title: Identification of Potent and Selective Inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PfM18AAP) of Human Malaria via High-Throughput Screening.
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 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 counter screen 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 K(i) assay, both compounds demonstrated micromolar noncompetitive inhibition in the PfM18AAP enzyme assay. Both CID 6852389 and CID 23724194 demonstrated potency in malaria growth assays (IC504 µM and 1.3 µM, respectively).
authors.affiliation: The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, FL, USA.
authors.forename: Timothy
authors.initials: T
authors.lastname: Spicer
authors.affiliation: The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, FL, USA.
authors.forename: Virneliz
authors.initials: V
authors.lastname: Fernandez-Vega
authors.affiliation: The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, FL, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Chase
authors.affiliation: The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, FL, USA.
authors.forename: Louis
authors.initials: L
authors.lastname: Scampavia
authors.affiliation: Institute for Biotechnology of Infectious Diseases, University of Technology Sydney, Sydney, Australia.
authors.forename: Joyce
authors.initials: J
authors.lastname: To
authors.affiliation: Institute for Biotechnology of Infectious Diseases, University of Technology Sydney, Sydney, Australia Institute of Parasitology, McGill University, Quebec, Canada.
authors.forename: John P
authors.initials: JP
authors.lastname: Dalton
authors.affiliation: Malaria Biology Laboratory, The Queensland Institute of Medical Research, Brisbane, Australia.
authors.forename: Fabio L
authors.initials: FL
authors.lastname: Da Silva
authors.affiliation: Malaria Biology Laboratory, The Queensland Institute of Medical Research, Brisbane, Australia.
authors.forename: Tina S
authors.initials: TS
authors.lastname: Skinner-Adams
authors.affiliation: Malaria Biology Laboratory, The Queensland Institute of Medical Research, Brisbane, Australia.
authors.forename: Donald L
authors.initials: DL
authors.lastname: Gardiner
authors.affiliation: Malaria Biology Laboratory, The Queensland Institute of Medical Research, Brisbane, Australia.
authors.forename: Katharine R
authors.initials: KR
authors.lastname: Trenholme
authors.affiliation: School of Biomolecular and Physical Sciences, Griffith University, Brisbane, Australia.
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Brown
authors.affiliation: The University of Kansas Specialized Chemistry Center, Lawrence, KS, USA.
authors.forename: Partha
authors.initials: P
authors.lastname: Ghosh
authors.affiliation: The University of Kansas Specialized Chemistry Center, Lawrence, KS, USA.
authors.forename: Patrick
authors.initials: P
authors.lastname: Porubsky
authors.affiliation: The University of Kansas Specialized Chemistry Center, Lawrence, KS, USA.
authors.forename: Jenna L
authors.initials: JL
authors.lastname: Wang
authors.affiliation: The University of Kansas Specialized Chemistry Center, Lawrence, KS, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Whipple
authors.affiliation: The University of Kansas Specialized Chemistry Center, Lawrence, KS, USA.
authors.forename: Frank J
authors.initials: FJ
authors.lastname: Schoenen
authors.affiliation: The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, FL, USA hodderp@scripps.edu.
authors.forename: Peter
authors.initials: P
authors.lastname: Hodder
mesh_terms: D000626:Aminopeptidases / Q000037:antagonists & inhibitors*; D000818:Animals; D000962:Antimalarials / Q000737:chemistry* / Q000494:pharmacology; D056668:Cathepsin L / Q000737:chemistry; D016000:Cluster Analysis; D015195:Drug Design; D004912:Erythrocytes / Q000469:parasitology; D005210:Fasciola hepatica / Q000201:enzymology; D043384:Glutamyl Aminopeptidase / Q000037:antagonists & inhibitors* / Q000737:chemistry; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D016778:Malaria, Falciparum / Q000188:drug therapy*; D010455:Peptides / Q000378:metabolism; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry; D054852:Small Molecule Libraries / Q000737:chemistry; D012984:Software; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
chemical_list: D000962:Antimalarials; D010455:Peptides; D011994:Recombinant Proteins; D054852:Small Molecule Libraries; D000626:Aminopeptidases; D043384:Glutamyl Aminopeptidase; D056668:Cathepsin L
keywords:     PfM18AAP; 1536 well; Plasmodium falciparum; QFRET; aspartyl aminopeptidase; exopeptidase; malaria; parasite