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. 2017 Mar 13;2:17031.
doi: 10.1038/nmicrobiol.2017.31.

Mefloquine Targets the Plasmodium Falciparum 80S Ribosome to Inhibit Protein Synthesis

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Free PMC article

Mefloquine Targets the Plasmodium Falciparum 80S Ribosome to Inhibit Protein Synthesis

Wilson Wong et al. Nat Microbiol. .
Free PMC article

Abstract

Malaria control is heavily dependent on chemotherapeutic agents for disease prevention and drug treatment. Defining the mechanism of action for licensed drugs, for which no target is characterized, is critical to the development of their second-generation derivatives to improve drug potency towards inhibition of their molecular targets. Mefloquine is a widely used antimalarial without a known mode of action. Here, we demonstrate that mefloquine is a protein synthesis inhibitor. We solved a 3.2 Å cryo-electron microscopy structure of the Plasmodium falciparum 80S ribosome with the (+)-mefloquine enantiomer bound to the ribosome GTPase-associated centre. Mutagenesis of mefloquine-binding residues generates parasites with increased resistance, confirming the parasite-killing mechanism. Furthermore, structure-guided derivatives with an altered piperidine group, predicted to improve binding, show enhanced parasiticidal effect. These data reveal one possible mode of action for mefloquine and demonstrate the vast potential of cryo-electron microscopy to guide the development of mefloquine derivatives to inhibit parasite protein synthesis.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1. MFQ inhibits cytosolic translation in P. falciparum.
a, Translation inhibitory activity of antimalarial compounds, cycloheximide (CHX) at 1.3 µM, doxycycline (DOX) at 17 µM, chloroquine (CQ) at 110 nM, emetine (EME) at 105 nM and mefloquine (MFQ) at 90 nM. Asterisks indicate significant differences observed compared to CQ treatment. Mean ± SD are shown. Each assay was undertaken in triplicate of four independent occasions. b, Chemical structure of MFQ.
Fig. 2
Fig. 2. MFQ interacts with the GTPase-associated center of the P. falciparum large ribosomal subunit.
a-b, Cryo-EM density map of the primary MFQ binding pocket in the absence (a) and presence of MFQ (b). MFQ is represented as yellow sticks and binding residues are in purple. Oxygen is in red, nitrogen in blue, fluorine in cyan, and magnesium in green. c, Magnified EM density of (+)-MFQ depicted in various orientations. d, Ribosomal protein PfuL13 and rRNA ES13 form the MFQ binding pocket. Hydrophobic residues are colored in gray. Structure is derived from 112,347 particles from 829 micrographs (see Supplementary information). e, Atomic model of the Pf80S-MFQ complex is shown from the A-site entry side. Magnified inset shows the composition of the GTPase-associated center (GAC) with bound MFQ.
Fig. 3
Fig. 3. The primary binding site for (+)-MFQ.
a, Amino acid residues from the protein PfuL13 and bases from ES13 of the 28S rRNA involved in binding to (+)-MFQ. b, Residues that interact with (+)-MFQ with inter-atomic distances indicated. c, Mefloquine-mediated growth inhibition of control P. falciparum parasites carrying an integrated, wild type copy of uL13 gene and four transgenic parasite lines carrying single amino acid substitutions at the uL13 MFQ binding pocket. Data are shown as the mean ± SD of three biological replicates with each biological replicate representing three experimental replicates. d, Divergence in the ES13 part of the MFQ binding pocket between the P. falciparum A type (blood stage) and S type (sexual stage) ribosomes. A single nucleotide C1440 in the A-type 28S rRNA is deleted in the S-type 28S rRNA. MFQ binding residues are highlighted in a box. e, Sequence alignment of uL13 from P. falciparum, P. vivax, T. gondii and T. brucei. Residues involved in binding to MFQ are highlighted with asterisks.
Fig. 4
Fig. 4. Structure based design of MFQ-derivatives.
a, Chemical structure of MFQ_D1. b, MFQ_D1 docked into the MFQ binding pocket. c, Chemical structure of MFQ_D2. d, MFQ_D2 docked into the MFQ pocket. e, Parasite growth inhibition assay measuring the inhibitory activity of MFQ derivatives on 3D7 parasites. Data are shown as the mean ± SD of three biological replicates with each biological replicate representing three experimental replicates.

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