Artemisinins are extracted from sweet wormwood (Artemisia annua) and are the most potent antimalarials available1, rapidly killing all asexual stages of Plasmodium falciparum2. Artemisinins are sesquiterpene lactones widely used to treat multidrug-resistant malaria1, a disease that annually claims 1 million lives. Despite extensive clinical and laboratory experience3, 4, 5 their molecular target is not yet identified. Activated artemisinins form adducts with a variety of biological macromolecules, including haem, translationally controlled tumour protein (TCTP) and other higher-molecular-weight proteins6. Here we show that artemisinins, but not quinine or chloroquine, inhibit the SERCA orthologue (PfATP6) of Plasmodium falciparum in Xenopus oocytes with similar potency to thapsigargin (another sesquiterpene lactone and highly specific SERCA inhibitor). As predicted, thapsigargin also antagonizes the parasiticidal activity of artemisinin. Desoxyartemisinin lacks an endoperoxide bridge and is ineffective both as an inhibitor of PfATP6 and as an antimalarial. Chelation of iron by desferrioxamine abrogates the antiparasitic activity of artemisinins and correspondingly attenuates inhibition of PfATP6. Imaging of parasites with BODIPY-thapsigargin labels the cytosolic compartment and is competed by artemisinin. Fluorescent artemisinin labels parasites similarly and irreversibly in an Fe2+-dependent manner. These data provide compelling evidence that artemisinins act by inhibiting PfATP6 outside the food vacuole after activation by iron.

Artemisinins target the SERCA of Plasmodium falciparum

Artemisinin: mechanisms of action, resistance and toxicity.

Despite international efforts to ‘roll back malaria’ the 2008 World Malaria Report revealed the disease still affects approximately 3 billion people in 109 countries; 45 within the WHO African region. The latest report however does provide some ‘cautious optimism’; more than one third of malarious countries have documented greater than 50% reductions in malaria cases in 2008 compared to 2000. The goal of the Member States at the World Health Assembly and ‘Roll Back Malaria’ (RBM) partnership is to reduce the numbers of malaria cases and deaths recorded in 2000 by 50% or more by the end of 2010. Although malaria is preventable it is most prevalent in poorer countries where prevention is difficult and prophylaxis is generally not an option. The burden of disease has increased by the emergence of multi drug resistant (MDR) parasites which threatens the use of established and cost effective antimalarial agents. After a major change in treatment policies, artemisinins are now the frontline treatment to aid rapid clearance of parasitaemia and quick resolution of symptoms. Since artemisinin and its derivatives are eliminated rapidly, artemisinin combination therapies (ACT’s) are now recommended to delay resistance mechanisms. In spite of these precautionary measures reduced susceptibility of parasites to the artemisinin-based component of ACT’s has developed at the Thai-Cambodian border, a historical ‘hot spot’ for MDR parasite evolution and emergence. This development raises serious concerns for the future of the artemsinins and this is not helped by controversy related to the mode of action. Although a number of potential targets have been proposed the actual mechanism of action remains ambiguous. Interestingly, artemisinins have also shown potent and broad anticancer properties in cell lines and animal models and are becoming established as anti-schistosomal agents. In this review we will discuss the recent evidence explaining bioactivation and potential molecular targets in the chemotherapy of malaria and cancer.

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The molecular mechanism of action of artemisinin - the debate continues.

ion and allylic carbon radical formation with subsequent triplet ground-state oxygen capture result ultimately in the formation of lipid hydroperoxides. The explicit mechanism depicted in Scheme 1 is supported by the work of Berman and Adams and others, and it was proposed that the damage caused to the parasite’s FV membrane leads to vacuolar rupture and parasite autodigestion.41 This mechanism is consistent with the observed oxygen dependence of antimalarial action of artemisinin and the morphological changes seen following artemisinin administration to parasites in vitro.44 The biological significance of hydroperoxides in relation to biological hydroxylation and autoxidation of, for example, lipids and membrane bilayers is well established. The generation of unsaturated lipid hydroperoxides provides a means of initiation of such processes. In contrast to these proposals, other workers in the field have suggested that membrane-bound heme may have a role to play in reducing the effectiveness of endoperoxides such as dihydroartemisinin. Further work is required to clarify the role of vacuolar membrane bound heme in the mechanism of action of endoperoxide antimalarials.45 An alternative nonlipid “artemisinin derived source of hydroperoxide” is discussed below. Although Scheme 1 would appear chemically plausible, several workers have proposed that the parasite death in the presence of artemisinin is probably not due to nonspecific or random cell damage caused by freely diffusing oxygen radical species but might involve specific radicals and targets, some of which are described later in this review. The following section details the specific “transitory” species that may be responsible for the antimalarial mechanism of action of artemisinin. Targets of these species are discussed and will conclude with the most recent studies by Eckstein-Ludwig who suggest that artemisinin derivatives target the PfATP6, the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) of the parasite.46 Transitory Species Mediating the Antimalarial Activity of Artemisinin: Carbon Radicals, Carbocations, Hydroperoxides, and High-Valent IronOxo Species. On the basis of the seminal work of Posner and co-workers in the early 1990s, the free radical chemistry of artemisinin is now very welldefined and has been shown to involve an initial chemical decomposition induced by heme Fe(II) (reduced hemin) or other sources of ferrous iron within the malaria parasite to produce initially an oxy radical that subsequently rearranges into one or both of two distinctive carbon-centered radical species.47 Scheme 2a summarizes the main radical pathways available for artemisinin following endoperoxide-mediated bioactivation. Since artemisinin is an unsymmetrical endoperoxide, the oxygen atoms of the peroxide linkage can associate with reducing ferrous ions in two ways. Association of Fe(II) with oxygen 1 provides an oxy radical that goes on to produce a primary carbon-centered radical (5a). A surrogate marker for the intermediacy of this radical species is the ring-contracted tetrahydrofuran (RCT) product 5b. Alternatively, association with oxygen 2 provides an oxy radical species that, via a 1,5-H shift, can produce a secondary carbon-centered radical (5c). Again, like the previous route, a stable end-product, hydroxydeoxoartemisinin (HDA) (5d), functions as a surrogate marker for this secondary carbon-centered radical species. It has been proposed that final alkylation by these reactive intermediates of biomacromolecules such as Scheme 1. Proposed Chemical Mechanism for the Observed Artemisinin Mediated Lipid Peroxidation of Cell Membranesa a Carbon radicals, generated from artemisinin, abstract allylic hydrogen atoms from unsaturated lipid bilayers to set in motion the downstream generation of a variety of reactive oxygen species by a classic lipid peroxidation mechanism. Heme or hematin/thiols associated with the lipid bilayer may be the catalysts responsible for initiation of these processes. Perspective Journal of Medicinal Chemistry, 2004, Vol. 47, No. 12 2947

A medicinal chemistry perspective on artemisinin and related endoperoxides.

Carvone: Why and how should one bother to produce this terpene

The potent antimalarial activity of chloroquine against chloroquine-sensitive strains can be attributed, in part, to its high accumulation in the acidic environment of the heme-rich parasite food vacuole. A key component of this intraparasitic chloroquine accumulation mechanism is a weak base "ion-trapping" effect whereupon the basic drug is concentrated in the acidic food vacuole in its membrane-impermeable diprotonated form. By the incorporation of amino functionality into target artemisinin analogues, we hoped to prepare a new series of analogues that, by virtue of increased accumulation into the ferrous-rich vacuole, would display enhanced antimalarial potency. The initial part of the project focused on the preparation of piperazine-linked analogues (series 1 (7-16)). Antimalarial evaluation of these derivatives demonstrated potent activity versus both chloroquine-sensitive and chloroquine-resistant parasites. On the basis of these observations, we then set about preparing a series of C-10 carba-linked amino derivatives. Optimization of the key synthetic step using a newly developed coupling protocol provided a key intermediate, allyldeoxoartemisinin (17) in 90% yield. Further elaboration, in three steps, provided nine target C-10 carba analogues (series 2 (21-29)) in good overall yields. Antimalarial assessment demonstrated that these compounds were 4-fold more potent than artemisinin and about twice as active as artemether in vitro versus chloroquine-resistant parasites. On the basis of the products obtained from biomimetic Fe(II) degradation of the C-10 carba analogue (23), we propose that these analogues may have a mode of action subtly different from that of the parent drug artemisinin (series 1 (7-16)) and other C-10 ether derivatives such as artemether. Preliminary in vivo testing by the WHO demonstrated that four of these compounds are active orally at doses of less than 10 mg/kg. Since these analogues are available as water-soluble salts and cannot form dihydroartemisinin by P450-catalyzed oxidation, they represent useful leads that might prove to be superior to the currently used derivatives, artemether and artesunate.

Mechanism-based design of parasite-targeted artemisinin derivatives: synthesis and antimalarial activity of new diamine containing analogues.

Ten novel, second-generation, fluorinated ether and ester analogues of the potent first-generation analogues artemether (4a) and arteether (4b) have been designed and synthesized. All of the compounds demonstrate high antimalarial potency in vitro against the chloroquine-sensitive HB3 and -resistant K1 strains of Plasmodium falciparum. The most potent derivative 8 was 15 times more potent than artemisinin (2) against the HB3 strain of P. falciparum. In vivo, versus Plasmodium berghei in the mouse, selected derivatives were generally less potent than dihydroartemisinin with ED 50 values of between 5 and 8 mg/kg. On the basis of the products obtained from the in vitro biomimetic Fe(II)-mediated decomposition of 8, the radical mediator of biological activity of this series may be different from that of the parent drug, artemisinin (2).

Novel, potent, semisynthetic antimalarial carba analogues of the first-generation 1,2,4-trioxane artemether

Biomimetic Fe(II)-mediated degradation of arteflene (Ro-42-1611). The first EPR spin-trapping evidence for the previously postulated secondary carbon-centered cyclohexyl radical.

A carbonyl oxide route to antimalarial yingzhaosu a analogues : synthesis and antimalarial activity

The biomimetic iron-mediated degradation of arteflene (Ro-42-1611),an endoperoxide antimalarial: Implications for the mechanism of antimalarial activity

Natalie L. Searle

Papers

Mechanism-based design of parasite-targeted artemisinin derivatives: synthesis and antimalarial activity of new diamine containing analogues.

Novel, potent, semisynthetic antimalarial carba analogues of the first-generation 1,2,4-trioxane artemether

Biomimetic Fe(II)-mediated degradation of arteflene (Ro-42-1611). The first EPR spin-trapping evidence for the previously postulated secondary carbon-centered cyclohexyl radical.

A carbonyl oxide route to antimalarial yingzhaosu a analogues : synthesis and antimalarial activity

The biomimetic iron-mediated degradation of arteflene (Ro-42-1611),an endoperoxide antimalarial: Implications for the mechanism of antimalarial activity