title: Characterization of amino acid variation at strategic positions in parasite and human proteases for selective inhibition of falcipains in Plasmodium falciparum.
abstract: Falcipains (FP) of Plasmodium falciparum are important virulence factors marked as potential targets for antimalarial drug discovery. In this study, the previously uncharacterized fp2B (PF11_0161) was shown to be highly expressed as an active enzyme during the erythrocytic stage. With three related proteases in the FP family and the existence of human homologues, it is prudent to identify clusters of residues unique to the parasite proteases that can be targeted selectively for drug design. Using bioinformatic tools, we have carefully mapped out a highly conserved and unique region constituted by I85, S149, and A151 in the plasmodial proteases that can influence the development of compounds capable of inhibiting the entire FP family. Taking drug interactions with the human homologues into consideration, these residues in FP2B were replaced with the cognate residues found in human cathepsin L (catL) for evaluation. Despite the high sequence similarity between the FP2 isozymes (97.5%), FP2B is found to be more tolerant to amino acid substitution at position 149 than FP2A. This structural disparity implied that residues mediating peptide substrate interactions are not fully conserved across the FP family and warrant attention in the design and evaluation of protease inhibitors focused on the FPs. The simultaneous substitution of the neighboring residues (I85 or A151) rendered the double mutants (S149A/I85M and S149A/A151D) completely inactive. Significantly, the mutations did not result in 'catL-like' specificity, suggesting that substrate-based inhibitors could be rationally designed against these important parasite-specific structural determinants.
authors.affiliation: Department of Microbiology, Faculty of Medicine, National University of Singapore, MD4A, 5 Science Drive 2, Singapore 117597, Singapore.
authors.forename: Liuh Ling
authors.initials: LL
authors.lastname: Goh
authors.forename: Tiow Suan
authors.initials: TS
authors.lastname: Sim
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000378:metabolism; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014158:Transcription, Genetic
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D010447:Peptide Hydrolases; D003546:Cysteine Endopeptidases; C099607:falcipain