Prostate-specific membrane antigen (PSMA) is a well-characterized tumor marker associated with prostate cancer and neovasculature of most solid tumors. PSMA-specific ligands are thus being developed to deliver imaging or therapeutic agents to cancer cells. Here, we report on a crystal structure of human PSMA in complex with A9g, a 43-bp PSMA-specific RNA aptamer, that was determined to the 2.2 Å resolution limit. The analysis of the PSMA/aptamer interface allows for identification of key interactions critical for nanomolar binding affinity and high selectivity of A9g for human PSMA. Combined with in silico modeling, site-directed mutagenesis, inhibition experiments and cell-based assays, the structure also provides an insight into structural changes of the aptamer and PSMA upon complex formation, mechanistic explanation for inhibition of the PSMA enzymatic activity by A9g as well as its ligand-selective competition with small molecules targeting the internal pocket of the enzyme. Additionally, comparison with published protein-RNA aptamer structures pointed toward more general features governing protein-aptamer interactions. Finally, our findings can be exploited for the structure-assisted design of future A9g-based derivatives with improved binding and stability characteristics.

• MeSH
• Antigens, Surface chemistry   MeSH
• Aptamers, Nucleotide chemistry   MeSH
• PC-3 Cells MeSH
• Glutamate Carboxypeptidase II chemistry   MeSH
• HEK293 Cells MeSH
• Protein Interaction Domains and Motifs MeSH
• Humans MeSH
• Ligands MeSH
• Molecular Structure MeSH
• Biomarkers, Tumor chemistry   MeSH
• Prostatic Neoplasms metabolism   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• A9g RNA aptamer MeSH Browser
• Antigens, Surface MeSH
• Aptamers, Nucleotide MeSH
• FOLH1 protein, human MeSH Browser
• Glutamate Carboxypeptidase II MeSH
• Ligands MeSH
• Biomarkers, Tumor MeSH

Glutamate carboxypeptidase II (GCPII), also known as prostate-specific membrane antigen (PSMA) or folate hydrolase, is a metallopeptidase expressed predominantly in the human brain and prostate. GCPII expression is considerably increased in prostate carcinoma, and the enzyme also participates in glutamate excitotoxicity in the brain. Therefore, GCPII represents an important diagnostic marker of prostate cancer progression and a putative target for the treatment of both prostate cancer and neuronal disorders associated with glutamate excitotoxicity. For the development of novel therapeutics, mouse models are widely used. However, although mouse GCPII activity has been characterized, a detailed comparison of the enzymatic activity and tissue distribution of the mouse and human GCPII orthologs remains lacking. In this study, we prepared extracellular mouse GCPII and compared it with human GCPII. We found that mouse GCPII possesses lower catalytic efficiency but similar substrate specificity compared with the human protein. Using a panel of GCPII inhibitors, we discovered that inhibition constants are generally similar for mouse and human GCPII. Furthermore, we observed highest expression of GCPII protein in the mouse kidney, brain, and salivary glands. Importantly, we did not detect GCPII in the mouse prostate. Our data suggest that the differences in enzymatic activity and inhibition profile are rather small; therefore, mouse GCPII can approximate human GCPII in drug development and testing. On the other hand, significant differences in GCPII tissue expression must be taken into account when developing novel GCPII-based anticancer and therapeutic methods, including targeted anticancer drug delivery systems, and when using mice as a model organism.

• Keywords
• glutamate carboxypeptidase II, mouse animal model, neuronal disorders, prostate cancer, prostate‐specific membrane antigen,
• Publication type
• Journal Article MeSH

BACKGROUND: Prostate-specific membrane antigen (PSMA) is a validated target for the imaging and therapy of prostate cancer. Here, we report the detailed characterization of four novel murine monoclonal antibodies (mAbs) recognizing human PSMA as well as PSMA orthologs from different species. METHODS: Performance of purified mAbs was assayed using a comprehensive panel of in vitro experimental setups including Western blotting, immunofluorescence, immunohistochemistry, ELISA, flow cytometry, and surface-plasmon resonance. Furthermore, a mouse xenograft model of prostate cancer was used to compare the suitability of the mAbs for in vivo applications. RESULTS: All mAbs demonstrate high specificity for PSMA as documented by the lack of cross-reactivity to unrelated human proteins. The 3F11 and 1A11 mAbs bind linear epitopes spanning residues 226-243 and 271-288 of human PSMA, respectively. 3F11 is also suitable for the detection of PSMA orthologs from mouse, pig, dog, and rat in experimental setups where the denatured form of PSMA is used. 5D3 and 5B1 mAbs recognize distinct surface-exposed conformational epitopes and are useful for targeting PSMA in its native conformation. Most importantly, using a mouse xenograft model of prostate cancer we show that both the intact 5D3 and its Fab fragment are suitable for in vivo imaging. CONCLUSIONS: With apparent affinities of 0.14 and 1.2 nM as determined by ELISA and flow cytometry, respectively, 5D3 has approximately 10-fold higher affinity for PSMA than the clinically validated mAb J591 and, therefore, is a prime candidate for the development of next-generation theranostics to target PSMA. Prostate 77:749-764, 2017. © 2017 Wiley Periodicals, Inc.

• Keywords
• NAALADase, glutamate carboxypeptidase II, in vivo imaging, monoclonal antibody, prostate cancer,
• MeSH
• Antigens, Surface * immunology   MeSH
• Glutamate Carboxypeptidase II * antagonists & inhibitors   immunology   MeSH
• Humans MeSH
• Antibodies, Monoclonal immunology   pharmacology   MeSH
• Antibodies, Monoclonal, Murine-Derived immunology   pharmacology   MeSH
• Mice MeSH
• Prostatic Neoplasms * drug therapy   immunology   MeSH
• Prostate * immunology   pathology   MeSH
• Theranostic Nanomedicine methods   MeSH
• Xenograft Model Antitumor Assays methods   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antigens, Surface * MeSH
• FOLH1 protein, human MeSH Browser
• Glutamate Carboxypeptidase II * MeSH
• J591 monoclonal antibody MeSH Browser
• Antibodies, Monoclonal MeSH
• Antibodies, Monoclonal, Murine-Derived MeSH

Human diseases are often diagnosed by determining levels of relevant enzymes and treated by enzyme inhibitors. We describe an assay suitable for both ultrasensitive enzyme quantification and quantitative inhibitor screening with unpurified enzymes. In the DNA-linked Inhibitor ANtibody Assay (DIANA), the target enzyme is captured by an immobilized antibody, probed with a small-molecule inhibitor attached to a reporter DNA and detected by quantitative PCR. We validate the approach using the putative cancer markers prostate-specific membrane antigen and carbonic anhydrase IX. We show that DIANA has a linear range of up to six logs and it selectively detects zeptomoles of targets in complex biological samples. DIANA's wide dynamic range permits determination of target enzyme inhibition constants using a single inhibitor concentration. DIANA also enables quantitative screening of small-molecule enzyme inhibitors using microliters of human blood serum containing picograms of target enzyme. DIANA's performance characteristics make it a superior tool for disease detection and drug discovery.

• MeSH
• Biological Assay * MeSH
• DNA * MeSH
• Enzymes metabolism   MeSH
• Enzyme Inhibitors pharmacology   MeSH
• Humans MeSH
• Drug Discovery * MeSH
• Reproducibility of Results MeSH
• Sensitivity and Specificity MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA * MeSH
• Enzymes MeSH
• Enzyme Inhibitors MeSH