A partial convergence in action of methylene blue and artemisinins: Antagonism with chloroquine, a reversal with verapamil, and an insight into the antimalarial activity of chloroquine

Artemisinins rapidly oxidize leucomethylene blue (LMB) to methylene blue (MB); they also oxidize dihydroflavins such as the reduced conjugates RFH ₂ of riboflavin (RF), and FADH ₂ of the cofactor flavin adenine dinucleotide (FAD), to the corresponding flavins. Like the artemisinins, MB oxidizes FADH ₂, but unlike artemisinins, it also oxidizes NAD(P)H. Like MB, artemisinins are implicated in the perturbation of redox balance in the malaria parasite by interfering with parasite flavoenzyme disulfide reductases. The oxidation of LMB by artemisinin is inhibited by chloroquine (CQ), an inhibition that is abruptly reversed by verapamil (VP). CQ also inhibits artemisinin-mediated oxidation of RFH ₂ generated from N-benzyl-1,4-dihydronicotinamide (BNAH)-RF, or FADH ₂ generated from NADPH or NADPH-Fre, an effect that is also modulated by verapamil. The inhibition likely proceeds by the association of LMB or dihydroflavin with CQ, possibly involving donor-acceptor or πcomplexes that hinder oxidation by artemisinin. VP competitively associates with CQ, liberating LMB or dihydroflavin from their respective CQ complexes. The observations explain the antagonism between CQ-MB and CQ-artemisinins invitro, and are reconcilable with CQ perturbing intraparasitic redox homeostasis. They further suggest that a VP-CQ complex is a means by which VP reverses CQ resistance, wherein such a complex is not accessible to the putative CQ-resistance transporter (PfCRT).