Increased efforts in antimalarial drug discovery are urgently needed. The goal must be to develop safe and affordable new drugs to counter the spread of malaria parasites that are resistant to existing agents. Drug efficacy, pharmacology and toxicity are important parameters in the selection of compounds for development, yet little attempt has been made to review and standardize antimalarial drug-efficacy screens. Here, we suggest different in vitro and in vivo screens for antimalarial drug discovery and recommend a streamlined process for evaluating new compounds on the path from drug discovery to development.

https://www.mmv.org/sites/default/files/uploads/docs/publications/7_-_ScreeningArticlePDF_7.pdf

Antimalarial drug discovery: efficacy models for compound screening

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.

Diseases caused by tropical parasites affect hundreds of millions of people worldwide but have been largely neglected for drug development because they affect poor people in poor regions of the world. Most of the current drugs used to treat these diseases are decades old and have many limitations, including the emergence of drug resistance. This review will summarize efforts to reinvigorate the drug development pipeline for these diseases, which is driven in large part by support from major philanthropies. The organisms responsible for these diseases have a fascinating biology, and many potential biochemical targets are now apparent. These neglected diseases present unique challenges to drug development that are being addressed by new consortia of scientists from academia and industry.

Drug discovery and development for neglected parasitic diseases

New drugs against malaria are greatly needed. Many approaches to antimalarial drug discovery are available. These approaches must take into account specific concerns, in particular the requirement for very inexpensive and simple to use new therapies and the need to limit the cost of drug discovery. Among important efforts that are currently ongoing are the optimization of therapy with available drugs, including the use of combination therapy, the development of analogs of existing agents, the discovery of natural products, the use of compounds that were originally developed against other diseases, the evaluation of drug resistance reversers, and the consideration of new chemotherapeutic targets. The last category benefits from recent advances in malaria research technologies and genomics and is most likely to identify new classes of drugs. A number of new antimalarial therapies will likely be needed over the coming years, so it is important to pursue multiple strategies for drug discovery.

Antimalarial drug discovery: old and new approaches

Artemisinin contains an endoperoxide moiety that can react with iron to form cytotoxic free radicals. Cancer cells contain significantly more intracellular free iron than normal cells and it has been shown that artemisinin and its analogs selectively cause apoptosis in many cancer cell lines. In addition, artemisinin compounds have been shown to have anti-angiogenic, anti-inflammatory, anti-metastasis, and growth inhibition effects. These properties make artemisinin compounds attractive cancer chemotherapeutic drug candidates. However, simple artemisinin analogs are less potent than traditional cancer chemotherapeutic agents and have short plasma half-lives, and would require high dosage and frequent administration to be effective for cancer treatment. More potent and target-selective artemisinin-compounds are being developed. These include artemisinin dimers and trimers, artemisinin hybrid compounds, and tagging of artemisinin compounds to molecules that are involved in the intracellular iron-delivery mechanism. These compounds are promising potent anticancer compounds that produce significantly less side effect than traditional chemotherapeutic agents.

Development of artemisinin compounds for cancer treatment

Sixteen alkyl-substituted dispiro-1,2,4,5-tetraoxanes (7,8,15, 16-tetraoxadispiro[5.2.5.2]hexadecanes) were synthesized to explore dispiro-1,2,4,5-tetraoxane SAR and to identify tetraoxanes with better oral antimalarial activity than prototype tetraoxane 1 (WR 148999). The tetraoxanes were prepared either by peroxidation of the corresponding cyclohexanone derivatives in H(2)SO(4)/CH(3)CN or by ozonolysis of the corresponding cyclohexanone methyl oximes. Those tetraoxanes with alkyl substituents at the 1 and 10 positions were formed as single stereoisomers, whereas the five tetraoxanes formed without the stereochemical control provided by alkyl groups at the 1 and 10 positions were isolated as mixtures of diastereomers. Three of the sixteen tetraoxanes were inactive (IC(50)'s > 1000 nM), but five (2, 6, 10, 11, 12) had IC(50)'s between 10 and 30 nM against the chloroquine-sensitive D6 and chloroquine-resistant W2 clones of Plasmodium falciparum compared to corresponding IC(50)'s of 55 and 32 nM for 1 and 8.4 and 7.3 nM for artemisinin. We suggest that tetraoxanes 13, 16, and 17 were inactive and tetraoxanes 4 and 7 were weakly active due to steric effects preventing or hindering peroxide bond access to parasite heme. Tetraoxanes 1, 10, 11, and 14, along with artemisinin and arteether as controls, were administered po b.i.d. (128 mg/kg/day) to P. berghei-infected mice on days 3, 4, and 5 post-infection. At this dose, tetraoxanes 10, 11, and 14 cured between 40% and 60% of the infected animals. In comparison, artemisinin and tetraoxane 1 produced no cures, whereas arteether cured 100% of the infected animals. There was no apparent relationship between tetraoxane structure and in vitro neurotoxicity, nor was there any correlation between antimalarial activity and neurotoxicity for these seventeen tetraoxanes.

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W. G. Mclean

Papers

Synthesis and Antimalarial Activity of Sixteen Dispiro-1,2,4,5-tetraoxanes: Alkyl-Substituted 7,8,15,16-Tetraoxadispiro[5.2.5.2]hexadecanes