Research ArticleMalaria

The antimalarial MMV688533 provides potential for single-dose cures with a high barrier to Plasmodium falciparum parasite resistance

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Science Translational Medicine  21 Jul 2021:
Vol. 13, Issue 603, eabg6013
DOI: 10.1126/scitranslmed.abg6013
  • Fig. 1 The preclinical antimalarial candidate MMV688533 has a fast rate of antiplasmodial activity that offers potent single-dose activity against P. falciparum infection in a humanized mouse model.

    (A) Structural representation showing the optimization of the acylguanidine series from the initial hit MMV668603 and the lead MMV669851 to the candidate MMV688533. (B) Means ± SD values of viable P. falciparum parasites were determined daily for 5 days after in vitro incubation with MMV688533 at 10× the IC50. Dihydroartemisinin, chloroquine, pyrimethamine, and atovaquone were included as reference antimalarial drugs. (C) Means ± SD values of P. falciparum viability determined daily for 5 days after MMV688533 treatment at doses corresponding to 1×, 3×, 10×, or 30× the IC50. (D) Compound efficacy was assessed by measuring the initial clearance and time of recrudescence of P. falciparum in the peripheral blood of humanized mice that had been administered single doses of MMV688533 ranging from 0.5 to 75 mg/kg (two mice per dose). DHA (50 mg/kg) and vehicle were included as controls. (E) Concentration of MMV688533 in serial blood samples obtained after administering different doses to P. falciparum–infected humanized mice assayed in (D). LOQ, limit of quantification.

  • Fig. 2 MMV688533 antiplasmodial activity is unrelated to existing antimalarials and selects for low-grade resistance mediated in part by mutations in PfACG1 and PfEHD.

    (A) In vitro asexual blood-stage susceptibility assay showing MMV688533 activity in early and late rings, early and late trophozoites, and schizonts. IC50 values are shown as means ± SEM (N > 3, n = 2). (B) MMV688533 mean ± SEM IC50 values of selected (sel.) (533-CL1, 533-CL2, and 533-CL4), edited (ed.) (PfACG1G98V, PfACG1W286R, and PfEHDD218Y) lines, and the sel. ed. line 533-CL1EHD-D218Y compared to the 3D7-A10 parental line. N > 6, n = 2; **P < 0.01 and ***P < 0.0005; ns, not significant. (C) Western blot data showing effective reduction in PfACG1 and PfEHD protein levels upon removal of aTc, as detected using antibodies specific to the 2×HA tag added to the C terminus of each protein. Parasite survival was measured by quantifying expression of the integrated RLuc cassette (fig. S1), in the presence (50 nM) or absence of aTc. Data represent the mean of three biological replicates and are normalized to a fully inhibitory concentration of chloroquine (200 nM). (D) Dose-response curves for MMV688533 against PfACG1 and PfEHD ckD asexual blood-stage (ABS) parasites expressing wild-type or substantially reduced levels of each protein upon culturing with 500 nM aTc or no aTc, respectively. (E) G98V and W286R mutations in PfACG1 and a combination of both G98V in PfACG1 and D218Y in PfEHD in sel. ed. 533-CL1EHD-D218Y did not confer cross-resistance to a panel of known antimalarial drugs compared to the 3D7-A10 parent. Means ± SEM; N > 3, n = 2. (F) Fluorescence microscopy images of fixed NF543×HA-EHDattB-ACG1-eGFP parasites stained with either anti-GFP (green) antibodies or anti-HA (magenta) antibodies. Nuclei were stained with DAPI (blue). Scale bars, 2 μm. (G) Fluorescence microscopy image of fixed and doubly stained NF543×HA-EHDattB-ACG1-eGFP parasites using anti-GFP (green) and anti-HA (magenta) antibodies. Nuclei were stained with DAPI (blue). Scale bar, 2 μm. (H to L) Fluorescence microscopy images and 3D reconstructions of fixed NF543×HA-EHDattB-ACG1-eGFP parasites costained with antibodies to anti-GFP (green) and (H) anti-PfCRT antibodies, (I) LipidTOX neutral lipid stain, (J) anti-PDI, (K) anti-ERD2, or (L) anti-Rab5A (red) antibodies. Nuclei were stained with DAPI (blue). Scale bars, 2 μm. (M to Q) Fluorescence microscopy images and 3D reconstructions of fixed NF543×HA-EHDattB-ACG1-eGFP parasites costained with antibodies to anti-HA (magenta) and (M) anti-PfCRT antibodies, (N) LipidTOX neutral lipid stain, (O) anti-PDI, (P) anti-ERD2, or (Q) anti-coronin (cyan) antibodies. Nuclei were stained with DAPI (blue). Scale bars, 2 μm.

  • Table 1 MMV688533 activity (in nanomolar) against Plasmodium parasite lines and field isolates.

    In vitro activity against Plasmodium culture-adapted lines or field isolates was calculated from dose-response curves and is shown as median or mean half-maximal growth inhibition (IC50) in nanomolar concentrations. For the laboratory lines, numbers of independent repeats are shown in parentheses. 3D7 and FC27 are chloroquine sensitive, whereas Dd2 and K1 are chloroquine resistant. The potency of the other antimalarials was compared to MMV688533 using a Wilcoxon rank sum test.

    Laboratory
    lines
    Laboratory
    lines
    Laboratory
    lines
    Laboratory
    lines
    Clinical field
    isolates
    (Uganda)
    Clinical field
    isolates (Papua,
    Indonesia)
    Clinical field
    isolates (Papua,
    Indonesia)
    P. falciparumP. falciparumP. falciparumP. falciparumP. falciparumP. falciparumP. vivax
    Antimalarial3D7
    (Median, N)
    Dd2
    (Median, N)
    FC27
    (Mean, N)
    K1
    (Mean, N)
    Median
    (N; range)
    Median
    (N; range)
    Median
    (N; range)
    MMV6885331.9 (4)3.0 (4)9.7 (2)19 (2)1.3
    (143; 0.02–6.3)
    18.9
    (15; 5.3–39.2)
    12.0
    (6; 5.4–19.9)
    Chloroquine11 (11)347 (10)10.9 (2)100.3 (2)17***
    (143; 2.1–346)
    64.8***
    (15; 38.3–283)
    36.4*
    (6; 11.6–114)
    Piperaquine4.4 (11)7.9 (10)25.8 (2)111.2 (2)5.1***
    (140; 0.3–26)
    60.8***
    (15; 17.6–130)
    46.6*
    (6; 15.0–135)
    Mefloquine4.8 (11)6.6 (10)37.2 (2)8 (2)8.3***
    (120; 0.5–24)
    10.0
    (15; 4.9–41.9)
    11.2
    (6; 8.1–20.7)
    DHA/artesunate1.9 (9)1.7 (9)0.6 (2)1.1 (2)1.5
    (142; 0.1–9.0)
    1.2***
    (15; 0.4–4.3)
    0.6*
    (6; 0.3–2.4)

    *P < 0.05.

    ***P < 0.001.

    †DHA was tested on 3D7, Dd2, and Ugandan parasites, whereas artesunate was tested on FC27, K1 and Papua/Indonesian parasites.

    • Table 2 Mutations identified in MMV688533-selected resistant P. falciparum clones and validated using CRISPR-Cas9 gene editing.

      Four parasite clones (sel. 533-CL1 from flask 1, sel. 533-CL2 and 533-CL3 from flask 2, and sel. 533-CL4 from flask 3) were generated from selections (sel.) and named after the last three digits of the selecting compound (MMV688533) followed by the clone number. These clones were then chosen for whole-genome sequencing. Fold IC50 increases compared to the parent 3D7-A10 are indicated below the clone names. P. falciparum ACG1W286R, ACG1G98V, and EHDD218Y strains were gene-edited (ed.) using CRISPR-Cas9 to introduce the designated mutation into 3D7-A10 parasites. The sel. ed. 533-CL1EHD-D218Y clone was generated by CRISPR-Cas9 editing the EHD D218Y mutation into the selected 533-CL1 clone. wt, wild-type; ATP, adenosine 5′-triphosphate.

      Gene productGene IDAmino acid substitution
      sel.
      533-CL2
      ed. 3D7
      ACG1W286R
      sel.
      533- CL3
      sel.
      533- CL4
      ed. 3D7
      ACG1G98V
      ed. 3D7
      EHDD218Y
      sel. 533-
      CL1
      sel. ed.
      533-CL1EHD-D218Y
      3.1 × IC501.7 × IC502.5 × IC504.6 × IC501.8 × IC501.2 × IC502.2 × IC506.2 × IC50
      Conserved Plasmodium
      protein (PfACG1)
      PF3D7_0910300W286RW286RT92*G98VG98VwtG98VG98V
      EHD-containing protein
      (PfEHD)
      PF3D7_0304200wtwtwtD218YwtD218YwtD218Y
      Conserved Plasmodium
      protein
      PF3D7_0510100wtwtwtwtwtwtN1042HN1042H
      RNA pseudouridylate
      synthase, putative
      PF3D7_0511500wtwtK2762Ewtwtwtwtwt
      ATP synthase (C/AC39)
      subunit, putative
      PF3D7_1464700L260Iwtwtwtwtwtwtwt

      *Stop mutation resulting from a deletion-induced frameshift.

      Supplementary Materials

      • The PDF file includes:

        • Materials and Methods
        • Figs. S1 to S8
        • Tables S1 to S3
        • References (5467)

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