Targeting Plasmodium PI(4)K to eliminate malaria (original) (raw)

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Acknowledgements

We thank E. Miller for critical review of the manuscript and figure design. We also thank A. Rodriguez and the insectary core facility team at New York University for reliable supplies of malaria-infected mosquitoes. We gratefully acknowledge translational research grants (WT078285 and WT096157) from the Wellcome Trust and funding from the Medicines for Malaria Venture (MMV) to the Genomics Institute of the Novartis Research Foundation, the Swiss Tropical and Public Health Institute, Columbia University, the Novartis Institute for Tropical Diseases, the Singapore Immunology Network and Horizontal Programme on Infectious Diseases under the Agency Science Technology and Research (A*STAR, Singapore), and the Wellcome Trust (UK). Shoklo Malaria Research Unit is sponsored by The Wellcome Trust (UK), as part of the Oxford Tropical Medicine Research Programme of Wellcome Trust-Mahidol University. E.A.W. and D.A.F. are supported by grants from the Bill and Melinda Gates Foundation, MMV, and the National Institutes of Health (R01AI090141 to E.A.W. and R01085584 and R01079709 to D.A.F.).

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Author notes

1. Case W. McNamara and Marcus C. S. Lee: These authors contributed equally to this work.

Authors and Affiliations

1. Genomics Institute of the Novartis Research Foundation, San Diego, 92121, California, USA
   Case W. McNamara, Jason Roland, Advait Nagle, Arnab K. Chatterjee, Susan L. McCormack, Kerstin Gagaring, Maureen Ibanez, Nobutaka Kato, Kelli L. Kuhen, David M. Plouffe, Badry Bursulaya, David C. Tully, Richard J. Glynne & Elizabeth A. Winzeler
2. Department of Microbiology & Immunology, Columbia University Medical Center, New York, 10032, New York, USA
   Marcus C. S. Lee, T. R. Santha Kumar, Philipp P. Henrich & David A. Fidock
3. Novartis Institutes for Tropical Disease, 138670 Singapore,
   Chek Shik Lim, Siau Hoi Lim, Oliver Simon, Bryan K. S. Yeung, Christophe Bodenreider & Thierry T. Diagana
4. Department of Pediatrics, School of Medicine, University of California, San Diego, La Jolla, California 92093, USA,
   Micah J. Manary, Stephan Meister & Elizabeth A. Winzeler
5. Department of Parasitology, Biomedical Primate Research Centre, PO Box 3306, 2280 GH Rijswijk, The Netherlands,
   Anne-Marie Zeeman & Clemens H. M. Kocken
6. TropIQ Health Sciences, 6525 GA Nijmegen, The Netherlands,
   Koen J. Dechering, Martijn Timmerman & Robert W. Sauerwein
7. Swiss Tropical and Public Health Institute, CH-4002 Basel, Switzerland,
   Christoph Fischli & Matthias Rottmann
8. University of Basel, CH-4003 Basel, Switzerland,
   Matthias Rottmann
9. Department of Medicine, School of Medicine, Boston University, Boston, 02118, Massachusetts, USA
   Lucia Rameh
10. Novartis Institutes for BioMedical Research, CH-4002 Basel, Switzerland,
    Joerg Trappe & Dorothea Haasen
11. Department of Medical Microbiology, Radboud University, Nijmegen Medical CentrePO Box 9101, 6500 HB Nijmegen, The Netherlands,
    Robert W. Sauerwein
12. Laboratory of Malaria Immunobiology, Singapore Immunology Network, Agency for Science Technology and Research (A*STAR), Biopolis, 138648 Singapore,
    Rossarin Suwanarusk, Bruce Russell & Laurent Renia
13. Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System, 117545 Singapore,
    Bruce Russell
14. Nuffield Department of Medicine, Centre for Tropical Medicine, University of Oxford, Oxford OX3 7BN, UK,
    Francois Nosten
15. Shoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Mae Sot 63110, Thailand,
    Francois Nosten
16. Division of Infectious Diseases, Department of Medicine, Columbia University Medical Center, New York, 10032, New York, USA
    David A. Fidock

Authors

1. Case W. McNamara
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2. Marcus C. S. Lee
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3. Chek Shik Lim
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4. Siau Hoi Lim
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5. Jason Roland
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6. Advait Nagle
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7. Oliver Simon
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8. Bryan K. S. Yeung
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9. Arnab K. Chatterjee
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10. Susan L. McCormack
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11. Micah J. Manary
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12. Anne-Marie Zeeman
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13. Koen J. Dechering
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14. T. R. Santha Kumar
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15. Philipp P. Henrich
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16. Kerstin Gagaring
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17. Maureen Ibanez
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18. Nobutaka Kato
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19. Kelli L. Kuhen
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20. Christoph Fischli
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21. Matthias Rottmann
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22. David M. Plouffe
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23. Badry Bursulaya
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24. Stephan Meister
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25. Lucia Rameh
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26. Joerg Trappe
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27. Dorothea Haasen
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28. Martijn Timmerman
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29. Robert W. Sauerwein
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30. Rossarin Suwanarusk
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31. Bruce Russell
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32. Laurent Renia
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33. Francois Nosten
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34. David C. Tully
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35. Clemens H. M. Kocken
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36. Richard J. Glynne
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37. Christophe Bodenreider
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38. David A. Fidock
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39. Thierry T. Diagana
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40. Elizabeth A. Winzeler
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Contributions

C.W.M., with assistance from S.L.M., M.I. and D.M.P., evolved and characterized drug-resistant parasite lines, analysed microarray data, and performed phenotypic studies. M.C.S.L. performed genome editing and other transgenic parasite studies, as well as the fluorescence microscopy imaging. Additional experimental contributions were as follows: PvPI(4)K assay (C.S.L., S.H.L. and C.B.); imidazopyrazine chemistry (J.R., A.N., A.K.C. and D.C.T.); imidazopyrazine structure–activity studies (K.G. and K.L.K.); BQR695 re-synthesis (O.S.); quinoxaline development and human kinase panels (J.T. and D.H.); mutant P. berghei strain generation (T.R.S.K. and P.P.H.); next-generation sequencing data analysis (M.J.M.); in vitro assay with P. cynomolgi (A.-M.Z. and C.H.M.K.); sexual-stage P. falciparum assays (M.T., K.J.D. and R.W.S.); ex vivo assays on P. falciparum and P. vivax clinical isolates (R.S., B.R., L.R. and F.N.); in vivo efficacy studies in the mouse model (blood stage, C.F. and M.R.; liver stage, N.K.); in vitro assay with P. yoelii (D.M.P., S.M., S.L.M. and K.G.); in silico docking studies (B.B.). L.R. quantified phosphatidylinositol phosphates. R.J.G. managed Genomics Institute of the Novartis Research Foundation (GNF) activities. B.K.S.Y. coordinated collaborative efforts. C.W.M., M.C.S.L., C.B., D.A.F., T.T.D. and E.A.W. designed experiments and co-wrote the manuscript. C.W.M. and M.C.S.L. contributed equally to the study. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence toThierry T. Diagana or Elizabeth A. Winzeler.

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Competing interests

C.W.M., C.S.L., S.H.L., J.R., O.S., B.K.S.Y., K.L.K., K.G., D.M.P., B.B., J.T., D.H., D.T., R.J.G., C.B. and T.T.D. are employed by Novartis. C.W.M., J.R., K.L.K., B.B., J.T., D.H., D.T., R.J.G., T.T.D. and E.A.W. own shares of Novartis AG stock. E.A.W. has received grants from Novartis.

Extended data figures and tables

Extended Data Figure 1 Imidazopyrazines are active against all liver- and blood-stage forms within the vertebrate host.

The imidazopyrazines KAI407, KDU691 and KAI715 were tested, as available, in a comprehensive array of in vitro assays comprised of diverse Plasmodium species at representative stages of the vertebrate life cycle. a, The imidazopyrazines retained potency within 1−2 log units of atovaquone (ATQ), a licensed antimalarial with dual-stage activity, in the P. falciparum asexual blood-stage assay. IC50 values for each compound are shown within an inset table above the dose-response curves (n = 4). b, The in vivo efficacy of KDU691 against the blood stages of P. berghei in a malaria mouse model. c, A survival curve for mice (n = 8 per group) tested in a causal prophylaxis assay with varying doses of KDU691 (5, 7.5 and 10 mg kg−1). A single oral dose of 7.5 mg kg−1 or greater, administered just before intravenous infection with 50,000 sporozoites, protected mice from malaria. Blood parasitaemia was monitored for all mice for up to 30 days, at which time the experiment was discontinued and surviving mice were deemed cured. Atovaquone was included as a control and is known to provide 100% causal prophylactic protection at 2.5 mg kg−1. d, The effect of KDU691 on P. falciparum stage III−IV gametocytes was determined by measuring parasite lactate dehydrogenase activity. Data are expressed as a percentage effect relative to the positive (1 μM dihydroartemisinin; DHA) and negative (vehicle) controls (mean ± s.d.; n = 4). DHA and KDU691 were found to have IC50 values of 15.8 nM and 220 nM, respectively. e, f, The results of KDU691 tested in the standard membrane feeding assay are shown after pre-exposure of P. falciparum mature gametocytes to compound for 24 h. Oocyst counts from the dissected mosquito midguts (mean ± s.d.; n = 20) are expressed as a percentage effect relative to the negative (vehicle) control. An IC50 value of 316 nM on oocyst densities (e) and 370 nM on oocyst prevalence (f), that is, the percentage of mosquitos with one or more oocysts, was determined for KDU691.

Extended Data Figure 2 Imidazopyrazine and quinoxaline compounds arrest parasites in late schizogony before completion of daughter cell formation.

a, The onset of action for KAI407 was investigated using a highly synchronized population of blood-stage parasites. Complete culture medium containing 125 nM KAI407 was replaced with drug-free medium at the time indicated in 2-h intervals. The resultant parasitaemia in the next life cycle (t = 72 h) was normalized to untreated parasites (mean ± s.d.; n = 4). The time course represents trophozoite-stage parasites (t = 34 h) through the maturation of schizonts (t = 48 h). b, c, The plasma membrane marker PfATP4–GFP was used to visualize plasma membrane ingression around developing daughter merozoites, with nuclei stained by Hoechst 33342. Parasites treated with DMSO (control) formed clearly defined daughter cells uniformly surrounded by the plasma membrane. Conversely, parasites treated with 500 nM KAI407 for 4 h (KAI407-treated) had a disorganized membrane structure. Representative images from a single experimental replicate are shown (n = 2). Scale bar, 5 μm. d, Microscopy of Giemsa-stained parasites treated with ∼5 × IC50 drug (125 nM KAI407, 15 nM KAI715, 150 nM KDU691 or 400 nM BQR695) or DMSO vehicle. Representative images from a single experimental replicate are shown (n = 3). e, Measurement of merozoite viability via the merozoite release assay. The ability of merozoites to reinvade fresh RBCs after mechanical rupture of drug-arrested schizonts treated with 125 nM KAI407, 15 nM KAI715, 150 nM KDU691 or 400 nM BQR695 was compared to parasite reinvasion of untreated (DMSO) parasites. E-64 (grey), a known inhibitor of merozoite egress, was used as a control at 1 μM (mean ± s.d.; n = 4).

Extended Data Figure 3 Domain organization of PfPI(4)K, amino acid alignment, and expression of GFP–PfPI(4)K.

a, Schematic of the domain organization of PfPI(4)K. ARM, Armadillo-type fold; BSM, beta-signature motif; CAT, PI(3)K/PI(4)K catalytic domain; LKU, lipid kinase unique domain. Amino acid boundaries are given below the schematic, and the locations of the resistance SNVs starred. The beta-signature motif is exclusive to PI(4)Ks. b, Amino acid alignment of PI(4)KIIIβ from P. falciparum (Pf), P. vivax (Pv), P. berghei (Pb), P. yoelii (Py) and the human (h) orthologue. Amino acids shaded in black are identical between species, and those shaded grey are highly conserved. Resistance SNVs are highlighted in red. c, Fluorescence microscopy shows apical enrichment of GFP–PI(4)K in late schizonts. Representative images from a single experimental replicate are shown (n = 3). Scale bar, 5 μm. d, Quantification of phosphatidylinositol phosphate (PIP) species in synchronized asexual blood-stage parasites treated with 500 nM KAI407 (+) or DMSO (−). PIP species were normalized to phosphatidylinositol, and representative data (mean; n = 2) are shown.

Extended Data Figure 4 Resistance to imidazopyrazine and quinoxaline compounds is mediated by genes encoding PfPI(4)K or PfRab11A.

DNA microarray analysis of the parasite genomic DNA extracted from clonal lines evolved-to-resistance against KAI407, KAI715 or BQR695. a, c, e, CNV analysis is shown for all 14 chromosomes (chr) and organelle-specific plasmids in the mitochondria (mito) and apicoplast (api) for KAI407-R lines (a) KAI715-R lines (c) and BQR695-R lines (e). The inset box is a zoomed view of chromosome 5 centred on pfpi4k (indicated by black arrows). b, d, f, Key SNVs detected in the remaining KAI407-R (b), KAI715-R (d) and BQR695-R (f) parasite lines lacking significant CNV events. Results of direct DNA sequencing are given next to each detected mutation, and the gene model is provided beneath the SNV analysis output for reference.

Extended Data Figure 5 IC50 values for ZFN-edited lines and genome editing strategy to introduce a stop codon within the PfPI(4)K catalytic domain.

a, IC50 values for PI(4)K mutants generated via ZFN editing. IC50 values are represented as mean ± s.d. and were calculated from three independent experiments performed in quadruplicate. KAF179 was previously referred to as GNF179 (ref. 29); KAF246 was previously referred to as NITD246 (ref. 12). b, The ZFN plasmid contains an expression cassette for the ZFNs, which target a 34-bp site on pfpi4k (arrow), and a 1.7-kb homologous donor sequence with a single SNV introducing a stop codon at amino acid residue 1356 in the protein coding sequence. Although the edited mutation was detected in the bulk culture, analysis of cloned lines continued to show a mixed T/A peak at position 4068 (see chromatogram), suggesting a potential gene duplication. c, Whole-genome sequencing confirmed the direct sequencing results, yielding approximately equivalent numbers of reads encoding the wild-type and stop codons. These result from a partial amplification (from 413,512 to 416,372 bp; green line in the normalized coverage) of the 3′ region of pfpi4k, with the edited stop codon within the downstream truncated copy.

Extended Data Figure 6 Amino acid alignment and localization of PfRab11A.

a, Comparison of amino acid identity (shaded in black) and similarity (shaded in grey) in ClustalW2 between the human (HsRAB11A), P. falciparum (PfRab11A), and S. cerevisiae (ScYpt31) Rab11A homologues. The phosphate-binding P-loop and the switch regions that respond conformationally to the nucleotide-bound state of the protein are labelled above the amino acid alignment, and consensus residues (100% identity) are indicated with an asterisk. Asp 139 (D139), which confers imidazopyrazine and quinoxaline resistance when mutated to Tyr (Y), is labelled with an arrow. A negatively charged residue, either Asp (D) or Glu (E), occupies this position in all three species. b, Mapping of the resistance-conferring mutation (Asp139Tyr; yellow) within the X-ray crystallographic structure of PfRab11A (slate blue cartoon representation; PDB accession 3BFK) in complex with GDP (green stick model). The structure was visualized in Pymol. c, d, Visualization of GFP–PfRab11A-WT (c) and GFP–PfRab11A(D139Y) (d) by fluorescence microscopy in early (top) or segmented (bottom) schizonts (n = 3). Hoechst 33342 was used to stain the nuclei of the daughter merozoites (blue) that develop during schizogony. A DIC image of the parasitized RBC is shown. For each, representative images from a single experimental replicate are shown. Scale bar, 5 μm.

Extended Data Table 1 Activity of imidazopyrazines against drug-resistant lines of P. falciparum

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Extended Data Table 2 Pharmacokinetic properties of KDU691

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Extended Data Table 3 In vitro activities of KAI407, KDU691 and BQR695 against recombinant human lipid and protein kinases

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Extended Data Table 4 Whole-genome sequencing results of ZFN-edited lines

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Supplementary information

Supplementary Information

This file contains Supplementary Methods describing the synthesis of imidazopyrazine and quinoxaline compounds, and Supplementary Table 1 listing primers. (PDF 567 kb)

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McNamara, C., Lee, M., Lim, C. et al. Targeting Plasmodium PI(4)K to eliminate malaria.Nature 504, 248–253 (2013). https://doi.org/10.1038/nature12782

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• Received: 23 July 2013
• Accepted: 15 October 2013
• Published: 27 November 2013
• Issue Date: 12 December 2013
• DOI: https://doi.org/10.1038/nature12782