PMID- 31847200 OWN - NLM STAT- MEDLINE DCOM- 20200428 LR - 20200428 IS - 1422-0067 (Electronic) IS - 1422-0067 (Linking) VI - 20 IP - 24 DP - 2019 Dec 13 TI - Reductive Activity and Mechanism of Hypoxia- Targeted AGT Inhibitors: An Experimental and Theoretical Investigation. LID - 10.3390/ijms20246308 [doi] LID - 6308 AB - O(6)-alkylguanine-DNA alkyltransferase (AGT) is the main cause of tumor cell resistance to DNA-alkylating agents, so it is valuable to design tumor-targeted AGT inhibitors with hypoxia activation. Based on the existing benchmark inhibitor O(6)-benzylguanine (O(6)-BG), four derivatives with hypoxia-reduced potential and their corresponding reduction products were synthesized. A reductase system consisting of glucose/glucose oxidase, xanthine/xanthine oxidase, and catalase were constructed, and the reduction products of the hypoxia-activated prodrugs under normoxic and hypoxic conditions were determined by high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The results showed that the reduction products produced under hypoxic conditions were significantly higher than that under normoxic condition. The amount of the reduction product yielded from ANBP (2-nitro-6-(3-amino) benzyloxypurine) under hypoxic conditions was the highest, followed by AMNBP (2-nitro-6-(3-aminomethyl)benzyloxypurine), 2-NBP (2-nitro-6-benzyloxypurine), and 3-NBG (O6-(3-nitro)benzylguanine). It should be noted that although the levels of the reduction products of 2-NBP and 3-NBG were lower than those of ANBP and AMNBP, their maximal hypoxic/normoxic ratios were higher than those of the other two prodrugs. Meanwhile, we also investigated the single electron reduction mechanism of the hypoxia-activated prodrugs using density functional theory (DFT) calculations. As a result, the reduction of the nitro group to the nitroso was proven to be a rate-limiting step. Moreover, the 2-nitro group of purine ring was more ready to be reduced than the 3-nitro group of benzyl. The energy barriers of the rate-limiting steps were 34-37 kcal/mol. The interactions between these prodrugs and nitroreductase were explored via molecular docking study, and ANBP was observed to have the highest affinity to nitroreductase, followed by AMNBP, 2-NBP, and 3-NBG. Interestingly, the theoretical results were generally in a good agreement with the experimental results. Finally, molecular docking and molecular dynamics simulations were performed to predict the AGT-inhibitory activity of the four prodrugs and their reduction products. In summary, simultaneous consideration of reduction potential and hypoxic selectivity is necessary to ensure that such prodrugs have good hypoxic tumor targeting. This study provides insights into the hypoxia-activated mechanism of nitro-substituted prodrugs as AGT inhibitors, which may contribute to reasonable design and development of novel tumor-targeted AGT inhibitors. FAU - Xiao, Weinan AU - Xiao W AD - Beijing Key Laboratory of Environmental & Viral Oncology, College of Life Science and Bioengineering, Beijing University of Technology, Beijing 100124, China. FAU - Sun, Guohui AU - Sun G AUID- ORCID: 0000-0003-2259-5431 AD - Beijing Key Laboratory of Environmental & Viral Oncology, College of Life Science and Bioengineering, Beijing University of Technology, Beijing 100124, China. FAU - Fan, Tengjiao AU - Fan T AD - Beijing Key Laboratory of Environmental & Viral Oncology, College of Life Science and Bioengineering, Beijing University of Technology, Beijing 100124, China. AD - Department of Medical Technology, Beijing Pharmaceutical University of Staff and Workers, Beijing 100079, China. FAU - Liu, Junjun AU - Liu J AD - Beijing Key Laboratory of Environmental & Viral Oncology, College of Life Science and Bioengineering, Beijing University of Technology, Beijing 100124, China. FAU - Zhang, Na AU - Zhang N AD - Beijing Key Laboratory of Environmental & Viral Oncology, College of Life Science and Bioengineering, Beijing University of Technology, Beijing 100124, China. FAU - Zhao, Lijiao AU - Zhao L AUID- ORCID: 0000-0002-4876-323X AD - Beijing Key Laboratory of Environmental & Viral Oncology, College of Life Science and Bioengineering, Beijing University of Technology, Beijing 100124, China. FAU - Zhong, Rugang AU - Zhong R AD - Beijing Key Laboratory of Environmental & Viral Oncology, College of Life Science and Bioengineering, Beijing University of Technology, Beijing 100124, China. LA - eng GR - 21778011/National Natural Science Foundation of China/ GR - CIT&TCD20180308/the Great Wall Scholars Program of Beijing Municipal Education Commission/ GR - PXM2015_014204_500175/Education Commission Science and Technology Project of Beijing Municipality/ GR - 7184192/Beijing Natural Science Foundation/ GR - 2017M620567/China Postdoctoral Science Foundation/ GR - 2018-ZZ-022/Beijing Postdoctoral Research Foundation/ GR - 2018ZZ-01-25/Chaoyang District Postdoctoral Research Foundation/ PT - Journal Article DEP - 20191213 PL - Switzerland TA - Int J Mol Sci JT - International journal of molecular sciences JID - 101092791 RN - 0 (Enzyme Inhibitors) RN - 0 (Prodrugs) RN - EC 2.1.1.63 (O(6)-Methylguanine-DNA Methyltransferase) SB - IM MH - Chromatography, High Pressure Liquid MH - *Drug Delivery Systems MH - Enzyme Inhibitors/*chemistry MH - Humans MH - Hypoxia MH - *Molecular Docking Simulation MH - *O(6)-Methylguanine-DNA Methyltransferase/antagonists & inhibitors/chemistry MH - Prodrugs/*chemistry MH - Tandem Mass Spectrometry PMC - PMC6941096 OTO - NOTNLM OT - HPLC-ESI-MS/MS OT - density functional theory OT - hypoxia-activated AGT inhibitors OT - molecular docking OT - reduction of nitro group OT - tumor targeting COIS- The authors declare no conflict of interest. EDAT- 2019/12/19 06:00 MHDA- 2020/04/29 06:00 PMCR- 2019/12/01 CRDT- 2019/12/19 06:00 PHST- 2019/11/01 00:00 [received] PHST- 2019/12/05 00:00 [revised] PHST- 2019/12/10 00:00 [accepted] PHST- 2019/12/19 06:00 [entrez] PHST- 2019/12/19 06:00 [pubmed] PHST- 2020/04/29 06:00 [medline] PHST- 2019/12/01 00:00 [pmc-release] AID - ijms20246308 [pii] AID - ijms-20-06308 [pii] AID - 10.3390/ijms20246308 [doi] PST - epublish SO - Int J Mol Sci. 2019 Dec 13;20(24):6308. doi: 10.3390/ijms20246308.