Research ArticleMalaria

Antimalarial efficacy of MMV390048, an inhibitor of Plasmodium phosphatidylinositol 4-kinase

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Science Translational Medicine  26 Apr 2017:
Vol. 9, Issue 387, eaad9735
DOI: 10.1126/scitranslmed.aad9735
  • Fig. 1. In vitro potency of the compound MMV390048.

    (A) Structure of 2-aminopyridine MMV390048. (B) In vitro PRR depicting the number of viable parasites (P. falciparum 3D7 strain) over time after treatment with 10× IC50 of MMV390048 compared to other antimalarial drugs. (C) IC50 speed assay using unsynchronized culture of the P. falciparum NF54 strain (mean ± SD of n ≥ 3 independent assays), indicating activity at different incubation times. (D) Parasitemia as a function of time following once daily dosing for 4 days in the P. falciparum humanized SCID mouse model (n = 1 per dose level). Dosing was started on day 3 after infection. (All data points are included in tables S2 to S4.)

  • Fig. 2. In vitro and in vivo transmission-blocking potential of MMV390048.

    (A) P. falciparum sexual stages and the various readouts used in drug screening assays, including assays against early- and late-stage gametocytes, the male gamete formation assay, and the SMFA. (B) Gametocyte viability assay was performed by measuring LDH activity of nonrecombinant stage IV and V gametocytes of P. falciparum. MMV390048 is in black (IC50, 285 nM) and dihydroartemisinin is in blue (IC50, 12.0 nM) (n = 2). (C) Gametocyte viability assay was performed by measuring luminescence of recombinant parasites expressing luciferase. Stage I to III gametocytes are in black (IC50, 214 nM) and stage IV and V gametocytes are in red (IC50, 140 nM) (n = 3). (D) Clearance rates for stage I to III (black) and stage IV and V (red) gametocytes treated with MMV390048 at its IC50 concentration over a 3-day period indicated by the slopes of −6.09 and −15.49, respectively (n = 3). (E) Inhibition of exflagellation and gamete formation in the male gamete formation assay (n = 4). (F) Dose-response curve for MMV390048 in the indirect SMFA. The assay measures the number of oocysts that developed in the midgut of mosquitoes after feeding on P. falciparum stage V gametocyte–infected blood that was exposed to different concentrations of MMV390048 for 24 hours before feeding. In the direct SMFA (inset), mosquitoes fed on infected blood directly after it was treated with different concentrations of MMV390048 (n = 2). (G) Number of oocysts in the midgut of mosquitoes after feeding on P. berghei–infected mice that were treated with either MMV390048, vehicle, or control drugs during the mouse-to-mouse transmission study. (All data points are included in tables S10 to S12, S15, and S16).

  • Fig. 3. MMV390048 efficacy in P. cynomolgi–infected rhesus macaques and the phenotype of treated P. berghei liver-stage parasites.

    (A) Daily parasitemias for individual P. cynomolgi–infected monkeys that were treated with MMV390048 on day −1 before infection (prophylactic study). The black arrow indicates when chloroquine treatment (10 mg base/kg orally for 7 days) was started to clear primary infection. Control group monkeys relapsed on days 26 and 29, whereas monkeys treated with MMV390048 remained clear of parasitemia for the 100 days of observation. (B) Mean plasma concentrations of MMV390048 over time for the treated monkeys in the prophylactic study after a single oral treatment (20 mg/kg) with MMV390048. The concentration of MMV390048 remained above its IC99 for 6.5 days (orange arrow). The in vivo IC99 was estimated to be 353 ± 32 nM (138 ± 13 ng/ml) based on the in vitro IC99 determined in the P. cynomolgi liver-stage assay and correcting for serum albumin (AlbuMAX) binding (54%) and plasma protein binding (86%). (C) Daily parasitemias for individual P. cynomolgi–infected monkeys in the radical cure study. Once infection was established, animals were treated with either MMV390048 or vehicle (20 mg/kg once daily for 5 days; brown arrows) with simultaneous treatment with chloroquine (10 mg base/kg orally for 7 days) to clear primary parasitemia (black arrow indicating start of chloroquine treatment). Control group monkeys relapsed on days 24 and 29, whereas MMV390048-treated monkeys relapsed on days 33 to 35. (D) Mean plasma concentrations of MMV390048 over time for the treatment group monkeys in the radical cure study. Brown arrows indicate treatment with MMV390048 (20 mg/kg oral dose daily for 5 days). The plasma concentration of MMV390048 remained above the P. cynomolgi liver-stage IC99 for 15.8 days (orange arrow) and above the blood-stage MIC (11.5 ng/ml free concentration) for 20 days (black line). The MIC was calculated from the in vivo P. falciparum SCID mouse model and corrected for protein binding (86.1%) and blood-to-plasma partitioning (0.86). The blue arrow represents the time at which monkeys 3, 4, and 5 relapsed. (E) Image of an early liver-stage P. berghei parasite treated with 460 nM MMV390048 from 2 to 8 hours after inoculation. (F) The parasite displayed mislocalization of the parasitophorous vacuole membrane protein UIS4 from the surface to the parasite interior (red; visualized with goat polyclonal antibody and Alexa Fluor–conjugated secondary antibody) when treated with MMV390048 compared to vehicle. For visualization, cytoplasmic P. berghei heat shock protein 70 is displayed in green (visualized with 2E6 monoclonal antibody and Alexa Fluor–conjugated secondary antibody). Images are single confocal sections. LLOQ, lower limit of quantification. (All data points are included in tables S17 to S20.)

  • Fig. 4. Chemoproteomic identification of Plasmodium PI4K as the target of MMV390048.

    (A) Binding of MMV390048 to P. falciparum PI4K in parasite extracts. An analog of MMV390048, MMV666845, was covalently immobilized on Sepharose beads and used for affinity capture of potential target proteins from a P. falciparum blood-stage extract. The addition of excess MMV390048 (10 μM) to the P. falciparum extract competitively inhibited the binding of P. falciparum PI4K to the beads. No other protein was reproducibly inhibited. The addition of MMV034137, a closely related structural analog devoid of antimalarial activity, had no effect on the capture of P. falciparum PI4K. (B) MMV390048 binds to the ATP binding site of Plasmodium PI4K but not to human PI4Kα or PI4Kβ. A set of seven promiscuous ATP-competitive kinase inhibitors was covalently immobilized to beads (Kinobeads) and used for affinity capture of potential kinase targets from a P. falciparum blood-stage extract and from a human erythroleukemia K562 cell line. The plot illustrates pIC50 values obtained in two independent replicates of the experiment (exp. 1 and 2). The addition of MMV390048 to the extract competitively inhibited the binding of P. falciparum PI4K to the beads. PIP4K2C was the only human kinase for which binding was also competitively inhibited by MMV390048 with an IC50 value in the same range as the P. falciparum PI4K. (C) The addition of MMV390048 to the P. falciparum extract over a range of concentrations yielded an apparent dissociation constant (Kdapp) of 0.3 μM using MMV666845 for affinity capture and a calculated Kdapp of 0.1 μM using Kinobeads for affinity capture. (D) Structure-activity relationship of MMV390048 analogs revealed excellent correlation of antimalarial activity and binding to P. falciparum PI4K in parasite extracts. Fourteen compounds from the aminopyridine (MMV390048) series with increasing activity against P. falciparum were subjected to profiling using immobilized MMV666845 beads [see also (A)]. With increased potency against P. falciparum, the compounds showed increased competition for binding to P. falciparum PI4K and thus reduced its binding to the bead matrix. r, Pearson’s correlation coefficient; P, P value (calculated probability). (All data points are included in tables S26 and S27.)

  • Table 1. Transmission-blocking effects of MMV390048 (2 mg/kg) measured in the mouse-to-mouse transmission model.
    Effect on transmission
    to the mosquito
    vector
    Effect on oocyst intensity
    (%) (95% CI)
    69.3% (65.7–73.1)
    Effect on oocyst
    prevalence
    (%) (95% CI)
    30.3% (28–32.6)
    Effect on sporozoite
    intensity (%)
    (95% CI)
    37.2% (23.6–48.4)
    Effect on sporozoite
    prevalence
    (%) (95% CI)
    46.5% (19.9–65.6)
    Effect on subsequent
    transmission to
    the vertebrate host
    Inhibition of infection
    of naïve mice (%)
    (P value, Fisher’s exact)
    10.1% (0.075)
    Prepatency (±SEM)
    Compared to
    6.1 days (±0.28)
    in control mice
    6.8 days (±0.28)
    Effect size
    (95% CI)
    28.5% (22.8–33.7)
  • Table 2. MMV390048 radical cure (≥24-hour addition) versus prophylactic (3-hour addition) effects in the P. cynomolgi liver-stage assay.

    EEF, exoerythrocytic forms.

    Approximate time of
    MMV390048 addition
    Small EEFs
    (hypnozoites)
    IC50 (μM)*
    Large EEFs
    (schizonts)
    IC50 (μM)*
    3 hours (day 0)0.0610.064
    24 hours (day 1)2.60.097
    72 hours (day 3)5.10.31
    96 hours (day 4)7.01.0
    120 hours (day 5)>101.5

    *Data from three independent biological replicates.

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/9/387/eaad9735/DC1

      Materials and Methods

      Fig. S1. Activity of MMV390048 against NF54 asexual blood-stage parasites.

      Fig. S2. Lack of growth of schizonts compared to rings after treatment with MMV390048.

      Fig. S3. Onset and recrudescence experiments in the P. berghei mouse model using a single dose of 100 mg/kg compound.

      Fig. S4. Whole-blood concentrations of MMV390048 after the first dose of treatment during the PfSCID mouse efficacy study.

      Fig. S5. Observed and predicted blood PK profiles from PfSCID mouse efficacy study.

      Fig. S6. Observed (symbols) and predicted (lines) parasite load in mouse based on the direct effect model and PfSCID mouse efficacy data.

      Fig. S7. Prophylactic efficacy of MMV390048 in the P. cynomolgi liver-stage in vitro assay.

      Fig. S8. MMV390048-resistant clones show a four- to fivefold shift in IC50 relative to the parental Dd2 strain.

      Fig. S9. CNV analysis using the program BIC-Seq (23).

      Fig. S10. IC50 of MMV390048 against ZFN-modified parasites with point mutations in PI4K or the parental Dd2 strain.

      Fig. S11. Phylogenetic tree of the human (blue) and Plasmodium (red) lipid kinase family.

      Fig. S12. Correlation between whole-cell NF54 activity and inhibition of PvPI4K across a selection of MMV390048 analogs.

      Fig. S13. Pharmacokinetic profiles after intravenous and oral administration of MMV390048 to mice, rats, dogs, and monkeys.

      Fig. S14. Allometric scaling of clearance from different species as a function of body weight.

      Fig. S15. Assessment of hemolytic toxicity.

      Table S1. Activity against a panel of resistant strains to determine potential for cross-resistance.

      Table S2. In vitro parasite reduction data in support of Fig. 1B.

      Table S3. Speed assay IC50 results in support of Fig. 1C.

      Table S4. Efficacy data in the PfSCID mouse model in support of Fig. 1D.

      Table S5. Summary of exposure data in the PfSCID mouse model after oral administration of MMV390048 once a day for four consecutive days.

      Table S6. Estimated PK parameters from PfSCID mouse exposure with Ka fixed at 0.5/hour.

      Table S7. Estimated PKPD parameters from PfSCID mouse parasitemia and exposure study.

      Table S8. Estimated PKPD parameters from PfSCID mouse parasitemia and exposure study indicating highest net kill rate (red).

      Table S9. Estimated EC50, EC90, and MIC based on modeling of highest net kill rate.

      Table S10. Late-stage gametocyte viability measured by pLDH activity in support of Fig. 2B.

      Table S11. Early- and late-stage gametocyte viability measured by luminescence in support of Fig. 2C.

      Table S12. Early- and late-stage gametocyte clearance rates in support of Fig. 2D.

      Table S13. Gametocytocidal activity.

      Table S14. Inhibition of exflagellation in support of Fig. 2E.

      Table S15. SMFA indirect mode results in support of Fig. 2F.

      Table S16. SMFA direct mode results in support of Fig. 2F (inset).

      Table S17. Parasitemias during the prophylactic P. cynomolgi–infected monkey study in support of Fig. 3A.

      Table S18. Plasma concentrations of MMV390048 during the prophylactic P. cynomolgi–infected monkey study in support of Fig. 3B.

      Table S19. Parasitemias during the radical cure P. cynomolgi–infected monkey study in support of Fig. 3C.

      Table S20. Plasma concentrations of MMV390048 during the radical cure P. cynomolgi–infected monkey study in support of Fig. 3D.

      Table S21. Number of triplicates at different inocula that became positive after MMV390048 treatment.

      Table S22. Whole-genome sequence analysis of three cloned Dd2 parasite lines selected for resistance to compound MMV390048.

      Table S23. Correlation between the degree of binding to PfPI4K, activity against PcPI4K, and antiparasitic activity of MMV390048 analogs.

      Table S24. Plasma PK parameters after a single intravenous dose of MMV390048 in different species.

      Table S25. Plasma PK parameters after a single oral dose of MMV390048 in different species.

      Table S26. Chemoproteomics data (provided in Excel).

      Table S27. Proteomics data (provided in Excel).

      References (3853)

    • Supplementary Material for:

      Antimalarial efficacy of MMV390048, an inhibitor of Plasmodium phosphatidylinositol 4-kinase

      Tanya Paquet, Claire Le Manach, Diego González Cabrera, Yassir Younis, Philipp P. Henrich, Tara S. Abraham, Marcus C. S. Lee, Rajshekhar Basak, Sonja Ghidelli-Disse, María José Lafuente-Monasterio, Marcus Bantscheff, Andrea Ruecker, Andrew M. Blagborough, Sara E. Zakutansky, Anne-Marie Zeeman, Karen L. White, David M. Shackleford, Janne Mannila, Julia Morizzi, Christian Scheurer, Iñigo Angulo-Barturen, María Santos Martínez, Santiago Ferrer, Laura María Sanz, Francisco Javier Gamo, Janette Reader, Mariette Botha, Koen J. Dechering, Robert W. Sauerwein, Anchalee Tungtaeng, Pattaraporn Vanachayangkul, Chek Shik Lim, Jeremy Burrows, Michael J. Witty, Kennan C. Marsh, Christophe Bodenreider, Rosemary Rochford, Suresh M. Solapure, María Belén Jiménez-Díaz, Sergio Wittlin, Susan A. Charman, Cristina Donini, Brice Campo, Lyn-Marie Birkholtz, Kirsten K. Hanson, Gerard Drewes, Clemens H. M. Kocken, Michael J. Delves, Didier Leroy, David A. Fidock, David Waterson, Leslie J. Street, Kelly Chibale*

      *Corresponding author. Email: kelly.chibale{at}uct.ac.za

      Published 26 April 2017, Sci. Transl. Med. 9, eaad9735 (2017)
      DOI: 10.1126/scitranslmed.aad9735

      This PDF file includes:

      • Materials and Methods
      • Fig. S1. Activity of MMV390048 against NF54 asexual blood-stage parasites.
      • Fig. S2. Lack of growth of schizonts compared to rings after treatment with MMV390048.
      • Fig. S3. Onset and recrudescence experiments in the P. berghei mouse model using a single dose of 100 mg/kg compound.
      • Fig. S4. Whole-blood concentrations of MMV390048 after the first dose of treatment during the PfSCID mouse efficacy study.
      • Fig. S5. Observed and predicted blood PK profiles from PfSCID mouse efficacy study.
      • Fig. S6. Observed (symbols) and predicted (lines) parasite load in mouse based on the direct effect model and PfSCID mouse efficacy data.
      • Fig. S7. Prophylactic efficacy of MMV390048 in the P. cynomolgi liver-stage in vitro assay.
      • Fig. S8. MMV390048-resistant clones show a four- to fivefold shift in IC50 relative to the parental Dd2 strain.
      • Fig. S9. CNV analysis using the program BIC-Seq (23).
      • Fig. S10. IC50 of MMV390048 against ZFN-modified parasites with point mutations in PI4K or the parental Dd2 strain.
      • Fig. S11. Phylogenetic tree of the human (blue) and Plasmodium (red) lipid kinase family.
      • Fig. S12. Correlation between whole-cell NF54 activity and inhibition of PvPI4K across a selection of MMV390048 analogs.
      • Fig. S13. Pharmacokinetic profiles after intravenous and oral administration of MMV390048 to mice, rats, dogs, and monkeys.
      • Fig. S14. Allometric scaling of clearance from different species as a function of body weight.
      • Fig. S15. Assessment of hemolytic toxicity.
      • Table S1. Activity against a panel of resistant strains to determine potential for cross-resistance.
      • Table S2. In vitro parasite reduction data in support of Fig. 1B.
      • Table S3. Speed assay IC50 results in support of Fig. 1C.
      • Table S4. Efficacy data in the PfSCID mouse model in support of Fig. 1D.
      • Table S5. Summary of exposure data in the PfSCID mouse model after oral administration of MMV390048 once a day for four consecutive days.
      • Table S6. Estimated PK parameters from PfSCID mouse exposure with Ka fixed at 0.5/hour.
      • Table S7. Estimated PKPD parameters from PfSCID mouse parasitemia and exposure study.
      • Table S8. Estimated PKPD parameters from PfSCID mouse parasitemia and exposure study indicating highest net kill rate (red).
      • Table S9. Estimated EC50, EC90, and MIC based on modeling of highest net kill rate.
      • Table S10. Late-stage gametocyte viability measured by pLDH activity in support of Fig. 2B.
      • Table S11. Early- and late-stage gametocyte viability measured by luminescence in support of Fig. 2C.
      • Table S12. Early- and late-stage gametocyte clearance rates in support of Fig. 2D.
      • Table S13. Gametocytocidal activity.
      • Table S14. Inhibition of exflagellation in support of Fig. 2E.
      • Table S15. SMFA indirect mode results in support of Fig. 2F.
      • Table S16. SMFA direct mode results in support of Fig. 2F (inset).
      • Table S17. Parasitemias during the prophylactic P. cynomolgi–infected monkey study in support of Fig. 3A.
      • Table S18. Plasma concentrations of MMV390048 during the prophylactic P. cynomolgi–infected monkey study in support of Fig. 3B.
      • Table S19. Parasitemias during the radical cure P. cynomolgi–infected monkey study in support of Fig. 3C.
      • Table S20. Plasma concentrations of MMV390048 during the radical cure P. cynomolgi–infected monkey study in support of Fig. 3D.
      • Table S21. Number of triplicates at different inocula that became positive after MMV390048 treatment.
      • Table S22. Whole-genome sequence analysis of three cloned Dd2 parasite lines selected for resistance to compound MMV390048.
      • Table S23. Correlation between the degree of binding to PfPI4K, activity against PcPI4K, and antiparasitic activity of MMV390048 analogs.
      • Table S24. Plasma PK parameters after a single intravenous dose of MMV390048 in different species.
      • Table S25. Plasma PK parameters after a single oral dose of MMV390048 in different species.
      • Legends for tables S26 and S27
      • References (3853)

      [Download PDF]

      Other Supplementary Material for this manuscript includes the following:

      • Table S26. Chemoproteomics data (provided in Excel).
      • Table S27. Proteomics data (provided in Excel).

      [Download Tables S26 and S27]

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