The Oretical Studies On The Formation Of DNA Interstrand Crosslinks Induced By Chloro-Ethylnitrosourea And The Repair Mechanism

Chloroethylnitrosoureas (CENUs) are an significant family of anticanceralkylating agents widely used in the clinical treatments of various humanmalignancies, including brain tumor, melanoma, lymphoma and other solid tumors.Several lines of evidences indicate that CENUs induce the formation of DNAinterstrand crosslinks between G-C base pair. However, the reaction mechanismsleading to G-C crosslinks and the repair mechanisms of DNA alkylation for G-Ccrosslinks with the participation of alkyltransferase have not yet been clearlydescribed. This thesis is mainly involved in the study of the mechanisms of theformation and repair of DNA interstrand crosslinks induced by CENUs by u singquantum mechanical methods combined with molecular modelling protocals.Three reaction pathways for the mechanisms of the formation of CENUs-inducedG-C crosslinks have been explored through DFT-B3LYP computation at the6-31+G(d,p) theoretical level. The geometric structures of all reactants, trasition states,intermediates and products were optimized and the potential energy profiles wereobtained. The results indicated that the optimal route for the formation ofG(N1)-C(N3) was through the intermediate O~6,N1-ethanoguanine (O~6,N1-EtG) whichwas started with the alkylation of guanine by chloroethyl diazonium ion on G-O~6siteto the formation of O~6-chloroethyl-guanine (O~6-ClEtG). The intermidiate O~6,N1-EtGwas formed by intramolecular cyclization of N1and Cβ. The Cβof O~6,N1-EtGattacked the N3site of cytosine to form the G(N1)-C(N3). The intramolecularcyclization was the ratedeterming step of the whole crosslinking process, which had aenergy barriers of20.59kcal/mol. G(N1)-C(N3) was the most favorable crosslinkingproduct in both dynamics and thermodynamics compared with the other two possibleproducts, G(O~6)-C(N~4) and G(N~2)-C(O~2). These results were consistent with ourstudies on BCNU-induced G-C crosslink in calf-thymus DNA by HPLC-MS/MS. Thework provided a convincing theoretical explanation for the experimental researcheson the DNA crosslinking mechanisms induced by CENUs.The resistance of tumor cells to anti-cancer agents imposes restrictions on furtherdevelopement and application in the clinical employment of nitrosourea. Meanwhile,the clarification of the mechanism of drug resistance to CENUs become a hot spot. Inthe researches of the mechanisms of resistance to CENUs, the repair of DNA lesionsby O~6-alkylguanine-DNA-methyltransferase (AGT) has received the greatest attention.Although there were experimental reports on the repair of DNA interstrand crosslinks by AGT that was also investigated in this work, the repairing mechanism was not yetbeen clartified. In this work, quantum mechanical (QM), molecular mechanical (MM)and QM/MM methods were employed to reveal the mechanism of the repair of DNAinterstrand crosslinks by AGT. Firstly, the repairing mechanisms of O~6-ClEtG andO~6,N1-EtG by His-Cys dipeptides were explored with DFT-B3LYP computation at the6-31+G(d,p) theoretical level. Secondly, the Amber molecular fields of O~6-ClEtG andO~6,N1-EtG were estabolished by the Amber program package. The moleculardynamic (MD) modeling of AGT-O~6-ClEtG and AGT-O~6,N1-EtG were performed toobtain the stable conformations of the two molecules. Finally, the active sites of thesetwo stabe conformations were picked out. The mechanisms of the repair of O~6-ClEtGand O~6,N1-EtG by AGT were explored by ONIOM computations atB3LYP/6-31+G(d,p):AM1theoretical level.The results showed that the formation of AGT-G crosslink was more favorablethan ClEtAGT，which suggested that the O~6,N1-EtG was easier to be repaired by AGTthan O~6-ClEtG. The O~6-ClEtG was more likely to form O~6,N1-EtG intermediate byintramolecular cyclization, and less likely to be repaired to form ClEtAGT. TheO~6,N1-EtG could be repaired by AGT to form DNA-protein crosslink, also could bereact with complementary cytosine to form G-C crosslink. The calculational resultsproved that the energy barriers of the two reactions above was close, which indicatedthat the formation of AGT-DNA crosslink by AGT repairing was competitive with theanticancer effect of G-C crosslink induced by CENUs. This work provided satisfyingexplanation for the existing conclusions obtained from experimental investigations.The topic revealed the mechanism of DNA interstrand crosslink induced byCENUs, and clarificated the repairing mechanism of DNA interstrand crosslinks byAGT. The The geometric structures of all reactants, trasition states, intermediates andproducts were optimized and the relative energies (E), enthalpies (H) and freeenergies (G) were obtained. By compairing the potential energy profiles of all theprobalble pathways, the most favorable pathway of crosslinking and repairing wasdetermined to reveal the mechanism. The research was of great significance fordesigning and developing efficient nitroso drugs and drug combination by depthunderstanding of the nitrosourea resistance induced by AGT.