Facile Oxidation of Leucomethylene Blue and Dihydroflavins by Artemisinins: Relationship with Flavoenzyme Function and Antimalarial Mechanism of Action

被引:72
作者
Haynes, Richard K. [1 ]
Chan, Wing-Chi [1 ]
Wong, Ho-Ning [1 ]
Li, Ka-Yan [1 ]
Wu, Wai-Keung [1 ]
Fan, Kit-Man [1 ]
Sung, Herman H. Y. [1 ]
Williams, Ian D. [1 ]
Prosperi, Davide [3 ]
Melato, Sergio [2 ]
Coghi, Paolo [2 ]
Monti, Diego [2 ]
机构
[1] Hong Kong Univ Sci & Technol, Dept Chem, Inst Mol Technol Drug Discovery & Synth, Kowloon, Hong Kong, Peoples R China
[2] CNR ISTM, Dept Organ & Ind Chem, I-20133 Milan, Italy
[3] Dept Biotechnol & Biosci, I-20133 Milan, Italy
关键词
artemisinins; flavins; flavoenzymes; malaria; oxidative stress; PARASITE PLASMODIUM-FALCIPARUM; METHYLENE-BLUE; GLUTATHIONE-REDUCTASE; AGENT ARTEMISININ; CENTERED RADICALS; MODEL SYSTEMS; ARTESUNATE; MALARIA; ANALOGS; FLAVIN;
D O I
10.1002/cmdc.201000225
中图分类号
R914 [药物化学];
学科分类号
100701 ;
摘要
The antimalarial drug methylene blue (MB) affects the redox behaviour of parasite flavin-dependent disulfide reductases such as glutathione reductase (GR) that control oxidative stress in the malaria parasite. The reduced flavin adenine dinucleotide cofactor FADH(2) initiates reduction to leucomethylene blue (LMB), which is oxidised by oxygen to generate reactive oxygen species (ROS) and MB. MB then acts as a subversive substrate for NADPH normally required to regenerate FADE!, for enzyme function. The synergism between MB and the peroxidic antimalarial artemisinin derivative artesunate suggests that artemisinins have a complementary mode of action. We find that artemisinins are transformed by LMB generated from MB and ascorbic acid (AA) or N-benzyldihydronicotinamide (BNAH) in situ in aqueous buffer at physiological pH into single electron transfer (SET) rearrangement products or two-electron reduction products, the latter of which dominates with BNAH. Neither AA nor BNAH alone affects the artemisinins. The AAMB SET reactions are enhanced under aerobic conditions, and the major products obtained here are structurally closely related to one such product already reported to form in an intracellular medium. A ketyl arising via SET with the artemisinin is invoked to explain their formation. Dihydroflavins generated from riboflavin (RF) and FAD by pretreatment with sodium dithionite are rapidly oxidised by artemisinin to the parent flavins. When catalytic amounts of RF, FAD, and other flavins are reduced in situ by excess BNAH or NAD(P)H in the presence of the artemisinins in the aqueous buffer, they are rapidly oxidised to the parent flavins with concomitant formation of two-electron reduction products from the artemisinins; regeneration of the reduced flavin by excess reductant maintains a catalytic cycle until the artemisinin is consumed. In preliminary experiments, we show that NADPH consumption in yeast GR with redox behaviour similar to that of parasite GR is enhanced by artemisinins, especially under aerobic conditions. Recombinant human GR is not affected. Artemisinins thus may act as antimalarial drugs by perturbing the redox balance within the malaria parasite, both by oxidising FADH(2) in parasite GR or other parasite flavoenzymes, and by initiating autoxidation of the dihydroflavin by oxygen with generation of ROS. Reduction of the artemisinin is proposed to occur via hydride transfer from LMB or the dihydroflavin to O1 of the peroxide. This hitherto unrecorded reactivity profile conforms with known structure-activity relationships of artemisinins, is consistent with their known ability to generate ROS in vivo, and explains the synergism between artemisinins and redox-active antimalarial drugs such as MB and doxorubicin. As the artemisinins appear to be relatively inert towards human GR, a putative model that accounts for the selective potency of artemisinins towards the malaria parasite also becomes apparent. Decisively, ferrous iron or carbon-centered free radicals cannot be involved, and the reactivity described herein reconciles disparate observations that are incompatible with the ferrous iron-carbon radical hypothesis for antimalarial mechanism of action. Finally, the urgent enquiry into the emerging resistance of the malaria parasite to artemisinins may now in one part address the possibilities either of structural changes taking place in parasite flavoenzymes that render the flavin cofactor less accessible to artemisinins or of an enhancement in the ability to use intra-erythrocytic human disulfide reuctases required for maintenance of parasite redox balance.
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收藏
页码:1282 / 1299
页数:18
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