Open Access

In vitro activity of anti-malarial ozonides against an artemisinin-resistant isolate

  • Fabian Baumgärtner1, 2,
  • Joëlle Jourdan1, 2,
  • Christian Scheurer1, 2,
  • Benjamin Blasco3,
  • Brice Campo3,
  • Pascal Mäser1, 2 and
  • Sergio Wittlin1, 2Email author
Malaria Journal201716:45

https://doi.org/10.1186/s12936-017-1696-0

Received: 5 November 2016

Accepted: 13 January 2017

Published: 25 January 2017

Abstract

Background

Recently published data suggest that artemisinin derivatives and synthetic peroxides, such as the ozonides OZ277 and OZ439, have a similar mode of action. Here the cross-resistance of OZ277 and OZ439 and four additional next-generation ozonides was probed against the artemisinin-resistant clinical isolate Plasmodium falciparum Cam3.I, which carries the K13-propeller mutation R539T (Cam3.IR539T).

Methods

The previously described in vitro ring-stage survival assay (RSA0–3h) was employed and a simplified variation of the original protocol was developed.

Results

At the pharmacologically relevant concentration of 700 nM, all six ozonides were highly effective against the dihydroartemisinin-resistant P. falciparum Cam3.IR539T parasites, showing a per cent survival ranging from <0.01 to 1.83%. A simplified version of the original RSA0–3h method was developed and gave similar results, thus providing a practical drug discovery tool for further optimization of next-generation anti-malarial peroxides.

Conclusion

The absence of in vitro cross-resistance against the artemisinin-resistant clinical isolate Cam3.IR539T suggests that ozonides could be effective against artemisinin-resistant P. falciparum. How this will translate to the human situation in clinical settings remains to be investigated.

Keywords

Ring-stage survival assay Artemisinin Ozonide Plasmodium falciparum Cam3.IR539T Drug resistance

Background

Malaria is one of the most important tropical diseases resulting in 214 million new cases and an estimated 438,000 malaria deaths worldwide in 2015 [1]. The discovery of artemisinin in the 1970s was an important step forward in anti-malarial drug therapy and was recognized with the Nobel Prize in Physiology or Medicine in 2015 [2, 3]. Artemisinin and its semi-synthetic derivatives, such as dihydroartemisinin (DHA) (Fig. 1), artesunate and artemether, contain a unique sesquiterpene lactone peroxide (1,2,4-trioxane) structure and artemisinin-based combination therapy (ACT) represents the current first-line treatment of uncomplicated Plasmodium falciparum malaria [46]. Since the starting material artemisinin is a natural product, its production is limited to the availability of the plant [4, 7], although several total syntheses of artemisinin have been described [8]. In 2004, Vennerstrom et al. reported the discovery of a completely synthetic peroxide anti-malarial containing a 1,2,4-trioxolane (ozonide) pharmacophore named OZ277 (arterolane) (Fig. 1) with anti-malarial activity comparable to the artemisinin derivatives [9, 10]. In combination with piperaquine, arterolane was registered for anti-malarial combination therapy in India in 2011 [1114]. The next-generation ozonide, OZ439 (artefenomel) (Fig. 1), exhibits an increased pharmacokinetic half-life and good safety profile and is now being tested in phase IIb clinical trials [12, 1417].
Fig. 1

Chemical structures of dihydroartemisinin (DHA) and the six ozonides investigated

The iron-dependent alkylation hypothesis is one of the proposed modes of action of artemisinin and synthetic peroxides [1821] where the peroxide is thought to be activated by the reductive cleavage in the presence of ferrous haem (or free Fe(II) derived from haem) released as a by-product of haemoglobin digestion in the food vacuole [20, 2227]. Thereby carbon-centred radicals are generated, which then alkylate haem and parasite proteins [2833]. The interaction of the artemisinin derivatives or ozonides with parasite targets is irreversible [31, 34]. Although the semi-synthetic artemisinins are highly effective, prolonged parasite clearance times were first reported along the Thai–Cambodian border in 2006, suggesting an emerging artemisinin resistance phenotype [35]. Today, delayed parasite clearance following treatment with artemisinin derivatives has been observed across Southeast Asia [3641]. It was found that mutations in the Kelch 13 propeller domain are associated with ring-stage parasites entering a quiescent state with delayed parasite clearance after exposure to artemisinins [4145]. When 50% inhibitory concentrations (IC50) were measured using conventional methods such as the [3H] hypoxanthine incorporation assay [46], no difference was observed between artemisinin-resistant and -susceptible strains after treatment with artemisinin or its derivatives [4750]. In an effort to correlate the delayed parasite clearance observed in vivo with in vitro parasite survival, Witkowski et al. [48, 49] developed a ring-stage survival assay (RSA0–3h) that exploited the differences in susceptibility observed between wild-type and K13 mutants at the early ring stage of the asexual blood cycle following a short pulse of artemisinin treatment. In the RSA0–3h, synchronized young ring stage parasites (0–3 h old) are exposed to drugs for 6 h, and then cultured in drug free culture medium for 66 h before relative growth is determined by microscopic analysis [48, 49]. Since the structural analogies between artemisinins and ozonides (Fig. 1) suggest that they share similar modes of action, and thus some level of cross resistance [9, 10, 51, 52], the per cent survival of an artemisinin-resistant clinical isolate (Cam3.IR539T) treated with DHA, OZ277, OZ439, and four additional next-generation ozonides (Fig. 1) using the RSA0–3h as described by Witkowski et al. [48, 49] was evaluated. Additionally, a sub-set of these compounds was tested in the RSA0–3h described by Xie et al. [53] that also uses tightly synchronized ring-stage cultures, but allows the assay to be performed routinely within a convenient time-frame.

Methods

Parasite cultivation

The artemisinin-resistant P. falciparum isolate Cam3.IR539T from Battambang, Cambodia was obtained from BEI Resources [54] with the accession number MRA-1240. The drug-sensitive P. falciparum strain NF54 (airport strain from The Netherlands) was provided by F. Hoffmann-La Roche Ltd. Parasites were cultivated in standard cultivation medium, consisting of hypoxanthine (50 mg/l), RPMI (10.44 g/l) supplemented with HEPES (5.94 g/l), albumax (5 g/l), sodium bicarbonate (2.1 g/l) and neomycin (100 mg/l) [55].

Ring-stage survival assays (RSA0–3h)

Ring-stage survival assays (RSA0–3h) were carried out essentially as previously described by Witkowski et al. [48], but with a few modifications in the drug-washing procedure to ensure that no residual peroxide was present during the 66-h post-treatment period [56]. Briefly, zero to 3 h post-invasion ring stages were adjusted to 1% parasitaemia and 2.5% haematocrit by adding uninfected erythrocytes, transferred in a total volume of 1 ml into 48-well plates and exposed for 6 h to a range of concentrations (700, 350, 175, 88, and 49 nM) of DHA or one of the six ozonides tested in this study. The synthesis of the four next-generation ozonides, OZ493, OZ609, OZ655 and OZ657, will be reported in due course by the laboratory of Prof. Jonathan Vennerstrom (pers. comm.). After 6 h, cultures were transferred to 15 ml conical tubes, centrifuged at 1400 rpm (400g) for 2 min and carefully washed two times with 12 ml of culture medium. The complete removal of compound after washing was verified by incubating the supernatant recovered after the last washing step with fresh cultures of NF54 parasites, ensuring that no growth inhibition was detected. After washing, blood pellets were resuspended in complete drug-free culture medium, transferred into new wells and cultured for 66 h under standard conditions.

Thin blood smears were prepared, methanol-fixed and stained with 10% Giemsa. Per cent survival was assessed using light microscopy, counting the number of parasitized cells in ≥10,000 red blood cells (RBCs) and comparing survival to that of the drug-free dimethylsulfoxide incubation. Microscopy analysis was performed independently by two microscopists, one having more than 15 years of work experience.

Alternative parasite synchronization method

Parasites were synchronized according to Xie et al. [53] with 5% D-sorbitol. After 30 and 43 h, parasites were synchronized a second and third time, respectively, resulting in zero to 1-h old ring-stage parasites. The RSA0–3h was initiated 2 h later.

Standard [3H] hypoxanthine incorporation assay

The in vitro anti-malarial activity was measured using the [3H]-hypoxanthine incorporation assay [55]. Results were expressed as the concentration resulting in 50% inhibition (IC50).

Results

The per cent survival of parasites exposed to a concentration range of DHA and six different ozonides (Fig. 1) was determined using the artemisinin-resistant P. falciparum Cambodian isolate Cam3.IR539T. As expected, DHA exposure gave a high survival rate ranging from 74 to 33% at concentrations of 49 and 700 nM, respectively (Fig. 2), which is comparable to the observed survival value of 40% at 700 nM published previously [44]. In contrast, when tested at 700 nM, the two ozonides OZ277 and OZ439 showed an approximate 18- to 45-fold increase in potency compared with DHA (Fig. 2). Full and equal potency was observed when DHA, OZ277 and OZ439 were tested in parallel in the RSA0–3h using the artemisinin-sensitive strain NF54 (Additional file 1: Table S1). At the lowest concentration (49 nM), OZ277 had poor activity, showing a similar per cent survival to that of DHA, whereas OZ439 was still about fivefold more potent. A possible explanation for OZ439 being more potent than OZ277 could be related to its improved stability in blood as previously described [15]. In those studies, OZ277 or OZ439 were incubated at 37 °C in P. falciparum-infected human blood. After 2 h more than 90% of OZ277 was degraded, whereas OZ439 was found to be about 10–20× more stable. A similar and more recent study found similar differences in stability for OZ277 and OZ439 [56]. The same compounds were also tested in a more convenient variation of the standard RSA0–3h that uses synchronized ring-stage cultures that can be easily produced during normal working hours [53]. As shown in Table 1, this alternative synchronization method gave results that were comparable to those obtained using the standard RSA0–3h.
Fig. 2

Mean per cent survival ± standard error (SE) of Plasmodium falciparum isolate Cam3.IR539T parasites after a 6-h exposure to a range of concentrations of dihydroartemisinin (DHA) or six different ozonides. Three biological replicates were performed per compound

Table 1

Mean per cent survival (individual values in brackets) of Cam3.IR539T isolate after 6 h exposure to a range of concentrations of DHA, OZ439 or OZ277 using the synchronization protocol from Xie et al. [53]

Compounds

RSA values (% survival) at different concentrations

175 nM

350 nM

700 nM

DHA

46 (49, 43)

42 (45, 39)

37 (39, 35)

OZ277

4.0 (4.4, 3.6)

2.3 (1.9, 2.7)

1.4 (1.7, 1.1)

OZ439

<0.01 (<0.01, <0.01)

<0.01 (<0.01, <0.01)

<0.01 (<0.01, <0.01)

Two biological replicates were performed per compound

To investigate further the level of cross-resistance between DHA and the ozonides, four additional next-generation ozonides (OZ493, OZ609, OZ655, OZ657) (Fig. 1) were tested against the Cam3.IR539T parasites. While all six ozonides had a similar IC50 value using a conventional 72-h [3H] hypoxanthine incorporation assay (Additional file 1: Table S2), the RSA0–3h showed that OZ493, OZ609 and OZ655 were highly potent and completely inhibited the growth of the artemisinin-resistant isolate at the two highest concentrations tested (Fig. 2). At the lowest concentration, potency was comparable to that for OZ439. The overall potency of OZ657 was comparable to that of OZ277.

The RSA0–3h was recently developed to provide an in vitro correlate of the longer in vivo parasite clearance times observed after artemisinin treatment in Southeast Asia, which is widely interpreted as a sign of potential artemisinin resistance [57, 58]. Provided that the RSA0–3h does indeed predict the potency of compounds against artemisinin-resistant parasites in malaria patients, the here described data suggest that all of the tested ozonides are highly potent against isolates such as P. falciparum Cam3.IR539T. These data are in line with the recent clinical observation that the parasite clearance rate following OZ439 treatment is not significantly affected by resistance-associated mutations in the Kelch 13 propeller region [17] and the recent data published by Siriwardana et al. [59], which showed no reduced susceptibility of OZ439 in a different delayed clearance phenotype parasite (Cam3.II) in vitro.

Conclusion

In the traditional RSA0–3h, as well as a more convenient variation of the original method, all of the tested ozonides, were highly potent against the artemisinin-resistant isolate P. falciparum Cam3.IR539T in contrast to results for DHA. These data indicate that artemisinin-resistant P. falciparum infections could be successfully treated with ozonide anti-malarial drugs.

Abbreviations

OZ277: 

arterolane

OZ439: 

artefenomel

DHA: 

dihydroartemisinin

ACT: 

artemisinin combination therapy

RBC: 

red blood cell

Declarations

Authors’ contributions

FB, JJ, CS, BB, BC, PM and SW designed the research. FB and CS performed the research. All authors analysed data. FB, JJ and SW wrote the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We are grateful to Jonathan L Vennerstrom, Hugues Matile and Susan A Charman for critically reading the manuscript and making valuable suggestions.

Competing interests

The use of OZ277 and OZ439 against malaria has been patented.

Availability of data and materials

All data generated or analysed during this study are included in this published article and its supplementary information files.

Funding

This work was financially supported by the Swiss National Science Foundation (Grant 310030_149896 to SW), the Medicines for Malaria Venture, and the Swiss Tropical and Public Health Institute.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Swiss Tropical and Public Health Institute
(2)
University of Basel
(3)
Medicines for Malaria Venture, ICC

References

  1. WHO. World Malaria Report 2015. Geneva: World Health Organization; 2015.Google Scholar
  2. Tu Y. The discovery of artemisinin (qinghaosu) and gifts from Chinese medicine. Nat Med. 2011;17:1217–20.View ArticlePubMedGoogle Scholar
  3. Su XZ, Miller LH. The discovery of artemisinin and the Nobel Prize in Physiology or Medicine. Sci China Life Sci. 2015;58:1175–9.View ArticlePubMedPubMed CentralGoogle Scholar
  4. Klayman DL. Qinghaosu (artemisinin): an antimalarial drug from China. Science. 1985;228:1049–55.View ArticlePubMedGoogle Scholar
  5. White NJ. Artemisinin: current status. Trans R Soc Trop Med Hyg. 1994;88:3–4.View ArticleGoogle Scholar
  6. White NJ. Qinghaosu (artemisinin): the price of success. Science. 2008;320:330–4.View ArticlePubMedGoogle Scholar
  7. Kumar S, Srivastava S. Establishment of artemisinin combination therapy as first line treatment for combating malaria: Artemisia annua cultivation in India needed for providing sustainable supply chain of artemisinin. Curr Sci. 2005;89:1097–102.Google Scholar
  8. Avery MA, Chong WKM, Jennings- White C. Stereoselective total synthesis of (+)- artemisinin, the antimalarial constituent of Artemisia annua L. J Am Chem Soc. 1992;114:974–9.View ArticleGoogle Scholar
  9. Vennerstrom JL, Arbe-Barnes S, Brun R, Charman SA, Chiu FC, Chollet J, et al. Identification of an antimalarial synthetic trioxolane drug development candidate. Nature. 2004;430:900–4.View ArticlePubMedGoogle Scholar
  10. Tang Y, Dong Y, Vennerstrom JL. Synthetic peroxides as antimalarials. Med Res Rev. 2004;24:425–48.View ArticlePubMedGoogle Scholar
  11. Anthony MP, Burrows JN, Duparc S, Moehrle JJ, Wells TN. The global pipeline of new medicines for the control and elimination of malaria. Malar J. 2012;11:316.View ArticlePubMedPubMed CentralGoogle Scholar
  12. Mäser P, Wittlin S, Rottmann M, Wenzler T, Kaiser M, Brun R. Antiparasitic agents: new drugs on the horizon. Curr Opin Pharmacol. 2012;12:562–6.View ArticlePubMedGoogle Scholar
  13. Patil CY, Katare SS, Baig MS, Doifode SM. Fixed dose combination of arterolane and piperaquine: a newer prospect in antimalarial therapy. Ann Med Health Sci Res. 2014;4:466–71.View ArticlePubMedPubMed CentralGoogle Scholar
  14. Wells TN, van Huijsduijnen RH, Van Voorhis WC. Malaria medicines: a glass half full? Nat Rev Drug Discov. 2015;14:424–42.View ArticlePubMedGoogle Scholar
  15. Charman SA, Arbe-Barnes S, Bathurst IC, Brun R, Campbell M, Charman WN, et al. Synthetic ozonide drug candidate OZ439 offers new hope for a single-dose cure of uncomplicated malaria. Proc Natl Acad Sci USA. 2011;108:4400–5.View ArticlePubMedPubMed CentralGoogle Scholar
  16. Moehrle JJ, Duparc S, Siethoff C, van Giersbergen PL, Craft JC, Arbe-Barnes S, et al. First-in-man safety and pharmacokinetics of synthetic ozonide OZ439 demonstrates an improved exposure profile relative to other peroxide antimalarials. Br J Clin Pharmacol. 2013;75:535–48.View ArticleGoogle Scholar
  17. Phyo AP, Jittamala P, Nosten FH, Pukrittayakamee S, Imwong M, White NJ, et al. Antimalarial activity of artefenomel (OZ439), a novel synthetic antimalarial endoperoxide, in patients with Plasmodium falciparum and Plasmodium vivax malaria: an open-label phase 2 trial. Lancet Infect Dis. 2016;16:61–9.View ArticlePubMedPubMed CentralGoogle Scholar
  18. Kaiser M, Wittlin S, Nehrbass-Stuedli A, Dong Y, Wang X, Hemphill A, et al. Peroxide bond-dependent antiplasmodial specificity of artemisinin and OZ277 (RBx11160). Antimicrob Agents Chemother. 2007;51:2991–3.View ArticlePubMedPubMed CentralGoogle Scholar
  19. Creek DJ, Charman WN, Chiu FCK, Prankerd RJ, Dong Y, Vennerstrom JL, et al. Relationship between antimalarial activity and haem alkylation for spiro- and dispiro-1,2,4-trioxolane antimalarials. Antimicrob Agents Chemother. 2008;52:1291–6.View ArticlePubMedPubMed CentralGoogle Scholar
  20. Meunier B, Robert A. Heme as trigger and target for trioxane-containing antimalarial drugs. Acc Chem Res. 2010;43:1444–51.View ArticlePubMedGoogle Scholar
  21. Tilley L, Straimer J, Gnädig NF, Ralph SA, Fidock DA. Artemisinin action and resistance in Plasmodium falciparum. Trends Parasitol. 2016;32:682–96.View ArticlePubMedGoogle Scholar
  22. Klonis N, Crespo-Ortiz MP, Bottova I, Abu-Bakar N, Kenny S, Rosenthal PJ, Tilley L. Artemisinin activity against Plasmodium falciparum requires hemoglobin uptake and digestion. Proc Natl Acad Sci USA. 2011;108:11405–10.View ArticlePubMedPubMed CentralGoogle Scholar
  23. Crespo MD, Avery TD, Hanssen E, Fox E, Robinson TV, Valente P, Taylor DK, Tilley L. Artemisinin and a series of novel endoperoxide antimalarials exert early effects on digestive vacuole morphology. Antimicrob Agents Chemother. 2008;52:98–109.View ArticleGoogle Scholar
  24. Hartwig CL, Rosenthal AS, D’Angelo J, Griffin CE, Posner GH, Cooper RA. Accumulation of artemisinin trioxane derivatives within neutral lipids of Plasmodium falciparum malaria parasites is endoperoxide-dependent. Biochem Pharmacol. 2009;77:322–36.View ArticlePubMedGoogle Scholar
  25. Robert A, Claparols C, Witkowski B, Benoit-Vical F. Correlation between Plasmodium yoelii nigeriensis susceptibility to artemisinin and alkylation of heme by the drug. Antimicrob Agents Chemother. 2013;57:3998–4000.View ArticlePubMedPubMed CentralGoogle Scholar
  26. Klonis N, Creek DJ, Tilley L. Iron and heme metabolism in Plasmodium falciparum and the mechanism of action of artemisinins. Curr Opin Microbiol. 2013;16:722–7.View ArticlePubMedGoogle Scholar
  27. Uhlemann AC, Wittlin S, Matile H, Bustamante LY, Krishna S. Mechanism of antimalarial action of the synthetic trioxolane RBX11160 (OZ277). Antimicrob Agents Chemother. 2007;51:667–72.View ArticlePubMedGoogle Scholar
  28. Asawamahasakda W, Ittarat I, Pu YM, Ziffer H, Meshnick SR. Reaction of antimalarial endoperoxides with specific parasite proteins. Antimicrob Agents Chemother. 1994;38:1854–8.View ArticlePubMedPubMed CentralGoogle Scholar
  29. Meshnick SR. Artemisinin: mechanisms of action, resistance and toxicity. Int J Parasitol. 2002;32:1655–60.View ArticlePubMedGoogle Scholar
  30. Tang Y, Dong Y, Wang X, Sriraghavan K, Wood JK, Vennerstrom JL. Dispiro-1,2,4-trioxane analogs of a prototype dispiro-1,2,4-trioxolane: mechanistic comparators for artemisinin in the context of reaction pathways with iron (II). J Org Chem. 2005;70:5103–10.View ArticlePubMedGoogle Scholar
  31. Fügi MA, Wittlin S, Dong Y, Vennerstrom JL. Probing the antimalarial mechanism of artemisinin and OZ277 (arterolane) with nonperoxidic isosteres and nitroxyl radicals. Antimicrob Agents Chemother. 2010;54:1042–6.View ArticlePubMedGoogle Scholar
  32. Hartwig CL, Lauterwasser EMW, Mahajan SS, Hoke JM, Cooper RA, Renslo AR. Investigating the antimalarial action of 1,2,4-trioxolanes with fluorescent chemical probes. J Med Chem. 2011;54:8207–13.View ArticlePubMedPubMed CentralGoogle Scholar
  33. Tilley L, Charman S, Vennerstrom JL. Semisynthetic artemisinin and synthetic peroxide antimalarials. In: Palmer MJ, Wells TNC, editors. RSC Drug Discovery Series No. 14. London: Neglected Diseases and Drug Discovery; 2011. p. 33–64.Google Scholar
  34. Abiodun OO, Brun R, Wittlin S. In vitro interaction of artemisinin derivatives or the fully synthetic peroxidic anti-malarial OZ277 with thapsigargin in Plasmodium falciparum strains. Malar J. 2013;12:43.View ArticlePubMedPubMed CentralGoogle Scholar
  35. Noedl H, Se Y, Schaecher K, Smith BL, Socheat D, Fukuda MM. Artemisinin resistance in Cambodia 1 (ARC1) study consortium evidence of artemisinin-resistant malaria in western Cambodia. N Engl J Med. 2008;359:2619–20.View ArticlePubMedGoogle Scholar
  36. Noedl H, Socheat D, Satimai W. Artemisinin-resistant malaria in Asia. N Engl J Med. 2009;361:540–1.View ArticlePubMedGoogle Scholar
  37. Dondorp AM, Nosten F, Yi P, Das D, Phyo AP, Tarning J, et al. Artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med. 2009;38:455–67.View ArticleGoogle Scholar
  38. Amaratunga C, Sreng S, Suon S, Phelps ES, Stepniewska K, Lim P, et al. Artemisinin-resistant Plasmodium falciparum in Pursat province, western Cambodia: a parasite clearance rate study. Lancet Infect Dis. 2012;12:851–8.View ArticlePubMedPubMed CentralGoogle Scholar
  39. Phyo AP, Nkhoma S, Stepniewska K, Ashley EA, Nair S, McGready R, et al. Emergence of artemisinin-resistant malaria on the western border of Thailand: a longitudinal study. Lancet. 2012;379:1960–6.View ArticlePubMedPubMed CentralGoogle Scholar
  40. Ashley EA, Dhorda M, Fairhurst RM, Amaratunga C, Lim P, Suon S, et al. Spread of artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med. 2014;371:411–23.View ArticlePubMedPubMed CentralGoogle Scholar
  41. Paloque L, Ramadani AP, Mercereau-Puijalon O, Augereau JM, Benoit-Vical F. Plasmodium falciparum: multifaceted resistance to artemisinins. Malar J. 2016;15:149.View ArticlePubMedPubMed CentralGoogle Scholar
  42. Ariey F, Witkowski B, Amaratunga C, Beghain J, Langlois AC, Khim N, et al. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature. 2014;505:50–5.View ArticlePubMedGoogle Scholar
  43. Mok S, Ashley EA, Ferreira PE, Zhu L, Lin Z, Yeo T, et al. Population transcriptomics of human malaria parasites reveals the mechanism of artemisinin resistance. Science. 2015;347:431–5.View ArticlePubMedGoogle Scholar
  44. Straimer J, Gnädig NF, Witkowski B, Amaratunga C, Duru V, Ramadani AP, et al. Drug resistance. K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates. Science. 2015;347:428–31.View ArticlePubMedGoogle Scholar
  45. Mbengue A, Bhattacharjee S, Pandharkar T, Liu H, Estiu G, Stahelin RV, et al. A molecular mechanism of artemisinin resistance in Plasmodium falciparum malaria. Nature. 2015;520:683–7.View ArticlePubMedPubMed CentralGoogle Scholar
  46. Desjardins RE, Canfield CJ, Haynes JD, Chulay JD. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob Agents Chemother. 1979;16:710–8.View ArticlePubMedPubMed CentralGoogle Scholar
  47. Witkowski B, Lelièvre J, Barragán MJ, Laurent V, Su XZ, Berry A, Benoit-Vical F. Increased tolerance to artemisinin in Plasmodium falciparum is mediated by a quiescence mechanism. Antimicrob Agents Chemother. 2010;54:1872–7.View ArticlePubMedPubMed CentralGoogle Scholar
  48. Witkowski B, Khim N, Chim P, Kim S, Ke S, Kloeung N, et al. Reduced artemisinin susceptibility of Plasmodium falciparum ring stages in western Cambodia. Antimicrob Agents Chemother. 2013;57:914–23.View ArticlePubMedPubMed CentralGoogle Scholar
  49. Witkowski B, Amaratunga C, Khim N, Sreng S, Chim P, Kim S, et al. Novel phenotypic assays for the detection of artemisinin-resistant Plasmodium falciparum malaria in Cambodia: in vitro and ex vivo drug-response studies. Lancet Infect Dis. 2013;13:1043–9.View ArticlePubMedPubMed CentralGoogle Scholar
  50. Teuscher F, Gatton ML, Chen N, Peters J, Kyle DE, Cheng Q. Artemisinin-induced dormancy in Plasmodium falciparum: duration, recovery rates, and implications in treatment failure. J Infect Dis. 2010;202:1362–8.View ArticlePubMedPubMed CentralGoogle Scholar
  51. Jourdan J, Matile H, Reift E, Biehlmaier O, Dong Y, Wang X, et al. Monoclonal antibodies that recognize the alkylation signature of antimalarial ozonides OZ277 (Arterolane) and OZ439 (Artefenomel). ACS Infect Dis. 2015;2:54–61.View ArticlePubMedPubMed CentralGoogle Scholar
  52. Ismail HM, Barton VE, Panchana M, Charoensutthivarakul S, Biagini GA, Ward SA, et al. A click chemistry-based proteomic approach reveals that 1,2,4-trioxolane and artemisinin antimalarials share a common protein alkylation profile. Angew Chem Int Ed Engl. 2016;55:1–6.View ArticleGoogle Scholar
  53. Xie S, Dogovski C, Kenny S, Tilley L, Klonis N. Optimal assay design for determining the in vitro sensitivity of ring stage Plasmodium falciparum to artemisinins. Int J Parasitol. 2014;44:893–9.View ArticlePubMedGoogle Scholar
  54. B E I Resources. https://www.beiresources.org/Catalog/BEIParasiticProtozoa/MRA-1240.aspx. Accessed 26 Nov 2014.
  55. Snyder C, Chollet J, Santo-Tomas J, Scheurer C, Wittlin S. In vitro and in vivo interaction of synthetic peroxide RBx11160 (OZ277) with piperaquine in Plasmodium models. Exp Parasitol. 2007;115:296–300.View ArticlePubMedGoogle Scholar
  56. Yang T, Xie SC, Cao P, Giannangelo C, McCaw J, Creek DJ, Charman SA, Klonis N, Tilley L. Comparison of the exposure time dependence of the activities of synthetic ozonide antimalarials and dihydroartemisinin against K13 wild-type and mutant Plasmodium falciparum strains. Antimicrob Agents Chemother. 2016;60:4501–10.View ArticlePubMedPubMed CentralGoogle Scholar
  57. Phyo AP, Ashley EA, Anderson TJ, Bozdech Z, Carrara VI, Sriprawat K, et al. Declining efficacy of artemisinin combination therapy against P. falciparum malaria on the Thai-Myanmar border (2003–2013): the role of parasite genetic factors. Clin Infect Dis. 2016;63:784–91.View ArticlePubMedPubMed CentralGoogle Scholar
  58. Meshnick SR. Artemisinin resistance in Southeast Asia. Clin Infect Dis. 2016;63:1527.View ArticlePubMedGoogle Scholar
  59. Siriwardana A, Iyengar K, Roepe PD. Endoperoxide drug cross resistance patterns for Plasmodium falciparum exhibiting an artemisinin delayed clearance phenotype. Antimicrob Agents Chemother. 2016;60:6952–6.View ArticlePubMedGoogle Scholar

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