Interactions between Artemisinins and other Antimalarial Drugs in Relation to the Cofactor Model-A Unifying Proposal for Drug Action

Artemisinins are proposed to act in the malaria parasite cytosol by oxidizing dihydroflavin cofactors of redox-active flavoenzymes, and under aerobic conditions by inducing their autoxidation. Perturbation of redox homeostasis coupled with the generation of reactive oxygen species (ROS) ensues. Ascorbic acid-methylene blue (MB), N-benzyl-1,4-dihydronicotinamide (BNAH)-MB, BNAH-lumiflavine, BNAH-riboflavin (RF), and NADPH-FAD-E. coli flavin reductase (Fre) systems at pH 7.4 generate leucomethylene blue (LMB) and reduced flavins that are rapidly oxidized in situ by artemisinins. These oxidations are inhibited by the 4-aminoquinolines piperaquine (PPQ), chloroquine (CQ), and others. In contrast, the arylmethanols lumefantrine, mefloquine (MFQ), and quinine (QN) have little or no effect. Inhibition correlates with the antagonism exerted by 4-aminoquinolines on the antimalarial activities of MB, RF, and artemisinins. Lack of inhibition correlates with the additivity/synergism between the arylmethanols and artemisinins. We propose association via pi complex formation between the 4-aminoquinolines and LMB or the dihydroflavins; this hinders hydride transfer from the reduced conjugates to the artemisinins. The arylmethanols have a decreased tendency to form pi complexes, and so exert no effect. The parallel between chemical reactivity and antagonism or additivity/synergism draws attention to the mechanism of action of all drugs described herein. CQ and QN inhibit the formation of hemozoin in the parasite digestive vacuole (DV). The buildup of heme-Fe-III results in an enhanced efflux from the DV into the cytosol. In addition, the lipophilic heme-Fe-III complexes of CQ and QN that form in the DV are proposed to diffuse across the DV membrane. At the higher pH of the cytosol, the complexes decompose to liberate heme-Fe-III. The quinoline or arylmethanol reenters the DV, and so transfers more heme-Fe-III out of the DV. In this way, the 4-aminoquinolines and arylmethanols exert antimalarial activities by enhancing heme-Fe-III and thence free Fe-III concentrations in the cytosol. The iron species enter into redox cycles through reduction of Fe-III to Fe-II largely mediated by reduced flavin cofactors and likely also by NAD(P)H-Fre. Generation of ROS through oxidation of Fe-II by oxygen will also result. The cytotoxicities of artemisinins are thereby reinforced by the iron. Other aspects of drug action are emphasized. In the cytosol or DV, association by pi complex formation between pairs of lipophilic drugs must adversely influence the pharmacokinetics of each drug. This explains the antagonism between PPQ and MFQ, for example. The basis for the antimalarial activity of RF mirrors that of MB, wherein it participates in redox cycling that involves flavoenzymes or Fre, resulting in attrition of NAD(P)H. The generation of ROS by artemisinins and ensuing Fenton chemistry accommodate the ability of artemisinins to induce membrane damage and to affect the parasite SERCA PfATP6 Ca2+ transporter. Thus, the effect exerted by artemisinins is more likely a downstream event involving ROS that will also be modulated by mutations in PfATP6. Such mutations attenuate, but cannot abrogate, antimalarial activities of artemisinins. Overall, parasite resistance to artemisinins arises through enhancement of antioxidant defense mechanisms.