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

N-acetylated α-linked acidic dipeptidase-like protein (NAALADase L), encoded by the NAALADL1 gene, is a close homolog of glutamate carboxypeptidase II, a metallopeptidase that has been intensively studied as a target for imaging and therapy of solid malignancies and neuropathologies. However, neither the physiological functions nor structural features of NAALADase L are known at present. Here, we report a thorough characterization of the protein product of the human NAALADL1 gene, including heterologous overexpression and purification, structural and biochemical characterization, and analysis of its expression profile. By solving the NAALADase L x-ray structure, we provide the first experimental evidence that it is a zinc-dependent metallopeptidase with a catalytic mechanism similar to that of glutamate carboxypeptidase II yet distinct substrate specificity. A proteome-based assay revealed that the NAALADL1 gene product possesses previously unrecognized aminopeptidase activity but no carboxy- or endopeptidase activity. These findings were corroborated by site-directed mutagenesis and identification of bestatin as a potent inhibitor of the enzyme. Analysis of NAALADL1 gene expression at both the mRNA and protein levels revealed the small intestine as the major site of protein expression and points toward extensive alternative splicing of the NAALADL1 gene transcript. Taken together, our data imply that the NAALADL1 gene product's primary physiological function is associated with the final stages of protein/peptide digestion and absorption in the human digestive system. Based on these results, we suggest a new name for this enzyme: human ileal aminopeptidase (HILAP).

• Keywords
• Aminopeptidase, DPP IV Activity, Human Ileal Aminopeptidase, Intestinal Metabolism, Metalloprotease, Molecular Evolution, PICS, Protein Degradation, X-ray Crystallography,
• MeSH
• Dipeptidyl Peptidase 4 metabolism   MeSH
• Endopeptidases metabolism   MeSH
• Glutamate Carboxypeptidase II chemistry   genetics   metabolism   MeSH
• Rats MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Models, Molecular MeSH
• Molecular Sequence Data MeSH
• Gene Expression Regulation, Enzymologic MeSH
• Amino Acid Sequence MeSH
• Intestines enzymology   MeSH
• Protein Structure, Tertiary MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Intramural MeSH
• Names of Substances
• Dipeptidyl Peptidase 4 MeSH
• Endopeptidases MeSH
• Glutamate Carboxypeptidase II 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

Urea-based inhibitors of human glutamate carboxypeptidase II (GCPII) have advanced into clinical trials for imaging metastatic prostate cancer. In parallel efforts, agents with increased lipophilicity have been designed and evaluated for targeting GCPII residing within the neuraxis. Here we report the structural and computational characterization of six complexes between GCPII and P1'-diversified urea-based inhibitors that have the C-terminal glutamate replaced by more hydrophobic moieties. The X-ray structures are complemented by quantum mechanics calculations that provide a quantitative insight into the GCPII/inhibitor interactions. These data can be used for the rational design of novel glutamate-free GCPII inhibitors with tailored physicochemical properties.

• Keywords
• GCPII, Metallopeptidase, PSMA, Prostate-specific membrane antigen, Structure-based drug design, Urea-based inhibitor, X-ray crystallography,
• MeSH
• Antigens, Surface chemistry   MeSH
• Glutamate Carboxypeptidase II antagonists & inhibitors   chemistry   MeSH
• Enzyme Inhibitors chemistry   pharmacology   MeSH
• Kinetics MeSH
• Protein Conformation MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Urea analogs & derivatives   chemistry   pharmacology   MeSH
• Molecular Conformation MeSH
• Models, Molecular MeSH
• Drug Design MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Antigens, Surface MeSH
• FOLH1 protein, human MeSH Browser
• Glutamate Carboxypeptidase II MeSH
• Enzyme Inhibitors MeSH
• Urea MeSH

Glutamate carboxypeptidase II (GCPII) is an important target for therapeutic and diagnostic interventions aimed at prostate cancer and neurologic disorders. Here we describe the development and optimization of a high-throughput screening (HTS) assay based on fluorescence polarization (FP) that facilitates the identification of novel scaffolds inhibiting GCPII. First, we designed and synthesized a fluorescence probe based on a urea-based inhibitory scaffold covalently linked to a Bodipy TMR fluorophore (TMRGlu). Next, we established and optimized conditions suitable for HTS and evaluated the assay robustness by testing the influence of a variety of physicochemical parameters (e.g., pH, temperature, time) and additives. Using known GCPII inhibitors, the FP assay was shown to be comparable to benchmark assays established in the field. Finally, we evaluated the FP assay by HTS of a 20 000-compound library. The novel assay presented here is robust, highly reproducible (Z' = 0.82), inexpensive, and suitable for automation, thus providing an excellent platform for HTS of small-molecule libraries targeting GCPII.

• MeSH
• Antigens, Surface genetics   metabolism   MeSH
• Fluorescent Dyes chemical synthesis   MeSH
• Fluorescence Polarization methods   MeSH
• Glutamate Carboxypeptidase II antagonists & inhibitors   genetics   metabolism   MeSH
• Small Molecule Libraries pharmacology   MeSH
• Humans MeSH
• Ligands MeSH
• High-Throughput Screening Assays methods   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Antigens, Surface MeSH
• Fluorescent Dyes MeSH
• FOLH1 protein, human MeSH Browser
• Glutamate Carboxypeptidase II MeSH
• Small Molecule Libraries MeSH
• Ligands MeSH

Glutamate carboxypeptidase II (GCPII) is a membrane-bound binuclear zinc metallopeptidase with the highest expression levels found in the nervous and prostatic tissue. Throughout the nervous system, glia-bound GCPII is intimately involved in the neuron-neuron and neuron-glia signaling via the hydrolysis of N-acetylaspartylglutamate (NAAG), the most abundant mammalian peptidic neurotransmitter. The inhibition of the GCPII-controlled NAAG catabolism has been shown to attenuate neurotoxicity associated with enhanced glutamate transmission and GCPII-specific inhibitors demonstrate efficacy in multiple preclinical models including traumatic brain injury, stroke, neuropathic and inflammatory pain, amyotrophic lateral sclerosis, and schizophrenia. The second major area of pharmacological interventions targeting GCPII focuses on prostate carcinoma; GCPII expression levels are highly increased in androgen-independent and metastatic disease. Consequently, the enzyme serves as a potential target for imaging and therapy. This review offers a summary of GCPII structure, physiological functions in healthy tissues, and its association with various pathologies. The review also outlines the development of GCPII-specific small-molecule compounds and their use in preclinical and clinical settings.

• MeSH
• Glutamate Carboxypeptidase II antagonists & inhibitors   metabolism   MeSH
• Humans MeSH
• Prostatic Neoplasms diagnosis   drug therapy   metabolism   MeSH
• Nervous System Diseases diagnosis   drug therapy   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Glutamate Carboxypeptidase II MeSH

Affinity purification is a useful approach for purification of recombinant proteins. Eukaryotic expression systems have become more frequently used at the expense of prokaryotic systems since they afford recombinant eukaryotic proteins with post-translational modifications similar or identical to the native ones. Here, we present a one-step affinity purification set-up suitable for the purification of secreted proteins. The set-up is based on the interaction between biotin and mutated streptavidin. Drosophila Schneider 2 cells are chosen as the expression host, and a biotin acceptor peptide is used as an affinity tag. This tag is biotinylated by Escherichia coli biotin-protein ligase in vivo. We determined that localization of the ligase within the ER led to the most effective in vivo biotinylation of the secreted proteins. We optimized a protocol for large-scale expression and purification of AviTEV-tagged recombinant human glutamate carboxypeptidase II (Avi-GCPII) with milligram yields per liter of culture. We also determined the 3D structure of Avi-GCPII by X-ray crystallography and compared the enzymatic characteristics of the protein to those of its non-tagged variant. These experiments confirmed that AviTEV tag does not affect the biophysical properties of its fused partner. Purification approach, developed here, provides not only a sufficient amount of highly homogenous protein but also specifically and effectively biotinylates a target protein and thus enables its subsequent visualization or immobilization.

• MeSH
• Antigens, Surface chemistry   genetics   isolation & purification   metabolism   MeSH
• Biotin chemistry   metabolism   MeSH
• Biotinylation MeSH
• Cell Line MeSH
• Drosophila cytology   MeSH
• Escherichia coli enzymology   genetics   MeSH
• Gene Expression MeSH
• Glutamate Carboxypeptidase II chemistry   genetics   isolation & purification   metabolism   MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Models, Molecular MeSH
• Molecular Sequence Data MeSH
• Recombinant Proteins chemistry   genetics   isolation & purification   metabolism   MeSH
• Amino Acid Sequence 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., Intramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Antigens, Surface MeSH
• Biotin MeSH
• FOLH1 protein, human MeSH Browser
• Glutamate Carboxypeptidase II MeSH
• Recombinant Proteins MeSH

Virtually all low molecular weight inhibitors of human glutamate carboxypeptidase II (GCPII) are highly polar compounds that have limited use in settings where more lipophilic molecules are desired. Here we report the identification and characterization of GCPII inhibitors with enhanced liphophilicity that are derived from a series of newly identified dipeptidic GCPII substrates featuring nonpolar aliphatic side chains at the C-terminus. To analyze the interactions governing the substrate recognition by GCPII, we determined crystal structures of the inactive GCPII(E424A) mutant in complex with selected dipeptides and complemented the structural data with quantum mechanics/molecular mechanics calculations. Results reveal the importance of nonpolar interactions governing GCPII affinity toward novel substrates as well as formerly unnoticed plasticity of the S1' specificity pocket. On the basis of those data, we designed, synthesized, and evaluated a series of novel GCPII inhibitors with enhanced lipophilicity, with the best candidates having low nanomolar inhibition constants and clogD > -0.3. Our findings offer new insights into the design of more lipophilic inhibitors targeting GCPII.

• MeSH
• Antigens, Surface genetics   MeSH
• Dipeptides chemical synthesis   chemistry   pharmacology   MeSH
• Glutamate Carboxypeptidase II antagonists & inhibitors   genetics   MeSH
• Kinetics MeSH
• Protein Conformation MeSH
• Crystallography, X-Ray MeSH
• Quantum Theory MeSH
• Humans MeSH
• Ligands MeSH
• Models, Molecular MeSH
• Mutagenesis, Site-Directed MeSH
• Substrate Specificity MeSH
• Thermodynamics MeSH
• Binding Sites MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, N.I.H., Intramural MeSH
• Names of Substances
• Antigens, Surface MeSH
• Dipeptides MeSH
• FOLH1 protein, human MeSH Browser
• Glutamate Carboxypeptidase II MeSH
• Ligands MeSH