A molecular mechanism of artemisinin resistance in Plasmodium falciparum malaria (original) (raw)

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Acknowledgements

This work was supported by NIH grants HL069630, AI039071, HL078826 (K.H.); AI081077 (R.V.S.); DK26263 (N.M.) and a grant from Notre Dame International. All parasite gene/protein sequences were obtained from PlasmoDB (http://www.plasmodb.org). This study is dedicated to the memory of Dr. Martin John Rogers, NIAID for his leadership in antimalarial drug research.

Author information

Author notes

  1. Alassane Mbengue and Souvik Bhattacharjee: These authors contributed equally to this work.
  2. Guillermina Estiu: Deceased.

Authors and Affiliations

  1. Boler-Parseghian Center for Rare and Neglected Diseases, University of Notre Dame, Notre Dame, 46556, Indiana, USA
    Alassane Mbengue, Souvik Bhattacharjee, Trupti Pandharkar, Haining Liu, Guillermina Estiu, Robert V. Stahelin, Shahir S. Rizk, Dieudonne L. Njimoh, Yana Ryan, Olaf Wiest & Kasturi Haldar
  2. Department of Biological Sciences, University of Notre Dame, Notre Dame, 46556, Indiana, USA
    Alassane Mbengue, Souvik Bhattacharjee, Trupti Pandharkar, Shahir S. Rizk, Dieudonne L. Njimoh, Yana Ryan, Michael Pfrender & Kasturi Haldar
  3. Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, 46556, Indiana, USA
    Haining Liu, Guillermina Estiu, Robert V. Stahelin & Olaf Wiest
  4. Department of Biochemistry & Molecular Biology, Indiana University School of Medicine-South Bend, 143 Raclin-Carmichael Hall, 1234 Notre Dame Avenue, South Bend, 46617, Indiana, USA
    Robert V. Stahelin
  5. Departmen of. Biochemistry and Molecular Biology, Faculty of Science University of Buea, P.O. Box 63 Buea, Southwest region, Cameroon
    Dieudonne L. Njimoh
  6. Faculty of Tropical Medicine, Mahidol University, 420/6 Ratchawithi Road, Ratchathewi, 10400, Bangkok, Thailand
    Kesinee Chotivanich & Arjen M. Dondorp
  7. National Center for Parasitology, Entomology and Malaria Control, 12302 Phnom Penh, Monivong Blvd, Phnom Penh, 12302, Cambodia
    Chea Nguon
  8. CNRS 5290/IRD 224/University Montpellier 1&2 (“MiVEGEC”), Montpellier, France
    Mehdi Ghorbal & Jose-Juan Lopez-Rubio
  9. Department of Computer Science and Engineering, University of Notre Dame, Notre Dame, 46556, Indiana, USA
    Scott Emrich
  10. New York Blood Center, New York, 10032, New York, USA
    Narla Mohandas
  11. Nuffield Department of Clinical Medicine, Centre for Tropical Medicine, University of Oxford, Oxford, OX3 7BN, UK
    Arjen M. Dondorp
  12. Laboratory of Computational Chemistry and Drug Design, Laboratory of Chemical Genomics, Peking University Shenzhen Graduate School, Shenzhen, 518055, China
    Olaf Wiest

Authors

  1. Alassane Mbengue
  2. Souvik Bhattacharjee
  3. Trupti Pandharkar
  4. Haining Liu
  5. Guillermina Estiu
  6. Robert V. Stahelin
  7. Shahir S. Rizk
  8. Dieudonne L. Njimoh
  9. Yana Ryan
  10. Kesinee Chotivanich
  11. Chea Nguon
  12. Mehdi Ghorbal
  13. Jose-Juan Lopez-Rubio
  14. Michael Pfrender
  15. Scott Emrich
  16. Narla Mohandas
  17. Arjen M. Dondorp
  18. Olaf Wiest
  19. Kasturi Haldar

Contributions

A.M., S.B., T.P., K.H., H.L., G.E., S.S.R., D.L.N., Y.R., O.W., M.P. and S.E. designed, performed and interpreted the experimental work. K.H. along with A.M., O.W. and S.B. wrote the manuscript. R.V.S. and N.M. provided intellectual insight into aspects of this study. K.C., C.N., M.G., J.L.R. and A.M.D. provided reagents and intellectual input into study design. All authors commented on the manuscript.

Corresponding author

Correspondence toKasturi Haldar.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Ring Parasite PI3P and PfPI3K: effect of inhibitors, and characterization of antibodies.

a, Rings at 6 h post-invasion, were either mock treated or exposed to 4 nM DHA for 4 h. An aliquot was washed in serum-free RPMI and cultured for 24 h. Parasitemia (ring and trophozoite stages) were determined by Giemsa staining and light microscopy. Mean (error bars s.d. <2) from three experimental replicates are shown. b, Effect of artemisinins, deoxyartemisinin, PI3K inhibitors wortmannin and LY294002 (and its inactive orthologue LY303511) on PI3P level, as observed by redistribution of the PI3P reporter in transgenic 3D7 parasites expressing SS-EEA1WT-mCherry (red). Blue, parasite nucleus, P, parasite, E, erythrocyte; scale bar, 5 μm. Fluorescence and phase-merged images are as indicated. c, Quantitative analysis of the data in b. Mean (error bars s.d. <21) from three experimental replicates are shown. d, Effect of anti-folates and chloroquine on PI3P production, as observed by redistribution of the PI3P reporter in transgenic 3D7 parasites expressing SS-EEA1WT-mCherry (red). Blue, parasite nucleus, P, parasite, E, erythrocyte; scale bar, 5 μm. Fluorescence and phase-merged images are as indicated. e, Quantitative analysis of the data in d. Mean (error bars s.d. <27) from three experimental replicates are shown. As indicated in Fig. 1, the PI3P reporter SS-EEA1WT-mCherry (red) detects PI3P in punctate, perinuclear ER domains in 6 h rings11 (top panel) or reduced PI3P when the reporter diffuses to the PV (periphery). f, Schematic showing structural features of PfPI3K. Custom antibodies were generated in rabbits and guinea pigs against the indicated peptides. Western blots indicate that both antibodies specifically recognize a ∼255 kDa protein in infected (IE) but not uninfected (U) erythrocytes and are representative of 10 experimental replicates; raw data in Supplementary Data 2. Indirect immunofluorescence assays using rabbit (top panel) and guinea pig (bottom panel) antibodies confirm the PfPI3K (green) is localized in the parasite. Exp-2 (red) marks the parasite boundary. Blue, parasite nucleus, P, parasite, E, erythrocyte; scale bar, 5 μm. Fluorescence and phase-merged images are shown. Data are representative of 10 experimental replicates.

Extended Data Figure 2 Structural analyses of PfPI3K.

Sequence alignment of the putative catalytic domain of PfPI3K and its closest human orthologues human VPS34 and Drosophila VPS34. Asterisk symbol indicates identity; dots indicate homology. D1889 and Y1915 residues are highlighted in yellow.

Extended Data Figure 3 Structural analyses of PfPI3K and human PI3K, with and without artemisinin derived compounds

a Snapshots from 20 ns MD simulations and chemical structure of artelinic acid (top right), artemisone (bottom left) and deoxyartemisinin (bottom right) bound to PfPI3K. Snapshot from MD simulation of DHA bound to PfPI3K is shown (at the top left) for reference. Due to the lack of the hydroxyl group in artelinic acid and artemisone, no interactions involving D1889 and Y1915 were observed. Instead, the carboxylic group of artelinic acid forms a salt bridge with R1368 and the sulfone moiety of artemisone interacts with R1368. Deoxyartemisinin lacks hydrogen bond donors, so D1889 is not involved in protein-ligand interaction. Instead, the carbonyl oxygen of deoxyartemisinin forms a hydrogen bond with Y1915. Boxed inset shows the change in the overall shape due to the replacement of the endoperoxide with an ether bridge in deoxyartemisinin (green) and compared to DHA (red), thus leading to loss of the shape complementarity and reorientation in the binding site. As shown in Fig. 1a, c, deoxyartemisinin failed to block PI3P production, in transgenic 3D7 parasites expressing SS-EEA1WT-mCherry (red) or purified PfPI3K. Thus, modelling studies explain a lack of effect of deoxyartemisinin. They would also suggest lack of effect of artelinic acid and artemisone compounds on PfPI3K. It should be noted the target of these compounds is unknown and it remains unclear whether they would share a ring stage target with DHA important for clinical resistance to artemisinins. b, Snapshots of MD simulation of DHA–human VPS34 complex. The initial coordinates of DHA–human VPS34 complex was obtained by the overlay of DHA…PfPI3K model (Extended Data Fig. 2b) with the human VPS34 crystal structure (PDB: 3IHY). The obtained complex was then subjected to 20 ns MD simulations. As can be seen, DHA does not interact with D644 and Y670, which correspond to D1889 and Y1915 in PfPI3K, respectively. Instead, the hydroxyl group of DHA now interacts with D761 (left). Snapshot at the right shows an overlay of DHA…human VPS34 complex with the PfPI3K model. In human VPS34 (grey), the loop that contains the D761 residue bends down to interact with DHA. In contrast, D2008 of PfPI3K (cyan), which is in the same position as D761 in human VPS34, has a different orientation which makes it unable to interact with DHA. c, Both D1889 and Y1915 in PfPI3K are conserved in human PI3K p110γ. 20 ns MD simulations of the DHA …p110γ complex (PDB code 4ANV), reveal that although the hydrogen bond between DHA and D841 still remains, the interaction between DHA and Y867 is lost. This suggests a weaker binding of DHA to p110γ compared to PfPI3K. The same argument can be made of p110δ based on sequence homology, but no crystal structure of human p110δ is available. d, Partial sequence alignment of PfPI3K, with human PI3K-C2α, PI3K-C2β, p110δ, p110γ and VPS34. The key residues of PfPI3K (D1889 and Y1915) that interact with DHA and similar positions in the human kinases are coloured red. While D1889 in PfPI3K is conserved in both PI3K-C2α and PI3K-C2β, Y1915 in PfPI3K is replaced by F1172 and F1117 in PI3K-C2α and PI3K-C2β, respectively. Therefore, the hydrogen bond of the hydroxyl group in Y1915 suggested to be crucial in the computational analysis of PfPI3K cannot be formed with phenylalanine (F). AS far as we are aware, no crystal structures of PI3K-C2α and PI3K-C2β have been reported. In p110δ, p110γ and VPS34, a proline N-terminal to Y1915 is conserved. It is likely the reason for the different position and low flexibility of this loop observed in the MD simulations of p110δ, and VPS34.

Extended Data Figure 4 Transgenic P. falciparum expressing PfKelch13C580Y mutation in two different strains of P. falciparum using distinct approaches and markers.

a, Strategy 1: CRISPR–Cas9 used to introduce a single point mutation in the P. falciparum NF54 genome as described elsewhere20. Both parent and mutant strains were sequenced to ensure that the only change detected was PfKelch13C580Y. Western blots using anti-PfKelch13 antibodies confirm that mutation does not change the level of PfKelch13 protein. b, Strategy 2 expresses a dominant negative PfKelch13C580Y and wild-type PfKelch13WT tagged with HA, and driven by the constitutive cam promoter in P. falciparum 3D7. Western blots confirm that anti-HA antibodies detected tagged forms of PfKelch13 in transfected but not non-transfected 3D7. Comparable levels of wild-type and mutant transgenes are expressed in resulting two strains of parasites (as shown). In a and b, BiP, an ER marker serves as a parasite loading control in western blots. Molecular weight standards (in kDa) are as indicated. c, Western blot indicate that antibodies to PfKelch13 specifically recognize a ∼83 kDa protein in infected (IE) but not uninfected erythrocytes (U). Molecular weight standards (in kDa) are as indicated. In a–c, data are representative of three experimental replicates; raw data are in Supplementary Data 2.

Extended Data Figure 5 An in vitro ring-stage survival assay (RSA) level is associated with PI3P elevation in the presence and absence of Kelch mutations.

a, Schematic of the RSA assay22. b, Summary of RSA, PI3P, PfKelch13 mutation and PfPI3K polymorphisms in clinical and laboratory strains used in this study. Means are shown for three experimental replicates. RSA s.d. <0.5; PI3P s.d. <8 (as shown by error bars in Fig. 3c). c, d, Construction of 3D7 transgenic parasites expressing myc- tagged forms of human VPS34, the mammalian orthologue of PfPI3K. The resulting PfVPS34-myc1/2 express the VPS34 transgene in ring stage parasites, as detected in indirect immunofluorescence assays (PfVPS34-MUTANT-myc data not shown in IFA). Exp-2 (red) marks the parasite boundary. Blue, parasite nucleus, P, parasite, E, erythrocyte; scale bar, 5 μm. Fluorescent and merged images are shown. Whole genome Illumina sequencing indicated VPS34-myc1 was inserted into PF3D7_1363900 and VPS34-myc2 is inserted in PF3D7_0718800. Both PF3D7_1363900 and PF3D7_0718800 encode for unknown function and neither has been reported in GWAS on artemisinin resistance. e, Construction of the strain 3D7-SS-EEA1R1374A-mCherry (used as transfection control) and western blot to confirm expression of SS- EEA1R1374A-mCherry (arrow). Molecular weight standards (in kDa) are as indicated. f, Construction of PfVPS34-myc1: PfKelch13WT-HA and PfVPS34-myc1: PfKelch13C580Y-HA parasites and western blots confirming expression of the transgenes. Raw data for western blots in e-f are shown in Supplementary Data 2. Molecular weight standards (in kDa) are as indicated. In d–f, data are representative of three experimental replicates.

Extended Data Figure 6 Evidence that the PI3K–AKT pathway is responsive to DHA.

a, Western blot shows specificity of anti-PfAKT antibody that recognizes a band of expected size in infected (IE) but not uninfected (U) erythrocytes (1 × 107 total cells loaded in each lane). Raw data are in Supplementary Data 2. Data are representative of three experimental replicates. b, Strategy and construct for generating transgenic 3D7 parasites expressing PfAKT–GFP.

Extended Data Table 1 Percentage inhibition of mammalian kinases by 10 μM DHA

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Extended Data Table 2 Primers used for cloning

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Mbengue, A., Bhattacharjee, S., Pandharkar, T. et al. A molecular mechanism of artemisinin resistance in Plasmodium falciparum malaria.Nature 520, 683–687 (2015). https://doi.org/10.1038/nature14412

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