Paul M. O'Neill

Artemisinins target the SERCA of Plasmodium falciparum

Artemisinins are extracted from sweet wormwood (Artemisia annua) and are the most potent antimalarials available, rapidly killing all asexual stages of Plasmodium falciparum. Artemisinins are sesquiterpene lactones widely used to treat multidrug-resistant malaria, a disease that annually claims 1 million lives. Despite extensive clinical and laboratory experience 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 proteins. 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.

The Molecular Mechanism of Action of Artemisinin—The Debate Continues

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.

4-Aminoquinolines--past, present, and future: a chemical perspective.

The 4-aminoquinoline chloroquine (1) can be considered to be one of the most important synthetic chemotherapeutic agents in history. Since its discovery, chloroquine has proved to be a highly effective, safe, and well-tolerated drug for the treatment and prophylaxis of malaria. However, the emergence of chloroquine-resistant strains of the malarial parasite has underlined the requirement for a synthetic alternative to chloroquine. This review describes structure-activity relationships for the 4-aminoquinolines, along with views on the mechanism of action and parasite resistance. A description of drug metabolism and toxicity also is included, with a brief description of potential approaches to the design of new synthetic derivatives.

Evidence for the Involvement of Carbon-centered Radicals in the Induction of Apoptotic Cell Death by Artemisinin Compounds *

Artemisinin and its derivatives are currently recommended as first-line antimalarials in regions where Plasmodium falciparum is resistant to traditional drugs. The cytotoxic activity of these endoperoxides toward rapidly dividing human carcinoma cells and cell lines has been reported, and it is hypothesized that activation of the endoperoxide bridge by an iron(II) species, to form C-centered radicals, is essential for cytotoxicity. The studies described here have utilized artemisinin derivatives, dihydroartemisinin, 10β-(p-bromophenoxy)dihydroartemisinin, and 10β-(p-fluorophenoxy)dihydroartemisinin, to determine the chemistry of endoperoxide bridge activation to reactive intermediates responsible for initiating cell death and to elucidate the molecular mechanism of cell death. These studies have demonstrated the selective cytotoxic activity of the endoperoxides toward leukemia cell lines (HL-60 and Jurkat) over quiescent peripheral blood mononuclear cells. Deoxy-10β-(p-fluorophenoxy)dihydroartemisinin, which lacks the endoperoxide bridge, was 50- and 130-fold less active in HL-60 and Jurkat cells, respectively, confirming the importance of this functional group for cytotoxicity. We have shown that chemical activation is responsible for cytotoxicity by using liquid chromatography-mass spectrometry analysis to monitor endoperoxide activation by measurement of a stable rearrangement product of endoperoxide-derived radicals, which was formed in sensitive HL-60 cells but not in insensitive peripheral blood mononuclear cells. In HL-60 cells the endoperoxides induce caspase-dependent apoptotic cell death characterized by concentration- and time-dependent mitochondrial membrane depolarization, activation of caspases-3 and -7, sub-G0/G1 DNA formation, and attenuation by benzyloxycarbonyl-VAD-fluoromethyl ketone, a caspase inhibitor. Overall, these results indicate that endoperoxide-induced cell death is a consequence of activation of the endoperoxide bridge to radical species, which triggers caspase-dependent apoptosis.

The Role of Heme and the Mitochondrion in the Chemical and Molecular Mechanisms of Mammalian Cell Death Induced by the Artemisinin Antimalarials

The artemisinin compounds are the frontline drugs for the treatment of drug-resistant malaria. They are selectively cytotoxic to mammalian cancer cell lines and have been implicated as neurotoxic and embryotoxic in animal studies. The endoperoxide functional group is both the pharmacophore and toxicophore, but the proposed chemical mechanisms and targets of cytotoxicity remain unclear. In this study we have used cell models and quantitative drug metabolite analysis to define the role of the mitochondrion and cellular heme in the chemical and molecular mechanisms of cell death induced by artemisinin compounds. HeLa ρ0 cells, which are devoid of a functioning electron transport chain, were used to demonstrate that actively respiring mitochondria play an essential role in endoperoxide-induced cytotoxicity (artesunate IC50 values, 48 h: HeLa cells, 6 ± 3 μm; and HeLa ρ0 cells, 34 ± 5 μm) via the generation of reactive oxygen species and the induction of mitochondrial dysfunction and apoptosis but do not have any role in the reductive activation of the endoperoxide to cytotoxic carbon-centered radicals. However, using chemical modulators of heme synthesis (succinylacetone and protoporphyrin IX) and cellular iron content (holotransferrin), we have demonstrated definitively that free or protein-bound heme is responsible for intracellular activation of the endoperoxide group and that this is the chemical basis of cytotoxicity (IC50 value and biomarker of bioactivation levels, respectively: 10β-(p-fluorophenoxy)dihydroartemisinin alone, 0.36 ± 0.20 μm and 11 ± 5%; and with succinylacetone, >100 μm and 2 ± 5%).

Changes in spinal cord regenerative ability through phylogenesis and development: Lessons to be learnt

Lower vertebrates, such as fish and amphibians, and developing higher vertebrates can regenerate complex body structures, including significant portions of their central nervous system. It is still poorly understood why this potential is lost with evolution and development and becomes very limited in adult mammals. In this review, we will discuss the current knowledge on the cellular and molecular changes after spinal cord injury in adult tailed amphibians, where regeneration does take place, and in developing chick and mammalian embryos at different developmental stages. We will focus on the recruitment of progenitor cells to repair the damage and discuss possible roles of changes in early response to injury, such as cell death by apoptosis, and of myelin‐associated proteins, such as Nogo, in the transition between regeneration‐competent and regeneration‐incompetent stages of development. A better understanding of the mechanisms underlying spontaneous regeneration of the spinal cord in vivo in amphibians and in the chick embryo will help to devise strategies for restoring function to damaged or diseased nervous tissues in mammals. Developmental Dynamics 226:245–256, 2003.© 2003 Wiley‐Liss, Inc.

Generation of quinolone antimalarials targeting the Plasmodium falciparum mitochondrial respiratory chain for the treatment and prophylaxis of malaria

There is an urgent need for new antimalarial drugs with novel mechanisms of action to deliver effective control and eradication programs. Parasite resistance to all existing antimalarial classes, including the artemisinins, has been reported during their clinical use. A failure to generate new antimalarials with novel mechanisms of action that circumvent the current resistance challenges will contribute to a resurgence in the disease which would represent a global health emergency. Here we present a unique generation of quinolone lead antimalarials with a dual mechanism of action against two respiratory enzymes, NADH:ubiquinone oxidoreductase (Plasmodium falciparum NDH2) and cytochrome bc1. Inhibitor specificity for the two enzymes can be controlled subtly by manipulation of the privileged quinolone core at the 2 or 3 position. Inhibitors display potent (nanomolar) activity against both parasite enzymes and against multidrug-resistant P. falciparum parasites as evidenced by rapid and selective depolarization of the parasite mitochondrial membrane potential, leading to a disruption of pyrimidine metabolism and parasite death. Several analogs also display activity against liver-stage parasites (Plasmodium cynomolgi) as well as transmission-blocking properties. Lead optimized molecules also display potent oral antimalarial activity in the Plasmodium berghei mouse malaria model associated with favorable pharmacokinetic features that are aligned with a single-dose treatment. The ease and low cost of synthesis of these inhibitors fulfill the target product profile for the generation of a potent, safe, and inexpensive drug with the potential for eventual clinical deployment in the control and eradication of falciparum malaria.

Functional Characterization and Target Validation of Alternative Complex I of Plasmodium falciparum Mitochondria

ABSTRACT This study reports on the first characterization of the alternative NADH:dehydrogenase (also known as alternative complex I or type II NADH:dehydrogenase) of the human malaria parasite Plasmodium falciparum, known as PfNDH2. PfNDH2 was shown to actively oxidize NADH in the presence of quinone electron acceptors CoQ1 and decylubiquinone with an apparent Km for NADH of approximately 17 and 5 μM, respectively. The inhibitory profile of PfNDH2 revealed that the enzyme activity was insensitive to rotenone, consistent with recent genomic data indicating the absence of the canonical NADH:dehydrogenase enzyme. PfNDH2 activity was sensitive to diphenylene iodonium chloride and diphenyl iodonium chloride, known inhibitors of alternative NADH:dehydrogenases. Spatiotemporal confocal imaging of parasite mitochondria revealed that loss of PfNDH2 function provoked a collapse of mitochondrial transmembrane potential (Ψm), leading to parasite death. As with other alternative NADH:dehydrogenases, PfNDH2 lacks transmembrane domains in its protein structure, and therefore, it is proposed that this enzyme is not directly involved in mitochondrial transmembrane proton pumping. Rather, the enzyme provides reducing equivalents for downstream proton-pumping enzyme complexes. As inhibition of PfNDH2 leads to a depolarization of mitochondrial Ψm, this enzyme is likely to be a critical component of the electron transport chain (ETC). This notion is further supported by proof-of-concept experiments revealing that targeting the ETCs Q-cycle by inhibition of both PfNDH2 and the bc1 complex is highly synergistic. The potential of targeting PfNDH2 as a chemotherapeutic strategy for drug development is discussed.

Identification of a 1,2,4,5-tetraoxane antimalarial drug-development candidate (RKA 182) with superior properties to the semisynthetic artemisinins.

Artemisinin (1) is an extract of the Chinese wormwood Artemisia annua and has been used since ancient times to treat malaria. Today, semisynthetic derivatives artesunate (2) and artemether (3) are used clinically in drug combinations (ACT; artemisinin-based combination therapy). However, first-generation analogues (e.g. 2 and 3) have a limited availability, high cost, and poor oral bioavailability (Scheme 1a). In addition to these drawbacks there have been recent reports of high failure rates associated with ACTs suggesting the possibility of clinical artemisinin resistance along the Thai–Cambodian border. In the light of these observations there is an urgent need to develop alternative endoperoxide-based therapies. The crucial structural functionality within artemisinin and synthetic 1,2,4-trioxanes is the endoperoxide bridge. Recently a series of molecules based on an ozonide structure were developed from which the candidate OZ277 was shown to have impressive antimalarial activity profiles in vitro and in rodent models of malaria. However, the recent

The role of drug accumulation in 4-aminoquinoline antimalarial potency: The influence of structural substitution and physicochemical properties☆

We have investigated a series of novel 4-aminoquinoline analogues related to amodiaquine, that possess side chain modifications designed to influence both drug pKa and lipophilicity. These compounds have been used to determine the influence of physicochemical properties on antimalarial activity against, and accumulation by, both chloroquine-susceptible and chloroquine-resistant isolates of Plasmodium falciparum. The compounds tested exhibited a 500-fold range of absolute antimalarial potency. Absolute drug potency and drug accumulation were found to be significantly correlated in each of the four isolates of Plasmodium falciparum studied. The level of accumulation was unrelated to lipophilicity and was significantly greater than the predicted levels of accumulation based on drug pKa, compartmental pH, and Henderson-Hasselbach considerations. Further analysis of the relationship between 4-aminoquinoline accumulation and activity implicated the involvement of additional forces in the accumulation process.