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

Spicer, T; Fernandez-Vega, V; Chase, P; Scampavia, L; To, J; Dalton, JP; Da Silva, FL; Skinner-Adams, TS; Gardiner, DL; Trenholme, KR; Brown, CL; Ghosh, P; Porubsky, P; Wang, JL; Whipple, DA; Schoenen, FJ; Hodder, P

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

Spicer, T Fernandez-Vega, V Chase, P Scampavia, L To, J

Dalton, JP Da Silva, FL Skinner-Adams, TS Gardiner, DL Trenholme, KR Brown, CL Ghosh, P Porubsky, P Wang, JL Whipple, DA Schoenen, FJ Hodder, P

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Publication Type:: Journal Article
Citation:: Journal of Biomolecular Screening, 2014, 19 (7), pp. 1107 - 1115
Issue Date:: 2014-01-01

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Field	Value	Language
dc.contributor.author	Spicer, T	en_US
dc.contributor.author	Fernandez-Vega, V	en_US
dc.contributor.author	Chase, P	en_US
dc.contributor.author	Scampavia, L	en_US
dc.contributor.author	To, J https://orcid.org/0000-0003-3482-4369	en_US
dc.contributor.author	Dalton, JP	en_US
dc.contributor.author	Da Silva, FL	en_US
dc.contributor.author	Skinner-Adams, TS	en_US
dc.contributor.author	Gardiner, DL	en_US
dc.contributor.author	Trenholme, KR	en_US
dc.contributor.author	Brown, CL	en_US
dc.contributor.author	Ghosh, P	en_US
dc.contributor.author	Porubsky, P	en_US
dc.contributor.author	Wang, JL	en_US
dc.contributor.author	Whipple, DA	en_US
dc.contributor.author	Schoenen, FJ	en_US
dc.contributor.author	Hodder, P	en_US
dc.date.available	2014-02-04	en_US
dc.date.issued	2014-01-01	en_US
dc.identifier.citation	Journal of Biomolecular Screening, 2014, 19 (7), pp. 1107 - 1115	en_US
dc.identifier.issn	1087-0571	en_US
dc.identifier.uri	http://hdl.handle.net/10453/116033
dc.description.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 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). © 2014 Society for Laboratory Automation and Screening.	en_US
dc.relation.ispartof	Journal of Biomolecular Screening	en_US
dc.relation.isbasedon	10.1177/1087057114525852	en_US
dc.subject.classification	Medicinal & Biomolecular Chemistry	en_US
dc.subject.mesh	Erythrocytes	en_US
dc.subject.mesh	Animals	en_US
dc.subject.mesh	Humans	en_US
dc.subject.mesh	Fasciola hepatica	en_US
dc.subject.mesh	Plasmodium falciparum	en_US
dc.subject.mesh	Malaria, Falciparum	en_US
dc.subject.mesh	Aminopeptidases	en_US
dc.subject.mesh	Glutamyl Aminopeptidase	en_US
dc.subject.mesh	Peptides	en_US
dc.subject.mesh	Recombinant Proteins	en_US
dc.subject.mesh	Antimalarials	en_US
dc.subject.mesh	Spectrometry, Fluorescence	en_US
dc.subject.mesh	Cluster Analysis	en_US
dc.subject.mesh	Inhibitory Concentration 50	en_US
dc.subject.mesh	Substrate Specificity	en_US
dc.subject.mesh	Drug Design	en_US
dc.subject.mesh	Kinetics	en_US
dc.subject.mesh	Software	en_US
dc.subject.mesh	Small Molecule Libraries	en_US
dc.subject.mesh	Cathepsin L	en_US
dc.title	Identification of potent and selective inhibitors of the plasmodium falciparum M18 aspartyl aminopeptidase (PfM18AAP) of human malaria via high-throughput screening	en_US
dc.type	Journal Article
utslib.citation.volume	7	en_US
utslib.citation.volume	19	en_US
utslib.for	0601 Biochemistry and Cell Biology	en_US
pubs.embargo.period	Not known	en_US
pubs.organisational-group	/University of Technology Sydney
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
pubs.organisational-group	/University of Technology Sydney/Faculty of Science/School of Life Sciences
pubs.organisational-group	/University of Technology Sydney/Strength - ithree - Institute of Infection, Immunity and Innovation
utslib.copyright.status	closed_access
pubs.issue	7	en_US
pubs.publication-status	Published	en_US
pubs.volume	19	en_US

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 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). © 2014 Society for Laboratory Automation and Screening.

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http://hdl.handle.net/10453/116033