Dual artemisinin action combats resistance

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Summary The broad long-term goal is to optimize critical artemisinin and quinoline malaria drug combinations for maximum killing of the P. falciparum parasite by defeating resistance. In the setting of ongoing drug resistance to currently deployed drugs, this work will quantify a novel quinoline-like mechanism of action for the heme-artemisinin adducts, define reversible or irreversible heme crystal inhibition correlated to level of drug resistance, and explore optimum heme crystal inhibition related to parasite killing with quinoline-artemisinin combinations. Preliminary data validate an additional mechanism of action for the artemisinins based on formation of abundant heme-artemisinin adduct, which inhibits heme crystallization with irreversible action. Exogenous heme-artemisinin adducts inhibit artemisinin ring-resistant mutant Kelch13 P. falciparum parasites with low nM IC50s. The experimental approach employs the synergy between experimental investigations with P. falciparum drug-sensitive and resistant parasites in vitro and physicochemical insights obtained by time-resolved in situ observations of crystal growth by atomic force microscopy in the presence of different drug combinations. The hypothesis is that the inhibition of heme crystal formation by the heme adduct of dihydroartemisinin (DHA, the product of most artemisinin-class drugs in vivo) renders trophozoites of any Plasmodium isolate sensitive, which defeats the artemisinin ring-stage resistance. We also hypothesize that certain combinations of quinolines and heme-dihydroartemisinin adduct (H-DHA) are superior in killing of parasites correlated to heme crystal inhibition as well as separately to the degree of reversible/irreversible heme crystal inhibition. Towards these objectives, we will pursue three specific aims: Aim 1. Establish the inhibition concentrations and mechanism of action of exogenous as well as bio-activated H-DHA in drug sensitive and resistant Plasmodium. Aim 2. Establish reversibility or irreversibility of H-DHA heme crystal inhibition in vivo and in vitro. Aim 3. Establish if double combinations of antimalarial quinolines and H-DHA adducts enhance, weaken, or are indifferent to their partner’s action on parasite killing and the rate of hematin crystallization. This proposed research will quantify the amount of parasite killing by artemisinin adduct metabolites which renders trophozoite stages sensitive to the artemisinin drug class. The work will also inform fundamental knowledge regarding mechanisms to defeat artemisinin resistance, degree of reversibility of hematin crystal growth, and optimum combinations of malaria drugs based upon interaction effects.