Prostate cancer (PCa) ranks as the second leading cause of cancer-related deaths among men in the United States. Prostate-specific membrane antigen (PSMA) represents a well-established biomarker of PCa, and its levels correlate positively with the disease progression, culminating at the stage of metastatic castration-resistant prostate cancer. Due to its tissue-specific expression and cell surface localization, PSMA shows superior potential for precise imaging and therapy of PCa. Antibody-based immunotherapy targeting PSMA offers the promise of selectively engaging the host immune system with minimal off-target effects. Here we report on the design, expression, purification, and characterization of a bispecific engager, termed 5D3-CP33, that efficiently recruits macrophages to the vicinity of PSMA-positive cancer cells mediating PCa death. The engager was engineered by fusing the anti-PSMA 5D3 antibody fragment to a cyclic peptide 33 (CP33), selectively binding the Fc gamma receptor I (FcγRI/CD64) on the surface of phagocytes. Functional parts of the 5D3-CP33 engager revealed a nanomolar affinity for PSMA and FcγRI/CD64 with dissociation constants of KD = 3 nM and KD = 140 nM, respectively. At a concentration as low as 0.3 nM, the engager was found to trigger the production of reactive oxygen species by U937 monocytic cells in the presence of PSMA-positive cells. Moreover, flow cytometry analysis demonstrated antibody-dependent cell-mediated phagocytosis of PSMA-positive cancer cells by U937 monocytes when exposed to 0.15 nM 5D3-CP33. Our findings illustrate that 5D3-CP33 effectively and specifically activates monocytes upon PSMA-positive target engagement, resulting in the elimination of tumor cells. The 5D3-CP33 engager can thus serve as a promising lead for developing new immunotherapy tools for the efficient treatment of PCa.

• MeSH
• Antigens, Surface * immunology   metabolism   MeSH
• Glutamate Carboxypeptidase II * immunology   metabolism   MeSH
• Immunotherapy methods   MeSH
• Humans MeSH
• Macrophages immunology   MeSH
• Monocytes * immunology   metabolism   MeSH
• Cell Line, Tumor MeSH
• Prostatic Neoplasms * immunology   therapy   pathology   MeSH
• Antibodies, Bispecific * immunology   pharmacology   MeSH
• Receptors, IgG metabolism   immunology   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antigens, Surface * MeSH
• FOLH1 protein, human MeSH Browser
• Glutamate Carboxypeptidase II * MeSH
• Antibodies, Bispecific * MeSH
• Receptors, IgG MeSH

The sulfonamide function is used extensively as a general building block in various inhibitory scaffolds and, more specifically, as a zinc-binding group (ZBG) of metalloenzyme inhibitors. Here, we provide biochemical, structural, and computational characterization of a metallopeptidase in complex with inhibitors, where the mono- and bisubstituted sulfamide functions are designed to directly engage zinc ions of a bimetallic enzyme site. Structural data showed that while monosubstituted sulfamides coordinate active-site zinc ions via the free negatively charged amino group in a canonical manner, their bisubstituted counterparts adopt an atypical binding pattern divergent from expected positioning of corresponding tetrahedral reaction intermediates. Accompanying quantum mechanics calculations revealed that electroneutrality of the sulfamide function is a major factor contributing to the markedly lower potency of bisubstituted compounds by considerably lowering their interaction energy with the enzyme. Overall, while bisubstituted uncharged sulfamide functions can bolster favorable pharmacological properties of a given inhibitor, their use as ZBGs in metalloenzyme inhibitors might be less advantageous due to their suboptimal metal-ligand properties.

• MeSH
• Protease Inhibitors * pharmacology   MeSH
• Ions MeSH
• Metalloproteins * chemistry   MeSH
• Zinc metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Protease Inhibitors * MeSH
• Ions MeSH
• Metalloproteins * MeSH
• Zinc MeSH

Human glutamate carboxypeptidase 2 (GCP2) from the M28B metalloprotease group is an important target for therapy in neurological disorders and an established tumor marker. However, its physiological functions remain unclear. To better understand general roles, we used the model organism Caenorhabditis elegans to genetically manipulate its three existing orthologous genes and evaluate the impact on worm physiology. The results of gene knockout studies showed that C. elegans GCP2 orthologs affect the pharyngeal physiology, reproduction, and structural integrity of the organism. Promoter-driven GFP expression revealed distinct localization for each of the three gene paralogs, with gcp-2.1 being most abundant in muscles, intestine, and pharyngeal interneurons, gcp-2.2 restricted to the phasmid neurons, and gcp-2.3 located in the excretory cell. The present study provides new insight into the unique phenotypic effects of GCP2 gene knockouts in C. elegans, and the specific tissue localizations. We believe that elucidation of particular roles in a non-mammalian organism can help to explain important questions linked to physiology of this protease group and in extension to human GCP2 involvement in pathophysiological processes.

• Keywords
• N-acetyl-aspartyl-glutamate, folate hydrolase 1, phenotyping, promoter-driven GFP expression, prostate-specific membrane antigen,
• MeSH
• Caenorhabditis elegans * genetics   MeSH
• Carboxypeptidases genetics   metabolism   MeSH
• Humans MeSH
• Promoter Regions, Genetic MeSH
• Caenorhabditis elegans Proteins * genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• glutamate carboxypeptidase MeSH Browser
• Carboxypeptidases MeSH
• Caenorhabditis elegans Proteins * MeSH

BACKGROUND: Glutamate carboxypeptidase 2 (GCP2) belongs to the M28B metalloprotease subfamily encompassing a variety of zinc-dependent exopeptidases that can be found in many eukaryotes, including unicellular organisms. Limited information exists on the physiological functions of GCP2 orthologs in mammalian tissues outside of the brain and intestine, and such data are completely absent for non-mammalian species. Here, we investigate GCP2 orthologs found in trematodes, not only as putative instrumental molecules for defining their basal function(s) but also as drug targets. METHODS: Identified genes encoding M28B proteases Schistosoma mansoni and Fasciola hepatica genomes were analyzed and annotated. Homology modeling was used to create three-dimensional models of SmM28B and FhM28B proteins using published X-ray structures as the template. For S. mansoni, RT-qPCR was used to evaluate gene expression profiles, and, by RNAi, we exploited the possible impact of knockdown on the viability of worms. Enzymes from both parasite species were cloned for recombinant expression. Polyclonal antibodies raised against purified recombinant enzymes and RNA probes were used for localization studies in both parasite species. RESULTS: Single genes encoding M28B metalloproteases were identified in the genomes of S. mansoni and F. hepatica. Homology models revealed the conserved three-dimensional fold as well as the organization of the di-zinc active site. Putative peptidase activities of purified recombinant proteins were assayed using peptidic libraries, yet no specific substrate was identified, pointing towards the likely stringent substrate specificity of the enzymes. The orthologs were found to be localized in reproductive, digestive, nervous, and sensory organs as well as parenchymal cells. Knockdown of gene expression by RNAi silencing revealed that the genes studied were non-essential for trematode survival under laboratory conditions, reflecting similar findings for GCP2 KO mice. CONCLUSIONS: Our study offers the first insight to our knowledge into M28B protease orthologs found in trematodes. Conservation of their three-dimensional structure, as well as tissue expression pattern, suggests that trematode GCP2 orthologs may have functions similar to their mammalian counterparts and can thus serve as valuable models for future studies aimed at clarifying the physiological role(s) of GCP2 and related subfamily proteases.

• Keywords
• Fasciola hepatica, Folate hydrolase, Immunohistochemistry, M28B metalloproteases, NAALADase, Platyhelminth, Prostate specific-membrane antigen, RNA in situ hybridization, Schistosoma mansoni,
• MeSH
• Fasciola hepatica * genetics   MeSH
• Mice MeSH
• Peptide Hydrolases MeSH
• Mammals MeSH
• Schistosoma mansoni MeSH
• Trematoda * genetics   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• glutamate carboxypeptidase MeSH Browser
• Peptide Hydrolases MeSH

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

A series of carbamate-based inhibitors of glutamate carboxypeptidase II (GCPII) were designed and synthesized using ZJ-43, N-[[[(1S)-1-carboxy-3-methylbutyl]amino]carbonyl]-l-glutamic acid, as a molecular template in order to better understand the impact of replacing one of the two nitrogen atoms in the urea-based GCPII inhibitor with an oxygen atom. Compound 7 containing a C-terminal 2-oxypentanedioic acid was more potent than compound 5 containing a C-terminal glutamic acid (2-aminopentanedioic acid) despite GCPII's preference for peptides containing an N-terminal glutamate as substrates. Subsequent crystallographic analysis revealed that ZJ-43 and its two carbamate analogs 5 and 7 with the same (S,S)-stereochemical configuration adopt a nearly identical binding mode while (R,S)-carbamate analog 8 containing a d-leucine forms a less extensive hydrogen bonding network. QM and QM/MM calculations have identified no specific interactions in the GCPII active site that would distinguish ZJ-43 from compounds 5 and 7 and attributed the higher potency of ZJ-43 and compound 7 to the free energy changes associated with the transfer of the ligand from bulk solvent to the protein active site as a result of the lower ligand strain energy and solvation/desolvation energy. Our findings underscore a broader range of factors that need to be taken into account in predicting ligand-protein binding affinity. These insights should be of particular importance in future efforts to design and develop GCPII inhibitors for optimal inhibitory potency.

• Keywords
• Crystal structure, Glutamate carboxypeptidase II, Metallopeptidase, Prostate-specific membrane antigen,
• MeSH
• Cell Line MeSH
• Drosophila genetics   MeSH
• Enzyme Assays MeSH
• Glutamate Carboxypeptidase II antagonists & inhibitors   chemistry   metabolism   MeSH
• Protease Inhibitors chemical synthesis   chemistry   metabolism   MeSH
• Carbamates chemical synthesis   chemistry   metabolism   MeSH
• Catalytic Domain MeSH
• Quantum Theory MeSH
• Humans MeSH
• Urea analogs & derivatives   chemical synthesis   chemistry   metabolism   MeSH
• Models, Molecular MeSH
• Stereoisomerism MeSH
• Protein Binding MeSH
• Hydrogen Bonding MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Glutamate Carboxypeptidase II MeSH
• Protease Inhibitors MeSH
• Carbamates MeSH
• Urea MeSH
• ZJ43 MeSH Browser

Calcium ions are required for proper function of a wide spectrum of proteins within cells. X-ray crystallography of human glutamate carboxypeptidase II (GCPII) revealed the presence of a Ca2+ -binding site, but its importance for the structure and function of this metallopeptidase has not been elucidated to date. Here, we prepared a panel of mutants targeting residues that form the Ca2+ coordination sphere of GCPII and analyzed their structural and enzymatic properties using an array of complementary biophysical and biochemical approaches. Our data unequivocally show that even a slight disruption of the Ca2+ -binding site destabilizes the three-dimensional fold of GCPII and is associated with impaired secretion, a high propensity to form nonphysiological oligomers, and an inability to bind active site-targeted ligands. Additionally, the Ca2+ -binding site is critical for maintenance of the native homodimeric quaternary arrangement of GCPII, which is indispensable for its enzymatic activity. Overall, our results offer a clear picture of the importance of Ca2+ for the structural integrity and hydrolytic activity of human GCPII and by extension homologous members of the M28 zinc-dependent metallopeptidase family.

• Keywords
• NAALADase, calcium ion, circular dichroism, differential scanning fluorimetry, dimerization, folate hydrolase, metallopeptidase, prostate-specific membrane antigen,
• MeSH
• Dimerization MeSH
• Glutamate Carboxypeptidase II chemistry   genetics   metabolism   MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Models, Molecular MeSH
• Protein Stability MeSH
• Temperature * MeSH
• Calcium chemistry   metabolism   MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Glutamate Carboxypeptidase II MeSH
• Calcium 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

In addition to its well-characterized role in the central nervous system, human glutamate carboxypeptidase II (GCPII; Uniprot ID Q04609) acts as a folate hydrolase in the small intestine, participating in the absorption of dietary polyglutamylated folates (folyl-n-γ-l-glutamic acid), which are the provitamin form of folic acid (also known as vitamin B9 ). Despite the role of GCPII as a folate hydrolase, nothing is known about the processing of polyglutamylated folates by GCPII at the structural or enzymological level. Moreover, many epidemiologic studies on the relationship of the naturally occurring His475Tyr polymorphism to folic acid status suggest that this polymorphism may be associated with several pathologies linked to impaired folate metabolism. In the present study, we report: (a) a series X-ray structures of complexes between a catalytically inactive GCPII mutant (Glu424Ala) and a panel of naturally occurring polyglutamylated folates; (b) the X-ray structure of the His475Tyr variant at a resolution of 1.83 Å; (c) the study of the recently identified arene-binding site of GCPII through mutagenesis (Arg463Leu, Arg511Leu and Trp541Ala), inhibitor binding and enzyme kinetics with polyglutamylated folates as substrates; and (d) a comparison of the thermal stabilities and folate-hydrolyzing activities of GCPII wild-type and His475Tyr variants. As a result, the crystallographic data reveal considerable details about the binding mode of polyglutamylated folates to GCPII, especially the engagement of the arene binding site in recognizing the folic acid moiety. Additionally, the combined structural and kinetic data suggest that GCPII wild-type and His475Tyr variant are functionally identical.

• Keywords
• H475Y(1561C→T) polymorphism, arene-binding site, crystal structure, folate hydrolase 1, zinc metalloprotease,
• MeSH
• Antigens, Surface chemistry   genetics   MeSH
• Glutamate Carboxypeptidase II chemistry   genetics   MeSH
• Kinetics MeSH
• Crystallography, X-Ray MeSH
• Polyglutamic Acid chemistry   metabolism   MeSH
• Humans MeSH
• Models, Molecular MeSH
• Polymorphism, Genetic MeSH
• Enzyme Stability MeSH
• Binding Sites genetics   MeSH
• Hot Temperature MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Intramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Antigens, Surface MeSH
• FOLH1 protein, human MeSH Browser
• folyl-n-gamma-L-glutamic acid MeSH Browser
• Glutamate Carboxypeptidase II MeSH
• Polyglutamic Acid MeSH