title: Use of methotrexate-based peptide substrates to characterize the substrate specificity of prostate-specific membrane antigen (PSMA).
abstract: Prostate-Specific Membrane Antigen (PSMA) is a glutamate carboxypeptidase II that is highly expressed by both normal and malignant prostate epithelial cells and by the neovasculature of many tumor types but is not expressed by endothelial cells in normal tissue. PSMA possesses the hydrolytic properties of an N-acetylated alpha-linked acidic dipeptidase (NAALADase) and also functions as a pteroyl poly-gamma-glutamyl carboxypeptidase (i.e., folate hydrolase). Therefore, PSMA can be targeted for activation of peptide-based prodrugs within the extracellular fluid of prostate cancers. In this study, methotrexate-based peptide analogs were evaluated to identify PSMA selective substrates that are also stable to nonspecific hydrolysis in human and mouse plasma. These methotrexate analogs were also characterized for in vitro toxicity against PSMA and nonPSMA producing human cancer cell lines. Analogs containing gamma-linked glutamate residues were most efficiently hydrolyzed by PSMA, but were unstable in plasma. Analogs containing both alpha- and gamma-linked acidic amino acids were less efficiently hydrolyzed by PSMA but were most stable in plasma. Analogs were 5-10 fold more selectively toxic in vitro in the presence of active PSMA. These studies have identified PSMA selective, plasma stable peptide substrates that can be incorporated into prodrugs targeted for activation by PSMA within prostate cancer sites.
authors.affiliation: The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, The Johns Hopkins School of Medicine, Baltimore, Maryland, USA.
authors.forename: Annastasiah
authors.initials: A
authors.lastname: Mhaka
authors.forename: Alyssa M
authors.initials: AM
authors.lastname: Gady
authors.forename: D Marc
authors.initials: DM
authors.lastname: Rosen
authors.forename: Kin-Ming
authors.initials: KM
authors.lastname: Lo
authors.forename: Steven D
authors.initials: SD
authors.lastname: Gillies
authors.forename: Samuel R
authors.initials: SR
authors.lastname: Denmeade
mesh_terms: D000818:Animals; D000954:Antigens, Surface / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism; D015153:Blotting, Western; D045604:Extracellular Fluid; D043425:Glutamate Carboxypeptidase II / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D006801:Humans; D007142:Immunoglobulin gamma-Chains / Q000235:genetics; D007145:Immunoglobulin kappa-Chains / Q000235:genetics; D008297:Male; D008727:Methotrexate / Q000378:metabolism*; D051379:Mice; D010446:Peptide Fragments / Q000378:metabolism*; D011355:Prodrugs; D011471:Prostatic Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured / Q000378:metabolism
chemical_list: D000954:Antigens, Surface; D007142:Immunoglobulin gamma-Chains; D007145:Immunoglobulin kappa-Chains; D010446:Peptide Fragments; D011355:Prodrugs; D011993:Recombinant Fusion Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; C473848:FOLH1 protein, human; D043425:Glutamate Carboxypeptidase II; D008727:Methotrexate