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

• DOI: 10.1002/cmdc.201000225
• Publication Date: 2010-07-13T21:14:38Z
• Authors: Richard K. Haynes; Wing‐Chi Chan; Ho‐Ning Wong; Ka‐Yan Li; Wai‐Keung Wu; Kit‐Man Fan; Herman H. Y. Sung; Ian D. Williams; Davide Prosperi; Sergio Melato; Paolo Coghi; Diego Monti
• Keywords: Oxocarbenium

Abstract 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 malaria parasite. reduced flavin adenine dinucleotide cofactor FADH 2 initiates reduction to leucomethylene (LMB), which is oxidised by oxygen generate reactive species (ROS) and MB. MB then acts a subversive substrate for NADPH normally required regenerate enzyme function. synergism between peroxidic artemisinin derivative artesunate suggests artemisinins have complementary mode action. We find are transformed LMB generated from ascorbic acid (AA) or N ‐benzyldihydronicotinamide (BNAH) situ aqueous buffer at physiological pH into single electron transfer (SET) rearrangement products two‐electron products, latter dominates with BNAH. Neither AA nor BNAH alone artemisinins. AA–MB SET reactions enhanced under aerobic conditions, major obtained here structurally closely related one product already reported form an intracellular medium. A ketyl arising via invoked explain their formation. Dihydroflavins riboflavin (RF) FAD pretreatment sodium dithionite rapidly parent flavins. When catalytic amounts RF, FAD, other flavins excess NAD(P)H presence buffer, they concomitant formation artemisinins; regeneration reductant maintains cycle until consumed. In preliminary experiments, we show consumption yeast GR similar artemisinins, especially conditions. Recombinant human not affected. Artemisinins thus may act drugs perturbing balance within parasite, both oxidising flavoenzymes, initiating autoxidation dihydroflavin generation ROS. Reduction proposed occur hydride O1 peroxide. This hitherto unrecorded reactivity profile conforms known structure–activity relationships consistent ability ROS vivo, explains redox‐active doxorubicin. As appear be relatively inert towards GR, putative model accounts selective potency also becomes apparent. Decisively, ferrous iron carbon‐centered free radicals cannot involved, described herein reconciles disparate observations incompatible iron–carbon radical hypothesis mechanism Finally, urgent enquiry emerging resistance now part address possibilities either structural changes taking place flavoenzymes render less accessible enhancement use intra‐erythrocytic maintenance balance.