Theoretical Study On The Mechanism Of Hypoxia-Activated Prodrug Of O~6-Benzylguanine

Chloroethylnitrosourea(CENUs) is an important family of anticancer alkylating agents and currently used as one of the chemotherapeutic drugs in the clinical treatment of malignant tumors. CENUs can induce the generation of DNA interstrand cross-links(d G-d C), which can prevent the normal DNA replication, leading to the apoptosis of tumor cells. However, the intracellular O~6-alkylguanine-DNA alkyltransferase(AGT) can repair the DNA damages induced by CENUs, which can inhibit the formation of d G-d C cross-links, leading to the resistance of tumor cells against CENUs. Therefore, it is great importance to inhibit the AGT activity of tumor cells to improve the chemotherapeutic effects of CENUs. At present, a series of AGT inhibitors have been synthesized for combination chemotherapy with CENUs in which the O~6-benzylguanine(O~6-BG), a potential AGT inhibitor, is the first inhibitor entered clinical trials.But clinical studies have shown that O~6-BG not have a target. Improving tumor cells sensitivity to CENUs, while AGT also suppresses the normal cell activity, which resulted in the bone marrow inhibition of CENUs significantly enhanced.Therefore, the development of tumor-targeted inhibitors of AGT for improving the chemotherapeutic effectiveness of alkylating chemotherapy drugs is of great significance.Hypoxia environment araising from the rapid proliferation of tumor cells is one of the important characteristics of solid tumor organization, which can be used as a drug designed target. Therefore, it depends on the hypoxic tumor microenvironment, activated under hypoxic conditions and targeting tumor cells to the design and synthesis of biologically reductive prodrug. It has become a new way to improve the efficacy of chemotherapy drugs. However, there is rarely reports about the mechanism of hypoxia activated AGT inhibitors.Thus, the topic of using the method of computational chemistry of AGT inhibitor prodrug nitrobenzyl-guanine compound(NBGs) hypoxiaactivated reaction mechanisms were studied, and clarify NBGs by nitroreductase(NTR) and coenzyme FMNH under hypoxic mediated single electron transfer process and the reduction reaction mechanism for the development of hypoxia-activated targeting anticancer drugs provides a reliable theoretical basis.First, Density functional theory(DFT) at the B3LYP/6-31G(d,p) level to nitrobenzene as reaction model were studied, calculate nitro compounds of single electron transfer mechanism under hypoxic. It is concluded that nitrobenzene can get an electronic activated into nitro free radicals, the reaction activation energy of 35.35 kcal/mol, and further activation of the nitro group is reduced to form ammonia and hydroxylamine. The nitro group under aerobic conditions, activation of nitrobenzene will be lost electrons to the oxygen molecues, the reaction activation energy of 21.27 kcal/mol. The results showed that only under hypoxic conditions nitrobenzene will be restored.Second, under the conditions of the same method and basis set for nitrobenzyl guanine compound(NBGs) prodrugs in nitroreductase reduction mechanism(NTR) have been studied, optimized all molecular structure of the reactants, products, intermediate, the transition state, get all the reaction paths the potential energy curve. The results showed that under hypoxic conditions the two NBGs potential trends of the transition state of the reaction bond lengths and angles is very similar, there are two speed control step, which nitroso are generated in the transition state TS2 and hydroxylamine reduction reaction transition state TS5. So nitro prodrugs can be reduced to form amino prodrugs under hypoxic conditions, which the release of a higher AGT inhibitory activity.Third, using the method of molecular docking studied interaction of AGT enzyme and O~6-BG prodrug. The results showed that compared with the nitro prodrug, amino compounds with the enzyme active site Cys145 binding mode, location, and distance from the hydrogen bond has advantages, this suggests that after the reduction of nitro prodrugs have a higher activity of AGT inhibitors.The topic revealed the mechanism O~6-benzylguanine prodrug targeting activated under hypoxic conditions and in the reduction reaction mechanism of hypoxic conditions, optimized the reaction of the reactants, products, intermediates and transition states, obtained activation energy barrier, Gibbs free energy and geometry parameters. Potential energy curve to elucidate the reaction mechanism. By molecular docking results illustrate the amine prodrugs is more active than the nitro prodrugs. This study will provide adequate theoretical basis for the low oxygen activation targeted antitumor AGT inhibitor study.