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

• Published in: The EMBO journal Vol. 37; no. 7
• Main Authors: Mukherjee, Budhaditya, Tessaro, Francesca, Vahokoski, Juha, Kursula, Inari, Marq, Jean‐Baptiste, Scapozza, Leonardo, Soldati‐Favre, Dominique
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 03.04.2018; Blackwell Publishing Ltd; John Wiley and Sons Inc
• Subjects: Antimalarials - chemistry; Antimalarials - pharmacology; Aspartic Acid Endopeptidases - chemistry; Aspartic Acid Endopeptidases - metabolism; Aspartic Acid Proteases - antagonists & inhibitors; Aspartic Acid Proteases - metabolism; Aspartic endopeptidase; Aspartyl protease; Catalysis; Cysteine; Determinants; Dose-Response Relationship, Drug; Drug Resistance - drug effects; Drug Resistance - physiology; Egress; EMBO23; EMBO31; EMBO40; Homology; Inhibitors; Inhibitory Concentration 50; modeling; Models, Molecular; Molecular docking; Molecular Docking Simulation; Mutation; Parasite resistance; Parasites; Parasitic Sensitivity Tests; Phenylalanine; Phenylalanine - drug effects; Phenylalanine - genetics; Phylogeny; Plasmepsin; Plasmodium; Plasmodium falciparum; Plasmodium falciparum - drug effects; Plasmodium falciparum - genetics; Protease; Protease Inhibitors - pharmacology; Proteases; Proteins; Protozoan Proteins - chemistry; Protozoan Proteins - genetics; Recognition; Recombinant Proteins - genetics; Residues; Selectivity; Sensitivity; Sequence Alignment; Substrates; Toxoplasma; Toxoplasma - drug effects; Toxoplasma - genetics; Toxoplasma gondii; Tyrosine; Aspartyl protease; Plasmepsin; modeling; Toxoplasma; Plasmodium
• ISSN: 0261-4189; 1460-2075
• DOI: 10.15252/embj.201798047

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 IC 50 values of 49c. Compellingly the selection of mutagenized T. gondii resistant strains to 49c reproducibly converted F344 to a cysteine residue. Synopsis The apicomplexan aspartyl proteases TgASP3, PfPMIX and PfPMX play pivotal roles in the lytic cycle of Toxoplasma gondii and Plasmodium falciparum . The anti‐malarial compound 49c is a selective peptidomimetic inhibitor of these proteases that blocks host cell invasion and egress. Molecular homology and substrate docking models of the TgASP3, PfPMIX and PfPMX catalytic domains uncover conformational determinants responsible for distinct substrate recognition. A conserved phenylalanine residue in the aspartyl protease flap region is responsible for the selective sensitivity to 49c. Mutation of the conserved phenylalanine to tyrosine confers parasite resistance to 49c. Chemically mutagenized parasites selected for resistance to 49c harbour a cysteine mutation at the conserved phenylalanine residue, confirming its critical role. Graphical Abstract A conserved phenylalanine residue in the flap region of apicomplexan aspartyl proteases TgASP3, PfPMIX and PfPMX confers selective sensitivity to the peptidomimetic inhibitor 49c.