Modeling and resistant alleles explain the selectivity of antimalarial compound 49c towards apicomplexan aspartyl proteases

EMBO J. 2018 Apr 3;37(7):e98047. doi: 10.15252/embj.201798047. Epub 2018 Mar 8.


Toxoplasma gondii aspartyl protease 3 (TgASP3) phylogenetically clusters with Plasmodium falciparum Plasmepsins IX and X (PfPMIX, PfPMX). These proteases are essential for parasite survival, acting as key maturases for secreted proteins implicated in invasion and egress. A potent antimalarial peptidomimetic inhibitor (49c) originally developed against Plasmepsin II selectively targets TgASP3, PfPMIX, and PfPMX To unravel the molecular basis for the selectivity of 49c, we constructed homology models of PfPMIX, PfPMX, and TgASP3 that were first validated by identifying the determinants of microneme and rhoptry substrate recognition. The flap and flap-like structures of several reported Plasmepsins are highly flexible and critically modulate the access to the binding cavity. Molecular docking of 49c to TgASP3, PfPMIX, and PfPMX models predicted that the conserved phenylalanine residues in the flap, F344, F291, and F305, respectively, account for the sensitivity toward 49c. Concordantly, phenylalanine mutations in the flap of the three proteases increase twofold to 15-fold the IC50 values of 49c. Compellingly the selection of mutagenized T. gondii resistant strains to 49c reproducibly converted F344 to a cysteine residue.

Keywords: Plasmodium; Toxoplasma; Aspartyl protease; Plasmepsin; modeling.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antimalarials / chemistry
  • Antimalarials / pharmacology*
  • Aspartic Acid Endopeptidases / chemistry
  • Aspartic Acid Endopeptidases / metabolism
  • Aspartic Acid Proteases / antagonists & inhibitors*
  • Aspartic Acid Proteases / metabolism*
  • Cysteine
  • Dose-Response Relationship, Drug
  • Drug Resistance / drug effects
  • Drug Resistance / physiology*
  • Inhibitory Concentration 50
  • Models, Molecular
  • Molecular Docking Simulation
  • Mutation
  • Parasitic Sensitivity Tests
  • Phenylalanine / drug effects
  • Phenylalanine / genetics
  • Plasmodium falciparum / drug effects
  • Plasmodium falciparum / genetics
  • Protease Inhibitors / pharmacology*
  • Protozoan Proteins / chemistry*
  • Protozoan Proteins / genetics
  • Recombinant Proteins / genetics
  • Sequence Alignment
  • Toxoplasma / drug effects
  • Toxoplasma / genetics


  • Antimalarials
  • Protease Inhibitors
  • Protozoan Proteins
  • Recombinant Proteins
  • Phenylalanine
  • Aspartic Acid Proteases
  • Aspartic Acid Endopeptidases
  • plasmepsin
  • Cysteine