Apicoplast, plasmodium, limonene and nerolidol

The basic concept of this document stems from Patrick Ogwang, Makerere University, Uganda. Additional inputs on limonene and nerolidol from Pierre Lutgen, Belherb.

The apicoplast is a plastid organelle, homologous to chloroplasts of plants or algae, that is found in apicomplexan parasites like Plasmodium or Toxoplasma. In hindsight it seems incredible that such an organelle could so long have concealed its identity in parasites that have received as much scientific attention as Plasmodium ( RF Waller et al., Curr Issues Mol Biol 7, 2005, 57-80). Apicoplast function is necessary for both intraerythrocytic and intrahepatic development. Recently it was found that the apicoplast is also present in the gametocytogenesis, in the sexual stage of Plasmodium falciparum. But only the female macrogametes have an apicoplast, the male microgametes don’t (N Okamoto et al., Eukaryot Cell 8(1) 2009, 128-132).
Plastids, including those of malaria parasites, have bacterial-type metabolic pathways all of which are vulnerable to antibacterial compounds. Indeed, many antibacterials kill malaria parasites by blocking essential processes in the plastid. It is logical that herbicides that target plastid metabolism of undesired plants are also parasitocidal, making them potential new leads for antimalarial drugs.
The effects of anti-bacterials on the malaria parasite are well known (CD Goodman et al., Molecular & Biochemical Parasitology, 152, 2007, 181-191). Some have an immediate effect: slowing parasite growth, retarding organellar growth and preventing nuclear division. Others don’t intervene in the first cycle, but lead to severe defects in the apicoplast in the second cycle and failure to complete this cycle. Inhibitors of apicoplast pathways can thus be explored as treatments for acute infections, as well as prophylactics with some degree of optimism.
Antibiotics are used for the prevention of malaria. Already in 2007 it was shown that antibiotic treated parasites are unable to complete schizogony ( E L Dahl et al., Antimicrobial Agents and Chemotherapy, Oct 2007, 3485-90). The authors relate this antimalarial effect to non-functional apicoplasts. Researchers from Heidelberg and Berlin (S Borrman et al., Sci Trans Med 2, no 40, 14 July 2010) have shown that if malaria infected mice are administered an antibiotic , no parasites appear in the blood and the mice are protected from this life threatening disease. The parasites which accumulate in the liver give the immune system sufficient stimulus to develop robust, long term immunity. A needle-free vaccination!
Many herbicides also have an activity against the malaria protozoan (S O Duke, Weed Science, 58, 2010, 334-339). A quote from this paper: “The Silent Spring image of pesticides is an anachronism that serves to obscure the pharmaceutical potential of herbicides. An opportunity missed!” Recently it was found that the inexpensive kitasamycin used for agricultural applications achieved an antimalarial IC50 in the 50 nM range by targeting the apicoplast (E H Ekland et al., Faseb Journal July 11, 2011).
Other well known drugs like statins have a detrimental effect on the apicoplast. Inhibition of the development of Plasmodium falciparum by mevastatin is clearly stage-dependent, occurring only when parasites are treated before differentiation into schizonts. These findings indicate that the isoprenoid pathway may be an important regulatory target in the intra-erythrocytic stages. ( A S Couto et al., Biochem J., 341, 1999, 629-637). Another drug, risedronate, a biphosphonate used in the treatment of oesteoporosis is a promising antiparasitic candidate. Recently it was demonstrated that lipophilic biphosphonates are effective against Plasmodium liver stages (A P Singh et al., Agents Chemother 54, 2010, 2987-93). In a very efficient way, and no merozoites reach the red blood cells for several weeks.
Isoprenoids, sometimes called terpenoids, play an important role in herbal remedies. Well known terpenoids include menthol, camphor, citral, 1.8-cineole. The human body cannot generate them. In Plasmodium the apicoplast is the only site of isoprenoid precursor synthesis. It even seems to have as only true essential function the production of one chemical, the isoprenoid isopentenyl pyrophosphate (IPP) which pays an essential role in protecting the Plasmodium against aggression by other molecules (E Yeh, JL DeRisi, PLoS Biol 9(8), 2011). These authors treated cells with antibiotics, which destroy the entire apicoplast, and then with IPP. The cells, usually rendered dead after a treatment with antibiotics, stayed alive, demonstrating the importance of IPP to malaria.
Limonene is known as inhibitor of isoprenylation of proteins in Plasmodium falciparum and arrests parasite development ( I C Moura et al, Antimicrob Agents Chemother. 45(9) 2001, 2553-58). This becomes very evident 48 hours after the treatment. The in vitro IC50 against Plasmodium in these trials was found to be 2.27 mM , significantly below the value of 15.5 mM which had been found previously in in vivo clinical trials in patients with advanced cancer. The pharmacokinetics are favourable: limonene and its metabolites stay at least for 48 hours in the plasma. This is important for the elimination of gametes and malaria transmission.
The monoterpene limonene is a byproduct of the orange juice industry, has a very favourable toxicity profile and is easily available at low prices. Limonene is also present in Artemisia annua at concentrations of 1 mg/kg (R Chougouo, Thèse, Université des Montagnes, Cameroon, 2011). So far studies have concentrated on this particular monoterpene but it is not excluded that others present in the essential oil of artemisia plants might have a similar detrimental action on the apicoplast, like eucalyptol ( V Su et al., Flavour Fragr J., 23, 2008, 315-18). And that the saponins which are present in Artemisia annua may facilitate the penetration of limonene and other drugs into the infected erythrocyte.
Nerolidol which is found in Artemisia herba alba and in lemongrass also is able to arrest development of the intraerythrocytic stages of the parasite. Indians of the Amazon basin in Brazil use the vapors of the leaves of Viola surinamensis. The sesquiterpene nerolidol was identified as the active constituent leading to 100 % growth inhibition at the schizont stage ( NP Lopes et al., J Ethnopharmacol 67, 1999, 313-19). Luteolin from Artemisia afra has similar properties.

ACTs and ITNs may fail, but Artemisia plants have not delivered all their herbal treasures.

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