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  • Identification of potent and selective inhibitors of the plasmodium falciparum M18 aspartyl aminopeptidase (PfM18AAP) of human malaria via high-throughput screening

    Author(s)
    Spicer, Timothy
    Fernandez-Vega, Virneliz
    Chase, Peter
    Scampavia, Louis
    To, Joyce
    Dalton, John P
    Da Silva, Fabio L
    Skinner-Adams, Tina S
    Gardiner, Donald L
    Trenholme, Katharine R
    Brown, Christopher L
    Ghosh, Partha
    Porubsky, Patrick
    Wang, Jenna L
    Whipple, David A
    Schoenen, Frank J
    Hodder, Peter
    Griffith University Author(s)
    Brown, Chris L.
    Trenholme, Katharine
    Skinner-Adams, Tina
    Gardiner, Donald
    Year published
    2014
    Metadata
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    Abstract
    The target of this study, the PfM18 aspartyl aminopeptidase (PfM18AAP), is the only AAP present in the genome of the malaria parasite Plasmodium falciparum. PfM18AAP is a metallo-exopeptidase that exclusively cleaves N-terminal acidic amino acids glutamate and aspartate. It is expressed in parasite cytoplasm and may function in concert with other aminopeptidases in protein degradation, of, for example, hemoglobin. Previous antisense knockdown experiments identified a lethal phenotype associated with PfM18AAP, suggesting that it is a valid target for new antimalaria therapies. To identify inhibitors of PfM18AAP function, a ...
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    The target of this study, the PfM18 aspartyl aminopeptidase (PfM18AAP), is the only AAP present in the genome of the malaria parasite Plasmodium falciparum. PfM18AAP is a metallo-exopeptidase that exclusively cleaves N-terminal acidic amino acids glutamate and aspartate. It is expressed in parasite cytoplasm and may function in concert with other aminopeptidases in protein degradation, of, for example, hemoglobin. Previous antisense knockdown experiments identified a lethal phenotype associated with PfM18AAP, suggesting that it is a valid target for new antimalaria therapies. To identify inhibitors of PfM18AAP function, a fluorescence enzymatic assay was developed using recombinant PfM18AAP enzyme and a fluorogenic peptide substrate (H-Glu-NHMec). This was screened against the Molecular Libraries Probe Production Centers Network collection of ~292,000 compounds (the Molecular Libraries Small Molecule Repository). A cathepsin L1 (CTSL1) enzyme-based assay was developed and used as a counterscreen to identify compounds with nonspecific activity. Enzymology and phenotypic assays were used to determine mechanism of action and efficacy of selective and potent compounds identified from high-throughput screening. Two structurally related compounds, CID 6852389 and CID 23724194, yielded micromolar potency and were inactive in CTSL1 titration experiments (IC50 >59.6 µM). As measured by the Ki assay, both compounds demonstrated micromolar noncompetitive inhibition in the PfM18AAP enzyme assay. Both CID 6852389 and CID 23724194 demonstrated potency in malaria growth assays (IC50 4 µM and 1.3 µM, respectively).
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    Journal Title
    Journal of Biomolecular Screening
    Volume
    19
    Issue
    7
    DOI
    https://doi.org/10.1177/1087057114525852
    Subject
    Medical Parasitology
    Biochemistry and Cell Biology
    Publication URI
    http://hdl.handle.net/10072/63938
    Collection
    • Journal articles

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