Identification of Potent and Selective Inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PfM18AAP) of Human Malaria via High Throughput Screening

摘要

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 anti-malaria 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 (MLPCN) collection of ~292,000 compounds (the Molecular Libraries Small Molecule Repository (MLSMR)). 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 HTS. Two structurally related compounds, CID 6852389 and CID 23724194, yield micromolar potency and are inactive in CTSL1 titration experiments (IC50 >59.6 μM). As measured by Ki assay, both compounds demonstrate micromolar non-competitive inhibition in the PfM18AAP enzyme assay. Both CID 6852389 and CID 23724194 demonstrate potency in malaria growth assays (IC50 4 μM and 1.3 μM, respectively).