A second mechanism employed by artemisinins to suppress Plasmodium falciparum hinges on inhibition of hematin crystallization

Abstract

Malaria is a pervasive disease that affects millions of lives each year in equatorial regions of the world. During the erythrocytic phase of the parasite life cycle, Plasmodium falciparum invade red blood cells, where they catabolize hemoglobin and sequester the released toxic heme as innocuous hemozoin crystals. Artemisinin-class drugs are activated in vivo by newly-released heme, which creates a carbon-centered radical that markedly reduces parasite density. Radical damage to parasite lipids and proteins is perceived to be artemisinins’ dominant mechanism of action. By contrast, quinoline-class antimalarials inhibit the formation of hemozoin and in this way suppress heme detoxification. Here, we combine malaria parasite assays and scanning probe microscopy of growing beta-hematin crystals to elucidate an unexpected mechanism employed by two widely administered antimalarials, artemisinin and artesunate, to subdue the erythrocytic phase of the parasite life cycle. We demonstrate that heme-drug adducts, produced after the radical activation of artemisinins and largely believed to be benign bystanders, potently kills P. falciparum at low concentrations. We show that these adducts inhibit b-hematin crystallization and heme detoxification, a pathway which complements the deleterious effect of radicals generated via parent drug activation. Our findings reveal an irreversible mechanism of heme-artemisinin adduct inhibition of heme crystallization, unique among antimalarials and common crystal growth inhibitors, that opens new avenues for evaluating drug dosing regimens and understanding growing resistance of P. falciparum to artemisinin.

The Role of the Iron Protoporphyrins Heme and Hematin in the Antimalarial Activity of Endoperoxide Drugs

“…A second mechanism, where a drug binds by adsorption mechanisms to the surface of growing β-hematin crystals and suppresses crystal elongation, also indicated that artemisinin does not interfere with β-hematin crystal elongation [35,39]. All these findings, validated by independent research groups [40,41], converged to the notion that antimalarial peroxides do not interfere with hematin mineralization. Subsequently, it became clear that the absence of β-hematin inhibitory activity for antimalarial peroxides is due to a lack of binding affinity of these drugs for hematin [9].…”

“…Hung et al [43] made use of a radiolabeled artemisinin and protocols for extracting Hz to show that artemisinin treatment was associated with the Hz content, likely in the form of hematin-artemisinin adducts, denoting a possible fate of artemisinin to interact with Hz. However, using mass spectroscopy, two independent research groups were unable to identify endoperoxide and its hematin adducts adsorbed on Hz from parasites treated with artemisinin, artesunate or dihydroartemisinin in a concentration range from 500 to 5000 nM [41,44]. Importantly, dihydroartemisinin treatment at 5000 nM resulted in hematin-drug adducts widely associated with the parasite cell lysate but not detected in the Hz fraction [44].…”

(−)-6-epi-Artemisinin, a Natural Stereoisomer of (+)-Artemisinin in the Opposite Enantiomeric Series, from the Endemic Madagascar Plant Saldinia proboscidea, an Atypical Source

“…As an example, in the antimalaria activity of these molecules, the two oxygens present in the peroxide group with their electron density is an attractive site for the iron positively charged in heme. Ma et al [ 15 ] had demonstrated that heme–drug adducts, produced after the radical activation of artemisinins, can inhibit β-hematin crystallization and heme detoxification, a pathway which complements the deleterious effect of radicals generated via parent drug activation.…”

AFM-Based Correlative Microscopy Illuminates Human Pathogens

“…AFM offers high content data for characterizing cell surfaces (Bhat et al, 2015), membranes and envelopes (Viljoen et al, 2020), cell division (Formosa et al, 2013;Bhat et al, 2018a) and adhesion (Mathelie-Guinlet et al, 2019). Systems studied by AFM range from viruses (Chen et al, 2013) to embryonic tissue (Chevalier et al, 2016;Thompson et al, 2019), parasites (Sinha et al, 2015;Perez-Guaita et al, 2018;Ma et al, 2020) and other pathogens (Alsteens et al, 2009;Deupree and Schoenfisch, 2009;Alsteens et al, 2013).…”