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A novel multiple-stage antimalarial agent that inhibits protein synthesis

A Corrigendum to this article was published on 25 May 2016

Abstract

There is an urgent need for new drugs to treat malaria, with broad therapeutic potential and novel modes of action, to widen the scope of treatment and to overcome emerging drug resistance. Here we describe the discovery of DDD107498, a compound with a potent and novel spectrum of antimalarial activity against multiple life-cycle stages of the Plasmodium parasite, with good pharmacokinetic properties and an acceptable safety profile. DDD107498 demonstrates potential to address a variety of clinical needs, including single-dose treatment, transmission blocking and chemoprotection. DDD107498 was developed from a screening programme against blood-stage malaria parasites; its molecular target has been identified as translation elongation factor 2 (eEF2), which is responsible for the GTP-dependent translocation of the ribosome along messenger RNA, and is essential for protein synthesis. This discovery of eEF2 as a viable antimalarial drug target opens up new possibilities for drug discovery.

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Figure 1: Chemical evolution of DDD107498 from the phenotypic hit.
Figure 2: Efficacy studies and parasite killing rate.
Figure 3: In vitro activity of DDD107498 against P. berghei liver stages.
Figure 4: DDD107498 targets protein synthesis via eEF2.

References

  1. World Health Organization . World Malaria Report 2014 (World Health Organization, 2014)

  2. Ariey, F. et al. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature 505, 50–55 (2014)

    Article  ADS  Google Scholar 

  3. Alonso, P. L. et al. A research agenda for malaria eradication: drugs. PLoS Med. 8, e1000402 (2011)

    Article  Google Scholar 

  4. Wells, T. N. C. & Gutteridge, W. E. in Neglected Diseases and Drug Discovery (eds Palmer, M. J. & Wells, T. N. C.) Ch. 1 1–32 (Royal Society of Chemistry, 2012)

    Google Scholar 

  5. Brenk, R. et al. Lessons learnt from assembling screening libraries for drug discovery for neglected diseases. ChemMedChem 3, 435–444 (2008)

    Article  CAS  Google Scholar 

  6. Delves, M. et al. The activities of current antimalarial drugs on the life cycle stages of Plasmodium: a comparative study with human and rodent parasites. PLoS Med. 9, e1001169 (2012)

    Article  Google Scholar 

  7. Russell, B. et al. Determinants of in vitro drug susceptibility testing of Plasmodium vivax . Antimicrob. Agents Chemother. 52, 1040–1045 (2008)

    Article  CAS  Google Scholar 

  8. Karyana, M. et al. Malaria morbidity in Papua Indonesia, an area with multidrug resistant Plasmodium vivax and Plasmodium falciparum . Malar. J. 7, 148 (2008)

    Article  Google Scholar 

  9. Angulo-Barturen, I. et al. A murine model of falciparum-malaria by in vivo selection of competent strains in non-myelodepleted mice engrafted with human erythrocytes. PLoS ONE 3, e2252 (2008)

    Article  ADS  Google Scholar 

  10. Sanz, L. M. et al. P. falciparum in vitro killing rates allow to discriminate between different antimalarial mode-of-action. PLoS ONE 7, e30949 (2012)

    Article  CAS  ADS  Google Scholar 

  11. Burrows, J. N., van Huijsduijnen, R. H., Mohrle, J. J., Oeuvray, C. & Wells, T. N. Designing the next generation of medicines for malaria control and eradication. Malar. J. 12, 187 (2013)

    Article  Google Scholar 

  12. Meister, S. et al. Imaging of Plasmodium liver stages to drive next-generation antimalarial drug discovery. Science 334, 1372–1377 (2011)

    Article  CAS  ADS  Google Scholar 

  13. Adjalley, S. H. et al. Quantitative assessment of Plasmodium falciparum sexual development reveals potent transmission-blocking activity by methylene blue. Proc. Natl Acad. Sci. USA 108, E1214–E1223 (2011)

    Article  Google Scholar 

  14. Bousema, T. & Drakeley, C. Epidemiology and infectivity of Plasmodium falciparum and Plasmodium vivax gametocytes in relation to malaria control and elimination. Clin. Microbiol. Rev. 24, 377–410 (2011)

    Article  Google Scholar 

  15. Delves, M. J. et al. Male and female Plasmodium falciparum mature gametocytes show different responses to antimalarial drugs. Antimicrob. Agents Chemother. 57, 3268–3274 (2013)

    Article  CAS  Google Scholar 

  16. Delves, M. J. et al. A high-throughput assay for the identification of malarial transmission-blocking drugs and vaccines. Int. J. Parasitol. 42, 999–1006 (2012)

    Article  CAS  Google Scholar 

  17. Blagborough, A. M. et al. Transmission-blocking interventions eliminate malaria from laboratory populations. Nature Commun. 4, 1812 (2013)

    Article  CAS  ADS  Google Scholar 

  18. Upton, L. M. et al. Lead clinical and preclinical antimalarial drugs can significantly reduce sporozoite transmission to vertebrate populations. Antimicrob. Agents Chemother. 59, 490–497 (2015)

    Article  CAS  Google Scholar 

  19. Janse, C. J. et al. High efficiency transfection of Plasmodium berghei facilitates novel selection procedures. Mol. Biochem. Parasitol. 145, 60–70 (2006)

    Article  CAS  Google Scholar 

  20. Flannery, E. L., Fidock, D. A. & Winzeler, E. A. Using genetic methods to define the targets of compounds with antimalarial activity. J. Med. Chem. 56, 7761–7771 (2013)

    Article  CAS  Google Scholar 

  21. Rottman, M. et al. Spiroindolones, a potent compound class for the treatment of malaria. Science 329, 1175–1180 (2010)

    Article  ADS  Google Scholar 

  22. McNamara, C. W. et al. Targeting Plasmodium PI(4)K to eliminate malaria. Nature 504, 248–253 (2013)

    Article  CAS  ADS  Google Scholar 

  23. Miotto, O. et al. Multiple populations of artemisinin-resistant Plasmodium falciparum in Cambodia. Nature Genet. 45, 648–655 (2013)

    Article  CAS  Google Scholar 

  24. Jorgensen, R., Merrill, A. R. & Andersen, G. R. The life and death of translation elongation factor 2. Biochem. Soc. Trans. 34, 1–6 (2006)

    Article  CAS  Google Scholar 

  25. Justice, M. C. et al. Elongation factor 2 as a novel target for selective inhibition of fungal protein synthesis. J. Biol. Chem. 273, 3148–3151 (1998)

    Article  CAS  Google Scholar 

  26. Capa, L., Mendoza, A., Lavandera, J. L., de las Heras, F. G. & Garcia-Bustos, J. F. Translation elongation factor 2 is part of the target for a new family of antifungals. Antimicrob. Agents Chemother. 42, 2694–2699 (1998)

    Article  CAS  Google Scholar 

  27. Shastry, M. et al. Species-specific inhibition of fungal protein synthesis by sordarin: identification of a sordarin-specificity region in eukaryotic elongation factor 2. Microbiology 147, 383–390 (2001)

    Article  CAS  Google Scholar 

  28. Jorgensen, R. et al. Two crystal structures demonstrate large conformational changes in the eukaryotic ribosomal translocase. Nature Struct. Biol. 10, 379–385 (2003)

    Article  CAS  Google Scholar 

  29. Nkrumah, L. J. et al. Efficient site-specific integration in Plasmodium falciparum chromosomes mediated by mycobacteriophage Bxb1 integrase. Nature Methods 3, 615–621 (2006)

    Article  CAS  Google Scholar 

  30. Biswas, S. et al. Interaction of apicoplast-encoded elongation factor (EF) EF-Tu with nuclear-encoded EF-Ts mediates translation in the Plasmodium falciparum plastid. Int. J. Parasitol. 41, 417–427 (2011)

    Article  CAS  Google Scholar 

  31. Cox, G. et al. Ribosome clearance by FusB-type proteins mediates resistance to the antibiotic fusidic acid. Proc. Natl Acad. Sci. USA 109, 2102–2107 (2012)

    Article  CAS  ADS  Google Scholar 

  32. Ding, X. C., Ubben, D. & Wells, T. N. A framework for assessing the risk of resistance for anti-malarials in development. Malar. J. 11, 292 (2012)

    Article  Google Scholar 

  33. Manske, M. et al. Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing. Nature 487, 375–379 (2012)

    Article  CAS  ADS  Google Scholar 

  34. Florens, L. et al. A proteomic view of the Plasmodium falciparum life cycle. Nature 419, 520–526 (2002)

    Article  CAS  ADS  Google Scholar 

  35. Jiménez-Díaz, M. B. et al. Quantitative measurement of Plasmodium-infected erythrocytes in murine models of malaria by flow cytometry using bidimensional assessment of SYTO-16 fluorescence. Cytometry A 75, 225–235 (2009)

    Article  Google Scholar 

  36. Charman, S. A. et al. Synthetic ozonide drug candidate OZ439 offers new hope for a single-dose cure of uncomplicated malaria. Proc. Natl Acad. Sci. USA 108, 4400–4405 (2011)

    Article  CAS  ADS  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from Medicines for Malaria Venture, the Wellcome Trust (100476 (I.H.G., A.H.F.), 091625 (R.N.P.) and 098051 (J.C.R., W.P., T.D.O.)), the Bill and Melinda Gates Foundation (OPP1043501 (M.D., R.S.)), the National Institutes of Health (R01 AI103058 to E.A.W. and D.A.F.) and the European Union (EVIMalaR (T.D.O.)). Drug Discovery Unit infrastructure was supported by the European Regional Development Fund 2007-2013 and UK Research Partnership Investment Fund awards to M. Ferguson, whom we also thank for continued support. We thank C. Sibley for discussions. We acknowledge the East Scotland Blood Transfusion Service, Ninewells Hospital, Dundee, for erythrocyte supply to Dundee. We thank L. D. Shultz and The Jackson Laboratory for providing access to non-obese diabetic SCID IL2Rγc null mice through their collaboration with the GlaxoSmithKline Tres Cantos Medicines Development Campus. The following are acknowledged for technical assistance: all members of the Drug Discovery Unit (Dundee), M. Berriman, J. Kamber, E. Kenangalem, A. LaCrue, O. Montagnat, J. Rini Poespoprodjo, M. Sanders, S. Sax, C. Scheurer, L. Trianty, M. Tunnicliff (detailed in Supplementary Information).

Author information

Authors and Affiliations

Authors

Contributions

The author contributions are detailed in the Supplementary Information.

Corresponding authors

Correspondence to Kevin D. Read or Ian H. Gilbert.

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Competing interests

A patent relating to this work has been filed (PCT/GB2009/002084). K.J.D. and R.W.S. have shares in TropIQ Health Sciences.

Extended data figures and tables

Extended Data Figure 1 Synthetic methodology.

a, Synthesis of DDD107498 (4). b, Synthesis of DDD102542 (6a) and DDD103679 (6b).

Extended Data Figure 2 In vitro activity.

a, In vitro activity against a panel of resistant and sensitive strains of P. falciparum. CQ, chloroquine; PYR, pyrimethamine; CYC, cycloguanil; QUI, quinine; SUL, sulfadoxine; MFQ, mefloquine. Resistance as follows: K1 (CQ, SUL, PYR, CYC); W2 (CQ, SUL, PYR, CYC); 7G8 (CQ, PYR, CYC); TM90C2A (CQ, PYR, MFQ, CYC); D6 (MFQ); V1/S (CQ, SUL, PYR, CYC). Data are the means ± s.d. of n = 3 independent [3H]hypoxanthine incorporation experiments (each run in duplicate). b, Ex vivo activity against P. falciparum and P. vivax clinical isolates from Papua (Indonesia). The lines on the scatter plots represent the median value for each drug. c, Effect of DDD107498 on P. falciparum 3D7, HepG2 and MRC5 cells. Data are the means ± s.d. of n reported independent experiments.

Extended Data Figure 3 Effect of DDD107498 on parasite morphology.

a, Phenotype of P. falciparum in peripheral blood of NOD-scid IL-2R_null mice engrafted with human erythrocytes. Blood samples were taken at days 5 and 7 of the assay (one and two asexual cycles, respectively) after the start of treatment with vehicle or DDD107498 at day 3. The bi-dimensional flow cytometry plots measure the murine (Ter-119-PE+) and human (Ter-119-PE−) erythrocytes, and the presence of nucleic acids (infected SYTO-16+ events). The blue circles indicate the region of infected erythrocytes. Vehicle-treated mice showed a characteristic pattern of staining with SYTO-16 (ref. 35), which correlated with the presence of healthy rings, trophozoites and schizonts in blood smears. Conversely, mice treated with DDD107498 at 50 mg per kg showed only trophozoites with condensed cytoplasm and some pyknotic cells at day 5 (red circle in flow cytometry plot and corresponding blood smears). By day 7, few infected erythrocytes were detected by flow cytometry and blood smears revealed parasites with a similar morphology to those at day 5. This suggests that trophozoites are the most sensitive population since the cycle is interrupted at this stage. The images displayed are taken from a mouse with high levels of parasitaemia. At least 50 parasites were counted per sample screened in the microscope. Of these, four photographs of representative parasite phenotypes were selected to represent the morphology of the most prevalent phenotype. Thus, this is a qualitative assessment. b, Stage specificity assays using synchronized cultures. For morphological analysis of antimalarial drug action, thin blood smears were prepared, fixed and stained with Giemsa followed by examination with an upright microscope using an oil-immersion lens (×100). For parasitaemia determination, a total number of 1,000 red blood cells (corresponding to five microscopic fields) were counted. R to T, abnormal trophozoites observed after exposure for 24 h of synchronized rings to DDD107498. T to S, trophozoites do not develop into schizonts after exposure for 24 h to DDD107498. S to R, no ring stages are observed 24 h after treatment of schizonts with DDD107498. c, Percentage parasitaemia in the red blood cells. R, ring stage; T, trophozoite; S, schizont.

Extended Data Figure 4 Prophylactic activity of DDD107498 against sporozoite challenge.

P. berghei (luciferase) sporozoite in vivo mouse model of chemoprotection. A dose of 3 mg per kg was fully protective. Data are the mean of n = 5 experiments.

Extended Data Figure 5 DDD107498 in vitro activity on the different life-cycle stages of Plasmodium spp.

For comparator data with known antimalarials, see figure 5 in ref. 6. DDD107498 also showed potent activity in vitro against P. berghei ookinetes16 (5.0 nM (95% CI 4.4–5.7 nM)), confirming that if DDD107498 was taken up during a blood meal, it could continue to kill parasites within the mosquito.

Extended Data Figure 6 In vivo P. berghei mouse-to-mouse assay.

a, Impact of DDD107498 treatment on in vivo mosquito infection. Mice infected with P. berghei (PbGFPCON507 clone 1)19 were dosed orally with DDD107489 at 3 mg per kg, atovaquone at 0.3 mg per kg, sulphadiazine at 8.4 mg per kg, or were not drug treated (negative control). After 24 h, populations of treated mice (n = 5) were exposed to 500 female Anopheles stephensi mosquitoes, and oocyst intensity and infection prevalence in the mosquito midgut was measured 10 days after feeding. Individual data points represent the number of oocysts found in individual mosquitoes. Two replicates were performed. Sulphadiazine (8.4 mg per kg, intraperitoneally) and atovaquone (0.3 mg per kg, intraperitoneally) were used as negative and positive transmission-blocking drug controls, respectively. Horizontal bars represent mean intensity of infection, and error bars s.e.m., within individual mosquito populations. b, Impact of treatment with DDD107498 over a complete transmission cycle in vivo. After drug treatment of infected mice and mosquito feeds, surviving potentially infectious mosquitoes were allowed to blood-feed on naive mice at a range of transmission settings (biting rates of 2, 5 and 10 bites per naive mouse) to assess the ability of drug treatment to reduce the number of new malarial cases after mosquito bite. Efficacy is expressed as the following: (1) impact on the mosquito population—expressed as reduction in both oocyst and sporozoite intensity and prevalence; (2) impact on subsequent transmission to new naive vertebrate hosts—expressed as reduction in infection of naive mice (reduction in number of new malarial cases after mosquito bite) and inhibition of subsequent parasitaemia (day 10 after bite) in mice that do become infected; (3) effect size generated—by fitting the data achieved within this assay to a chain-binomial model we could assess the ability of DDD107498 to reduce R0 (assuming 100% coverage). If R0 is reduced to <1, transmission is unsustainable and elimination will occur. Values of 95% CIs are shown in brackets. Efficacy is calculated in comparison with no-drug controls. TB, transmission blocking.

Extended Data Figure 7 ClustalWS alignment of eEF2 sequences from human, yeast and Plasmodium (P. falciparum).

Alignment made using Jalview ClustalX default colouring.

Extended Data Figure 8 Fitness phenotypes of DDD107498-resistant parasite lines.

Unmarked Dd2- and DDD107498-selected parasites with various levels of resistance were assessed for growth in a competition assay, relative to a Dd2–GFP reference line. a, Equal numbers of unmarked test lines were mixed with the Dd2–GFP reference, in triplicate wells, and the ratio of non-fluorescent and fluorescent cells assessed by flow cytometry over time. At day 0, all lines had a 1:1 ratio with the Dd2–GFP reference. Increased growth of the test line over the Dd2–GFP reference, which has a slower growth rate than unmarked WT Dd2, would result in an increased ratio of test:Dd2–GFP. b, Growth assay of four different test lines: (1) WT Dd2, (2) EF2-E134D, (3) EF2-L755F and (4) EF2-Y186N, relative to Dd2–GFP. A faster growth rate of WT Dd2 (DDD107498 IC50 0.14 nM) relative to the fluorescent Dd2–GFP line is reflected in an increased ratio over time. The low-level resistant line EF2-E134D (IC50 5.8 nM) did not attain a WT growth rate, and the high-level resistant lines EF2-L755F (IC50 660 nM) and EF2-Y186N (3,100 nM) were further impaired. Means ± s.d.; n = 4 independent experiments, each run in triplicate.

Extended Data Table 1 Pharmacokinetics and rodent efficacy of DDD107498
Extended Data Table 2 Summary of resistance selection experiments with DDD107498

Supplementary information

Supplementary Information

This file contains author contributions, chemical synthesis, erythrocyte parasite assays, chemoprotection and transmission blocking assays, in vitro and in vivo DMPK, selectivity index and safety profile, mode of action studies. It also contains Supplementary Tables 1-2. (PDF 1226 kb)

Supplementary Data 1

This file contains an overview of Genome Sequencing Data. Summary statistics from genome sequencing of DDD107498 resistant Plasmodium falciparum lines. Genome sequence generation and analysis is described in Supplementary Methods. This table summarises the sequencing output, along with the number of indel and single nucleotide polymorphisms found in each of ten resistant lines compared to their three parental clones. (XLSX 12 kb)

Supplementary Data 2

This file contains Single Nucleotide Polymorphism (SNP) in Resistant Clones Summary. It summarizes the single nucleotide polymorphisms found within Pf3D7_145110 (PfeEF2) in the DDD107498 resistant Plasmodium falciparum lines. This table includes IC50 data for each resistant line, the nature and position of PfeEF2 mutations found in each line, and the number of whole genome and capillary sequencing reads that validate each mutation. (XLSX 148 kb)

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Baragaña, B., Hallyburton, I., Lee, M. et al. A novel multiple-stage antimalarial agent that inhibits protein synthesis. Nature 522, 315–320 (2015). https://doi.org/10.1038/nature14451

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