The Effect Of Sulfur-containing Protein On DNA Platination By Trans-platinum Complexes

Platinum complexes have been used as antitumor drugs for more than30years, the interactions of cisplatin with biological molecules have been deeply studied. After platinum drugs are administrated, they can interact with many sulfur-containing biological molecules, and these interactions can influence the uptake, transport, activity, efflux and other biological processes of platinum drugs.In this work we studied the interactions of platinum complexes with sulfur-containing biological molecules, and the effect of the interaction on the DNA platination. The studies contain the following three parts:(1) Using L-Methionine or N-acetyl-L-Methionine as a model of sulfur-containing protein to study their effect on the reactions of trans-platinum complex with DNA, and comparing the difference results of trans-platinum complex with cis-platinum complexes.(2) Using a7AA model peptide and histone H1N90to study their reactions rate and products with cis and trans-platinum complexes, and studying the interactions of these products with DNA, to understand the role of protein in DNA platination.(3) Studying the effect of pH on the interaction of trans-platinum complex with DNA model molecule (GMP), studying the interaction of buffer anions with trans-platinum complex and the effect of the interactions on the GMP platination by trans-platinum complex.In chapter1, we provide a brief review about the interaction of trans-platinum complex with sulfur-containing molecules. It mainly contains the diversity of antitumor mechanism between cis and trans-platinum complexes, the interactions of cis and trans-platinum complexes with sulfur-containing molecules and proteins, the effect of these interactions on the DNA platination by platinum drugs, the functions of histone H1, the recognization of histone H1to DNA modified by trans-platinum drugs, the effect of pH and buffer anion on the reaction of cisplatin with GMP.In chapter2, the effect of methionine on the reaction of DNA with trans-platinum complex was studied. We found that trans-EE could form monofunctional adduct with methionine (Met), and this monofunctional adduct reacted much faster with DNA than trans-EE itself. But this enhancement was never found in the reaction of cisplatin with DNA. Moreover, the platination rate was found to be pH-dependent, which could correlate with the different drug sensitivity of various tumors. The binding of Met-containing peptides to trans-EE was also shown to occur readily in cellular systems. Taken together, the present results clearly demonstrate that S-donor molecules can play completely different roles in the cellular processing of trans-platinum drugs compared with antitumor drugs with cis geometry.In chapter3, we further studied the reaction of trans-EE with methionine. A detailed investigation has shown that in the reaction of trans-EE with methionine at low NaCl the bisadduct is formed rapidly and is the dominant species as long as free Met is present. In contrast, at high NaCl concentration the monoadduct is formed preferentially. These results suggest that different reaction products may be formed in the extra-or intracellular environment. The bisadduct frans-Pt(E-iminoether)2(AcMet)2, although completely deprived of leaving chlorido ligands, can react rather fast with GMP forming the coordinatively bound ternary adduct AcMeX/trans-EE/GMP. Altogether, these results indicate that coordination to DNA of platinum-protein adducts is highly feasible for trans-configuration platinum complexes. It has also been shown that trans-Pt(E-iminoether)2(Met)2is stable in acidic condition, whereas an intramolecular S to N isomerization of one Met occurs at neutral pH. Such an isomerization can be applied to explain the formation of cis-Pt(Met-N,S)2in the reaction of cis-DDP with excess methionine.In chapter4, the effect of buffer on the reaction of trans-EE with GMP was studied. The reaction rate of trans-EE with GMP is obviously affected by pH. Hepes buffer decreases the reaction rate, because of its weak interaction with platinum and its effect of ionic strength on electrostatic interaction. Carbonate can react with trans-EE, forming monofunctional adducts. So carbonate decreases the rate of platination of GMP, because of its competition with GMP. Phosphate can form ternary adduct with trans-EE and GMP, the adduct is {trans-Pt[(E-Iminoether)2(H2PO4)(GMP)]}+, so phosphate doesn’t compete for platinum. The actual effect of phosphate buffer is ionic strength, which is similar to Hepes and different from carbonate buffer. In conclusion, the reaction character of buffer, ionic strength and pH can influence the reaction rate of platinum drugs with biological molecules.In chapter5, we studied the reaction rates and products of cis and trans-platinum complexes with peptide H1N7and protein H1N90, and the further interactions of these products with DNA. The1/2n of reactions of H1N7with cis-DDP, trans-EE and trans-PTZ are1.8h,14min,<10min respectively. When reacting with H1N7, cis-DDP doesn’t lose its two NH3ligands. This means that its antitumor activity is still observed. The products of H1N7with trans-EE and trans-PTZ are both trans chelates. The interactions of these products with DNA are under further investigation. We also expressed and purified histone H1N90. We use ESI-MS to study the products of H1N90with cis-DDP, trans-EE and trans-PTZ. The major product of H1N90with cis-DDP is bidentate chelate, one NH3ligand is observed. The predominant product of H1N90with trans-EE is also bidentate chelate, two iminoether ligands are still observed. H1N90and trans-PTZ mainly form tridentate chelate, only one non-leaving group of trans-PTZ (NH3or thiazole) is observed. The products of H1N90with cis-DDP and trans-PTZ both can react with ss and ds-DNA, forming protein-Pt-DNA ternary product. This implies that protein may transport platinum to target DNA.