PSMA-617 is recognized as a benchmark ligand for prostate-specific membrane antigen (PSMA) owing to its broad utilization in prostate cancer (PCa) targeted radionuclide therapy. In this study, the structure-activity relationships (SAR) of PSMA-617 and two novel analogs featuring modified linkers were investigated. In compounds P17 and P18, the 2-naphthyl-l-Ala moiety was replaced with a less lipophilic 3-styryl-l-Ala moiety while the cyclohexyl ring in P18 was replaced with a phenyl group. The first ever crystal structure of the PSMA/PSMA-617 complex reported here revealed a folded conformation of the PSMA-617 linker while for the PSMA/P17 and PSMA/P18 complexes, the extended orientations of the linkers revealed linker flexibility within the PSMA cavity, a change in binding that can be exploited for the structure-guided design of PSMA-targeting agents. Despite structural differences from PSMA-617, the analogs maintained high PSMA inhibition potency, cellular binding, and internalization. In vivo biodistribution studies revealed comparable tumor uptake across all three compounds with P18 displaying higher spleen accumulation, likely due to phenyl ring lipophilicity. These SAR findings provide a strategic framework for the rational design of PSMA ligands, paving the way for the development of next-generation theranostic agents for PCa.

• Publication type
• Journal Article 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

INTRODUCTION: N-glycosylation is a ubiquitous and variable posttranslational modification that regulates physiological functions of secretory and membrane-associated proteins and the dysregulation of glycosylation pathways is often associated with cancer growth and metastasis. Prostate-specific membrane antigen (PSMA) is an established biomarker for prostate cancer imaging and therapy. METHODS: Mass spectrometry was used to analyze the distribution of the site-specific glycoforms of PSMA in insect, human embryonic kidney, and prostate cancer cells, and in prostate tissue upon immunoaffinity enrichment. RESULTS: While recombinant PSMA expressed in insect cells was decorated mainly by paucimannose and high mannose glycans, complex, hybrid, and high mannose glycans were detected in samples from human cells and tissue. We noted an interesting spatial distribution of the glycoforms on the PSMA surface-high mannose glycans were the dominant glycoforms at the N459, N476, and N638 sequons facing the plasma membrane, while the N121, N195, and N336 sites, located at the exposed apical PSMA domain, carried primarily complex glycans. The presence of high mannose glycoforms at the former sequons likely results from the limited access of enzymes of the glycosynthetic pathway required for the synthesis of the complex structures. In line with the limited accessibility of membrane-proximal sites, no glycosylation was observed at the N51 site positioned closest to the membrane. CONCLUSIONS: Our study presents initial descriptive analysis of the glycoforms of PSMA observed in cell lines and in prostate tissue. It will hopefully stimulate further research into PSMA glycoforms in the context of tumor staging, noninvasive detection of prostate tumors, and the impact of glycoforms on physicochemical and enzymatic characteristics of PSMA in a tissue-specific manner.

• Keywords
• N-glycosylation, NAALADase I, PSMA, folate hydrolase, glutamate carboxypeptidase II, site-specific glycoform,
• MeSH
• Antigens, Surface metabolism   MeSH
• Cell Line MeSH
• Glutamate Carboxypeptidase II metabolism   MeSH
• Glycosylation MeSH
• Mass Spectrometry methods   MeSH
• Humans MeSH
• Biomarkers, Tumor analysis   MeSH
• Prostatic Neoplasms * metabolism   pathology   MeSH
• Polysaccharides * classification   metabolism   MeSH
• Protein Processing, Post-Translational MeSH
• Prostate * enzymology   metabolism   pathology   MeSH
• Neoplasm Staging 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
• Antigens, Surface MeSH
• FOLH1 protein, human MeSH Browser
• Glutamate Carboxypeptidase II MeSH
• Biomarkers, Tumor MeSH
• Polysaccharides * MeSH

Surgery is an efficient way to treat localized prostate cancer (PCa), however, it is challenging to demarcate rapidly and accurately the tumor boundary intraoperatively, as existing tumor detection methods are seldom performed in real-time. To overcome those limitations, we develop a fluorescent molecular rotor that specifically targets the prostate-specific membrane antigen (PSMA), an established marker for PCa. The probes have picomolar affinity (IC50 = 63-118 pM) for PSMA and generate virtually instantaneous onset of robust fluorescent signal proportional to the concentration of the PSMA-probe complex. In vitro and ex vivo experiments using PCa cell lines and clinical samples, respectively, indicate the utility of the probe for biomedical applications, including real-time monitoring of endocytosis and tumor staging. Experiments performed in a PCa xenograft model reveal suitability of the probe for imaging applications in vivo.

• MeSH
• Antigens, Surface chemistry   metabolism   MeSH
• PC-3 Cells MeSH
• Endocytosis MeSH
• Spectrometry, Fluorescence methods   MeSH
• Glutamate Carboxypeptidase II chemistry   metabolism   MeSH
• Humans MeSH
• Models, Molecular MeSH
• Molecular Probes chemistry   metabolism   MeSH
• Mice, Inbred BALB C MeSH
• Mice, Nude MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Prostatic Neoplasms diagnosis   metabolism   MeSH
• Optical Imaging methods   MeSH
• Protein Domains MeSH
• Transplantation, Heterologous MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Mice 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
• Antigens, Surface MeSH
• FOLH1 protein, human MeSH Browser
• Glutamate Carboxypeptidase II MeSH
• Molecular Probes 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

A series of phosphoramidate-based prostate specific membrane antigen (PSMA) inhibitors of increasing lipophilicity were synthesized (4, 5, and 6), and their fluorine-18 analogs were evaluated for use as positron emission tomography (PET) imaging agents for prostate cancer. To gain insight into their modes of binding, they were also cocrystallized with the extracellular domain of PSMA. All analogs exhibited irreversible binding to PSMA with IC50 values ranging from 0.4 to 1.3 nM. In vitro assays showed binding and rapid internalization (80-95%, 2 h) of the radiolabeled ligands in PSMA(+) cells. In vivo distribution demonstrated significant uptake in CWR22Rv1 (PSMA(+)) tumor, with tumor to blood ratios of 25.6:1, 63.6:1, and 69.6:1 for [(18)F]4, [(18)F]5, and [(18)F]6, respectively, at 2 h postinjection. Installation of aminohexanoic acid (AH) linkers in the phosphoramidate scaffold improved their PSMA binding and inhibition and was critical for achieving suitable in vivo imaging properties, positioning [(18)F]5 and [(18)F]6 as favorable candidates for future prostate cancer imaging clinical trials.

• MeSH
• Amides chemical synthesis   chemistry   pharmacology   MeSH
• Antigens, Surface MeSH
• Neoplasms, Experimental diagnostic imaging   MeSH
• Glutamate Carboxypeptidase II antagonists & inhibitors   MeSH
• Phosphoric Acids chemical synthesis   chemistry   pharmacology   MeSH
• Humans MeSH
• Models, Molecular MeSH
• Molecular Structure MeSH
• Mice, Nude MeSH
• Mice MeSH
• Tumor Cells, Cultured MeSH
• Prostatic Neoplasms diagnostic imaging   MeSH
• Peptidomimetics chemical synthesis   chemistry   pharmacology   MeSH
• Positron-Emission Tomography * MeSH
• Fluorine Radioisotopes MeSH
• Dose-Response Relationship, Drug MeSH
• Structure-Activity Relationship MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Amides MeSH
• Antigens, Surface MeSH
• FOLH1 protein, human MeSH Browser
• Glutamate Carboxypeptidase II MeSH
• Phosphoric Acids MeSH
• Peptidomimetics MeSH
• phosphoramidic acid MeSH Browser
• Fluorine Radioisotopes MeSH

UNLABELLED: Inhibitors targeting human glutamate carboxypeptidase II (GCPII) typically consist of a P1' glutamate-derived binding module, which warrants the high affinity and specificity, linked to an effector function that is positioned within the entrance funnel of the enzyme. Here we present a comprehensive structural and computational study aimed at dissecting the importance of the effector function for GCPII binding and affinity. To this end we determined crystal structures of human GCPII in complex with a series of phosphoramidate-based inhibitors harboring effector functions of diverse physicochemical characteristics. Our data show that higher binding affinities of phosphoramidates, compared to matching phosphonates, are linked to the presence of additional hydrogen bonds between Glu424 and Gly518 of the enzyme and the amide group of the phosphoramidate. While the positioning of the P1' glutamate-derived module within the S1' pocket of GCPII is invariant, interaction interfaces between effector functions and residues lining the entrance funnel are highly varied, with the positively charged arginine patch defined by Arg463, Arg534 and Arg536 being the only 'hot-spot' common to several studied complexes. This variability stems in part from the fact that the effector/GCPII interfaces generally encompass isolated areas of nonpolar residues within the entrance funnel and resulting van der Waals contacts lack the directionality typical for hydrogen bonding interactions. The presented data unravel a complexity of binding modes of inhibitors within non-prime site(s) of GCPII and can be exploited for the design of novel GCPII-specific compounds. PDB ID CODES: Atomic coordinates of the present structures together with the experimental structure factor amplitudes were deposited at the RCSB Protein Data Bank under accession codes 4P44 (complex with JRB-4-81), 4P45 (complex with JRB-4-73), 4P4B (complex with CTT54), 4P4D (complex with MP1C), 4P4E (complex with MP1D), 4P4F (complex with NC-2-40), 4P4I (complex with T33) and 4P4J (complex with T33D).

• Keywords
• NAALADase, X-ray crystallography, molecular modeling, phosphoramidate, prostate-specific membrane antigen,
• MeSH
• Amides chemical synthesis   chemistry   pharmacology   MeSH
• Antigens, Surface metabolism   MeSH
• Glutamate Carboxypeptidase II antagonists & inhibitors   metabolism   MeSH
• Enzyme Inhibitors chemical synthesis   chemistry   pharmacology   MeSH
• Crystallography, X-Ray MeSH
• Phosphoric Acids chemical synthesis   chemistry   pharmacology   MeSH
• Humans MeSH
• Models, Molecular MeSH
• Molecular Structure MeSH
• Drug Design * MeSH
• Hydrogen Bonding MeSH
• Dose-Response Relationship, Drug MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Amides MeSH
• Antigens, Surface MeSH
• FOLH1 protein, human MeSH Browser
• Glutamate Carboxypeptidase II MeSH
• Enzyme Inhibitors MeSH
• Phosphoric Acids MeSH
• phosphoramidic acid MeSH Browser

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