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Antimicrobial Agents and Chemotherapy, August 2007, p. 2991-2993, Vol. 51, No. 8
0066-4804/07/$08.00+0     doi:10.1128/AAC.00225-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Peroxide Bond-Dependent Antiplasmodial Specificity of Artemisinin and OZ277 (RBx11160){triangledown}

Marcel Kaiser,1 Sergio Wittlin,1 Angela Nehrbass-Stuedli,1 Yuxiang Dong,2 Xiaofang Wang,2 Andrew Hemphill,4 Hugues Matile,3 Reto Brun,1 and Jonathan L. Vennerstrom2*

Swiss Tropical Institute, Socinstrasse 57, CH-4002 Basel, Switzerland,1 College of Pharmacy, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198-6025,2 F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, CH-4070 Basel, Switzerland,3 Institute of Parasitology, University of Bern, CH-3012 Bern, Switzerland4

Received 14 February 2007/ Returned for modification 30 April 2007/ Accepted 1 June 2007


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ABSTRACT
 
Using nonperoxidic analogs of artemisinin and OZ277 (RBx11160), the strong in vitro antiplasmodial activities of the latter two compounds were shown to be peroxide bond dependent. In contrast, the weak activities of artemisinin and OZ277 against six other protozoan parasites were peroxide bond independent. These data support the iron-dependent artemisinin alkylation hypothesis.


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TEXT
 
The antimalarial artemisinin contains a pharmacophoric peroxide bond within its 1,2,4-trioxane heterocycle. A long-standing hypothesis (17, 24, 29) to account for the antimalarial specificity of artemisinin is that the peroxide bond undergoes reductive activation by heme released by parasite hemoglobin (Hb) digestion. Although most (95%) of the heme released from parasite digestion is incorporated into the relatively inert hemozoin (12), it is estimated (8) that there is some 100 µM of "free" heme/hematin in malaria-infected red blood cells, a more than adequate quantity to react with artemisinin. The irreversible redox reaction between artemisinin and heme produces carbon-centered free radicals or carbocations that alkylate heme (25, 32, 33) and membrane-associated parasite proteins (2), one of which is the translationally controlled tumor protein (PfTCTP) (4) and another is likely to be PfATP6, a SERCA-type Ca2+-ATPase (11). Eckstein-Ludwig et al. (11) studied the latter protein in some detail and provided evidence that it may be an important target of the artemisinins.

The structural diversity of semisynthetic artemisinins (41) and synthetic peroxides (37) is quite remarkable and, we suggest, has implications for the mechanism of action for this class of antimalarial drugs. In this respect, we thought it might be useful to compare the activities of artemisinin and synthetic peroxide (1,2,4-trioxolane) OZ277 (RBx11160) (40) and their nonperoxidic counterparts deoxyartemisinin (6) and carbaOZ277 (Fig. 1) against a range of protozoan parasites. CarbaOZ277 (1,3-dioxolane) was prepared as a mixture of cis-trans isomers by an acid-catalyzed reaction (9) between the requisite adamantane diol and amino amide cyclohexanone. In vitro antiprotozoal assays for Plasmodium falciparum, Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani, Giardia duodenalis, Babesia divergens, and Neospora caninum were performed as previously described (1, 5, 16, 28, 35, 38, 40) with standard drug controls (Table 1).


Figure 1
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  		FIG. 1. Structures of artemisinin, deoxyartemisinin, OZ277, and carbaOZ277.


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  		TABLE 1. Activity of artemisinin, deoxyartemisinin, OZ277, and carbaOZ277 against six protozoan parasites

The data in Table 1 shows a clear demarcation in the antiplasmodial activities of artemisinin and OZ277 versus their nonperoxidic analogs deoxyartemisinin (6) and carbaOZ277, confirming the peroxide bond-dependent activity of the former. For artemisinin and OZ277, there were losses of activity greater than 3 orders of magnitude against all of the other protozoa compared to activities against P. falciparum. In contrast, for deoxyartemisinin and carbaOZ277, there were only small differences in activity between P. falciparum and the other protozoa. Only for T. b. rhodesiense was there a significant difference between artemisinin and deoxyartemisinin versus OZ277 and carbaOZ277, suggesting that the latter two compounds may be exerting a structurally specific, but peroxide-independent, inhibition of parasite growth by some unknown mechanism. None of the four compounds was active against N. caninum at concentrations of up to 1.0 µg/ml (data not shown).

The data in Table 1 are consistent with the weak activities reported for artemisinin against protozoa other than plasmodia. For example, artemisinin has 50% inhibitory concentrations (IC50s) ranging from 8 to 35 µg/ml against Leishmania major amastigotes (42) and L. donovani promastigotes (3, 27). In the study by Avery et al. (3), several artemisinin derivatives had IC50 values as low as 0.79 µg/ml; in comparison, their nonperoxidic counterparts had no activity at concentrations of up to 50 µg/ml. This result is consistent with our data showing that deoxyartemisinin was completely inactive against L. donovani amastigotes, suggesting some role for the peroxide bond in the activity of artemisinin against this protozoa species. Artemisinin has an IC50 of 2.3 µg/ml against Toxoplasma gondii in cell culture (18); at 4.0 µg/ml, artemisinin completely eliminates the parasite (30). That this very weak activity of artemisinin against T. gondii is peroxide-bond dependent is suggested by data (18) in which one semisynthetic artemisinin was shown to be 64-fold more potent than its deoxy derivative. Artemisinin at 0.14 µg/ml inhibits the growth of cultured Pneumocystis carinii by 55% (23) and has IC50 values of 3.8 and 5.8 µg/ml against T. cruzi and T. b. rhodesiense in cell culture (27). At doses as high as 400 mg/kg of body weight, artemisinin had no efficacy against Cryptosporidium parvum infections in a neonatal mouse model (14). Nonprotozoans are similarly unaffected by the artemisinins. For example, the semisynthetic artemisinin, artemether, had almost no inhibitory activity against 86 bacterial and 72 fungal strains with MICs ranging from 64 to >512 µg/ml (13).

The iron-dependent artemisinin alkylation hypothesis is supported by the peroxide bond-dependent activity of artemisinin and OZ277 against plasmodia and the minimal and peroxide bond-independent activity of these two compounds against other protozoa. The signal biochemical difference between plasmodia and all other protozoa is that the former degrade Hb and the latter do not. This is most clearly evident for the intraerythrocytic protozoan Babesia, which unlike Plasmodium, does not catabolize Hb (31). It has also been suggested (21) that artemisinin may undergo reductive activation in the mitochondria of P. falciparum and thereby inhibit the growth of the parasite. Indeed, in addition to Hb (19, 26, 34, 43), artemisinin alkylates other hemoproteins, such as cytochrome c (43). However, if the mitochondria were the primary plasmodial target of artemisinin (21), it is hard to explain why all other protozoa are so much less sensitive to artemisinin.

The iron-dependent artemisinin alkylation hypothesis is also supported by the antiplasmodial stage specificity of these drugs, that is, young ring stage malaria parasites are slightly less sensitive to the artemisinins and OZ277 than are the more mature parasites in the trophozoite and schizont stages (22). Hb digestion has been underway for only a short time in the ring stage compared to that in the mature parasite stages, and consequently less heme has been generated. Even so, hemozoin can already be detected in young ring stage parasites (15). Similarly, young gametocytes are more susceptible (20) to artemisinin than are more mature gametocytes, presumably a reflection of the greater heme content (7) of the former. Only the sporozoite, bereft of hemozoin (36), is unaffected by artemisinin (10).

Whether heme or protein alkylation is the more important pharmacodynamic event in the antimalarial activity of artemisinin and OZ277 is unknown. In this regard, it is interesting to note that artemisinin inhibits the putative target PfATP6 with an IC50 of 79 nM, a value 2 orders of magnitude more potent than the one reported for OZ277 (IC50 = 7,700 nM) (39). Deoxyartemisinin at concentrations up to 50 µM does not inhibit PfATP6 (11). It will be informative to ascertain whether PfATP6 homologs in other protozoan parasites are inhibited by artemisinin and deoxyartemisinin. Future studies are likely to reveal new plasmodial targets of the artemisinins and synthetic peroxides.


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ACKNOWLEDGMENTS
 
This investigation received financial support from Medicines for Malaria Venture.

We thank Christian Scheurer, Monica Cal, Christina Kunz, and Sacha Schneeberger for technical assistance with the antiprotozoal assays.


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FOOTNOTES
 
* Corresponding author. Mailing address: College of Pharmacy, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198-6025. Phone: (402) 559-5362. Fax: (402) 559-9543. E-mail: jvenners{at}unmc.edu Back

{triangledown} Published ahead of print on 11 June 2007. Back


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REFERENCES
 
    1
  1. Ankli, A., M. Heinrich, P. Bork, L. Wolfram, P. Bauerfeind, R. Brun, C. Schmid, C. Weiss, R. Bruggisser, J. Gertsch, M. Wasescha, and O. Sticher. 2002. Yucatec Mayan medicinal plants: evaluation based on indigenous uses. J. Ethnopharmacol. 79:43-52.[CrossRef][Medline]
    2. 2
  2. Asawamahasakda, W., I. Ittarat, Y. M. Pu, H. Ziffer, and S. R. Meshnick. 1994. Reaction of antimalarial endoperoxides with specific parasite proteins. Antimicrob. Agents Chemother. 38:1854-1858.[Abstract/Free Full Text]
    3. 3
  3. Avery, M. A., K. M. Muraleedharan, P. V. Desai, A. K. Bandyopadhyaya, M. M. Furtado, and B. L. Tekwani. 2003. Structure-activity relationships of the antimalarial agent artemisinin. 8. Design, synthesis, and CoMFA studies toward the development of artemisinin-based drugs against leishmaniasis and malaria. J. Med. Chem. 46:4244-4258.[CrossRef][Medline]
    4. 4
  4. Bhisutthibhan, J., X.-Q. Pan, P. A. Hossler, D. J. Walker, C. A. Yowell, J. Carlton, J. R. Dame, and S. R. Meshnick. 1998. The Plasmodium falciparum translationally controlled tumor protein homolog and its reaction with the antimalarial drug artemisinin. J. Biol. Chem. 273:16192-16198.[Abstract/Free Full Text]
    5. 5
  5. Brasseur, P., S. Lecoublet, N. Kapel, L. Favennec, and J. J. Ballet. 1998. In vitro evaluation of drug susceptibilities of Babesia divergens isolates. Antimicrob. Agents Chemother. 42:818-820.[Abstract/Free Full Text]
    6. 6
  6. Brossi, A., B. Venugopalan, L. Dominguez Gerpe, H. J. C. Yeh, J. L. Flippen-Anderson, P. Buchs, X. D. Luo, W. K. Milhous, and W. Peters. 1988. Arteether, a new antimalarial drug: synthesis and antimalarial properties. J. Med. Chem. 31:645-650.[CrossRef][Medline]
    7. 7
  7. Butcher, G. A. 1997. Antimalarial drugs and the mosquito transmission of Plasmodium. 1997. Int. J. Parasitol. 27:975-987.[CrossRef][Medline]
    8. 8
  8. Campanale, N., C. Nickel, C. A. Daubenberger, D. A. Wehlan, J. J. Gorman, N. Klonis, K. Becker, and L. Tilley. 2003. Identification and characterization of heme-interacting proteins in the malaria parasite, Plasmodium falciparum. J. Biol. Chem. 278:27354-27361.[Abstract/Free Full Text]
    9. 9
  9. Dong, Y., Y. Tang, J. Chollet, H. Matile, S. Wittlin, S. A. Charman, W. N. Charman, J. Santo Tomas, C. Scheurer, C. Snyder, B. Scorneaux, S. Bajpai, S. A. Alexander, X. Wang, M. Padmanilayam, S. R. Cheruku, R. Brun, and J. L. Vennerstrom. 2005. Effect of functional group polarity on the antimalarial activity of spiro and dispiro-1,2,4-trioxolanes. Bioorg. Med. Chem. 14:6368-6382.
    10. 10
  10. Dutta, G. P., R. Bajpai, and R. A. Vishwakarma. 1989. Artemisinin (qinghaosu)—a new gametocytocidal drug for malaria. Chemotherapy 35:200-207.[Medline]
    11. 11
  11. Eckstein-Ludwig, U., R. J. Webb, I. D. A. van Goethem, J. M. East, A. G. Lee, M. Kimura, P. M. O'Neill, P. G. Bray, S. A. Ward, and S. Krishna. 2003. Artemisinins target the SERCA of Plasmodium falciparum. Nature 424:957-961.[CrossRef][Medline]
    12. 12
  12. Egan, T. J., J. M. Combrinck, J. Egan, G. R. Hearne, H. M. Marques, S. Ntenteni, B. T. Sewells, P. J. Smith, D. Taylor, D. A. Van Schalkwyk, and J. C. Walden. 2002. Fate of haem iron in the malaria parasite Plasmodium falciparum. Biochem. J. 365:343-347.[CrossRef][Medline]
    13. 13
  13. Farina, C., P. Olliaro, and A. Goglio. 1997. Failure to detect significant in-vitro activity of artemether and ß-arteether against bacteria and fungi by an agar dilution test. J. Antimicrob. Chemother. 40:743-745.[Free Full Text]
    14. 14
  14. Fayer, R., and W. Ellis. 1994. Qinghaosu (artemisinin) and derivatives fail to protect neonatal BALB/c mice against Cryptosporidium parvum (Cp) infection. J. Euk. Microbiol. 41:41S.[Medline]
    15. 15
  15. Francis, S. E., D. J. Sullivan, Jr., and D. E. Goldberg. 1997. Hemoglobin metabolism in the malarial parasite Plasmodium falciparum. Annu. Rev. Microbiol. 51:97-123.[CrossRef][Medline]
    16. 16
  16. Hemphill, A., B. Gottstein, and H. Kaufmann. 1996. Adhesion and invasion of bovine endothelial cells by Neospora caninum. Parasitology 112:183-197.
    17. 17
  17. Jefford, C. W. 2001. Why artemisinin and certain synthetic peroxides are potent antimalarials. Implications for the mode of action. Curr. Med. Chem. 8:1803-1826.[Medline]
    18. 18
  18. Jones-Brando, L., J. D'Angelo, G. H. Posner, and R. Yolken. 2006. In vitro inhibition of Toxoplasma gondii by four new derivatives of artemisinin. Antimicrob. Agents Chemother. 50:4206-4208.[Abstract/Free Full Text]
    19. 19
  19. Kannan, R., K. Kumar, D. Sahal, S. Kukreti, and V. S. Chauhan. 2005. Reaction of artemisinin with haemoglobin: implications for antimalarial activity. Biochem. J. 385:409-418.[CrossRef][Medline]
    20. 20
  20. Kumar, N., and H. Zheng. 1990. Stage-specific gametocytocidal effect in vitro of the antimalaria drug qinghaosu on Plasmodium falciparum. Parasitol. Res. 76:214-218.[CrossRef][Medline]
    21. 21
  21. Li, W., W. Mo, D. Shen, L. Sun, J. Wang, S. Lu, J. M. Gitschier, and B. Zhou. 2005. Yeast model uncovers dual roles of mitochondria in the action of artemisinin. PLoS Genet. 1:0329-0334.
    22. 22
  22. Maerki, S., R. Brun, S. A. Charman, A. Dorn, H. Matile, and S. Wittlin. 2006. In vitro assessment of the pharmacodynamic properties and the partitioning of OZ277/RBx-11160 in cultures of Plasmodium falciparum. J. Antimicrob. Chemother. 58:52-58.[Abstract/Free Full Text]
    23. 23
  23. Merali, S., and S. R. Meshnick. 1991. Susceptibility of Pneumocystis carinii to artemisinin in vitro. Antimicrob. Agents Chemother. 35:1225-1227.[Abstract/Free Full Text]
    24. 24
  24. Meshnick, S. R. 2002. Artemisinin: mechanisms of action, resistance and toxicity. Int. J. Parasitol. 32:1655-1660.[CrossRef][Medline]
    25. 25
  25. Meshnick, S. R., A. Thomas, A. Ranz, C.-M. Xu, and H.-Z. Pan. 1991. Artemisinin (qinghaosu): the role of intracellular hemin in its mechanism of antimalarial action. Mol. Biochem. Parasitol. 49:181-190.[CrossRef][Medline]
    26. 26
  26. Messori, L., C. Gabbiani, A. Casini, M. Siragusa, F. F. Vincieri, and A. R. Bilia. 2006. The reaction of artemisinins with hemoglobin: a unified picture. Bioorg. Med. Chem. 14:2972-2977.[CrossRef][Medline]
    27. 27
  27. Mishina, Y. V., S. Krishna, R. K. Haynes, and J. C. Meade. 2007. Artemisinins inhibit Trypanosoma cruzi and Trypanosoma brucei rhodesiense in vitro growth. Antimicrob. Agents Chemother. 51:1852-1854.[Abstract/Free Full Text]
    28. 28
  28. Nguyen, C., G. Kasinathan, I. Leal-Cortijo, A. Musso-Buendia, M. Kaiser, R. Brun, L. M. Ruiz-Perez, N. G. Johansson, D. Gonzalez-Pacanowska, and I. H. Gilbert. 2005. Deoxyuridine triphosphate nucleotidohydrolase as a potential antiparasitic drug target. J. Med. Chem. 48:5942-5954.[CrossRef][Medline]
    29. 29
  29. O'Neill, P. M., and G. H. Posner. 2004. A medicinal chemistry perspective on artemisinin and related endoperoxides. J. Med. Chem. 47:2945-2964.[CrossRef][Medline]
    30. 30
  30. Ou-Yang, K., E. C. Krug, J. J. Marr, and R. L. Berens. 1990. Inhibition of growth of Toxoplasma gondii by qinghaosu and derivatives. Antimicrob. Agents Chemother. 34:1961-1965.[Abstract/Free Full Text]
    31. 31
  31. Richier, E., G. A. Biagini, S. Wein, F. Boudou, P. G. Bray, S. A. Ward, E. Precigout, M. Calas, J.-F. Dubremetz, and H. J. Vial. 2006. Potent antihematozoan activity of novel bisthiazolium drug T16: evidence for inhibition of phosphatidylcholine metabolism in erythrocytes infected with Babesia and Plasmodium spp. Antimicrob. Agents Chemother. 50:3381-3388.[Abstract/Free Full Text]
    32. 32
  32. Robert, A., F. Benoit-Vical, C. Claparols, and B. Meunier. 2005. The antimalarial drug artemisinin alkylates heme in infected mice. Proc. Natl. Acad. Sci. USA 102:13676-13680.[Abstract/Free Full Text]
    33. 33
  33. Robert, A., Y. Coppel, and B. Meunier. 2002. Alkylation of heme by the antimalarial drug artemisinin. Chem. Comm. 414-415.
    34. 34
  34. Selmeczi, K., A. Robert, C. Claparols, and B. Meunier. 2004. Alkylation of human hemoglobin A0 by the antimalarial drug artemisinin. FEBS Lett. 556:245-248.[CrossRef][Medline]
    35. 35
  35. Sperandeo, N. R., and R. Brun. 2003. Synthesis and biological evaluation of pyrazolylnaphthoquinones as new potential antiprotozoal and cytotoxic agents. Chembiochem 4:69-72.[CrossRef][Medline]
    36. 36
  36. Sullivan, D. J., Jr. 2003. Hemozoin: a biocrystal synthesized during the degradation of hemoglobin. Biopolymers 9:129-163.
    37. 37
  37. Tang, Y., Y. Dong, and J. L. Vennerstrom. 2004. Synthetic peroxides as antimalarials. Med. Res. Rev. 24:425-448.[CrossRef][Medline]
    38. 38
  38. Tasdemir, D., M. Kaiser, R. Brun, V. Yardley, T. J. Schmidt, F. Tosun, and P. Ruedi. 2006. Antitrypanosomal and antileishmanial activities of flavonoids and their analogues: in vitro, in vivo, structure-activity relationship, and quantitative structure-activity relationship studies. Antimicrob. Agents Chemother. 50:1352-1364.[Abstract/Free Full Text]
    39. 39
  39. Uhlemann, A.-C., S. Wittlin, H. Matile, L. Y. Bustamante, and S. Krishna. 2007. Mechanism of antimalarial action of synthetic trioxolane RBX11160 (OZ277). Antimicrob. Agents Chemother. 51:667-672.[Abstract/Free Full Text]
    40. 40
  40. Vennerstrom, J. L., S. Arbe-Barnes, R. Brun, S. A. Charman, F. C. K. Chiu, J. Chollet, Y. Dong, A. Dorn, D. Hunziker, H. Matile, K. McIntosh, M. Padmanilayam, J. Santo Tomas, C. Scheurer, B. Scorneaux, Y. Tang, U. Urwyler, W. Wittlin, and W. N. Charman. 2004. Identification of an antimalarial synthetic trioxolane drug development candidate. Nature 430:900-904.[CrossRef][Medline]
    41. 41
  41. Vroman, J. A., M. Alvim-Gaston, and M. A. Avery. 1999. Current progress in the chemistry, medicinal chemistry and drug design of artemisinin based antimalarials. Curr. Pharm. Design. 5:101-138.[Medline]
    42. 42
  42. Yang, D. M., and F. Y. Liew. 1993. Effects of qinghaosu (artemisinin) and its derivatives on experimental cutaneous leishmaniasis. Parasitology 106:7-11.[Medline]
    43. 43
  43. Yang, Y.-Z., B. Little, and S. R. Meshnick. 1994. Alkylation of proteins by artemisinin. Effects of heme, pH, and drug structure. Biochem. Pharmacol. 48:569-573.[CrossRef][Medline]

Antimicrobial Agents and Chemotherapy, August 2007, p. 2991-2993, Vol. 51, No. 8
0066-4804/07/$08.00+0     doi:10.1128/AAC.00225-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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