstrates that copper chaperone protein Cox17is involved in the transport of cisplatin to mitochondrial. This result further confirmed and supplemented the conclusions that copper transport proteins are involved in cell transfer process of cisplatin.In chapter3, the reactions of Cox172s-s with three platinum compounds cisplatin, transplatin, and oxaliplatin have been studied, and the influences of glutathione (GSH) on these reactions have been investigated. Results show that three platinum complexes are able to bind to Cox172s-s at the copper binding site. Transplatin and oxaliplatin bind to Cox17with mode different from cisplatin. Transplatin generates {Pt(NH3)2}-Cox17and {Pt2(NH3)4}-Coxl7adducts, and oxaliplatin generates {Pt(DACH)}-Coxl7and {Pt2(DACH)2} adducts. In addition, three platinum complexes can also react with CuICox172s-s, leading to the rapid release of copper from Cox17. It can be inferred that the binding of platinum complexes can leads to disruption of the function of Cox17. Glutathione shows different effects on the platination of Cox17by different platinum complexes. When the reactions were carried out in the presence of physiological concentration of glutathione, the platination of Cox17by cisplatin, transplatin and oxaliplatin were increased, decreased and unchanged respectively. Moreover, the cisplatin-GSH adducts still show high reactivity to Cox17while transplatin-GSH and oxaliplatin-GSH adducts almost can not react with Cox17. These findings suggest that the interaction of platinum drugs with cellular proteins will be highly affected by glutathione. Moreover, the influences of glutathione are very different depending on platinum species.In chapter4, the reactions of sodium arsenite with different ZFPs have been investigated. The single-domain and multi-domain zinc finger proteins of C2H2, C3H and C4types are expressed for the arsenite reactions. The binding affinity and reaction rate are determined using fluorescence specctroscopy. The product identities are characterized using ESI-MS, and the protein structure perturbations are verified using Circular dichroism and2D1H-15N HSQC NMR spectroscopy. The apparent dissociation constant and the pseudo first order rate constant of the reaction of arsenite and ZFPs are calculated. Results show that both the binding affinity and reaction rates of single-domain ZFPs follow the trend of C4> C3H>> C2H2. Compared with the C2H2motif ZFPs, the binding affinities of C3H and C4motif ZFPs are nearly two orders of magnitude higher and the reaction rates are about two-fold higher. The formation of multi-domain ZFPs significantly enhances the reactivity of C2H2type of ZFPs, but has negligible effects on C3H and C4ZFPs. Consequently, the reactivities of three types of multi-domain ZFPs are rather similar. This result indicates that the number of coordination sites of protein is the most critical factor determining the binding affinity.In chapter5, the NCp7-zf2is used as a model protein to investigate the competition of arsenic and zinc binding to ZFPs and the influence of solution conditions on the reaction. Spl-zf2was used as a model protein to investigate the influence of small thiol-containing biomolecules (GSH, Cys) on the interaction of arsenic with two-site protein. Results show that the interaction of arsenic with ZFPs is involved in the competitive binding of arsenic and zinc to ZFPs. Zinc competes overwhelmingly with arsenic for ZFPs in nomal neutral solution, and arsenite can not interferes with zinc finger structure. However, the reaction conditions (such as pH decreases, GSH, Cys, His, etc.) can change the competitive balance of arsenite and Zn2+. Under these conditions, arsenic could replace zinc on ZFPs. The affinity of zinc and ZFPs is greatly affected while the affinity of arsenic and ZFPs is less affected under these conditions. The presence of GSH or Cys not only improves the affinity but also promotes the reaction rate of arsenic binding to Spl-zf2. The mass spectrometry results suggest that GSH or Cys promotes the stability of adducts of arsenite by coodinating to the third site of arsenic. These results suggest that the biological mechanisms of arsenic largely depend on the intracellular environment.