Glutamate carboxypeptidase II (GCPII) is a metalloprotease implicated in neurological diseases and prostate oncology. While several classes of potent GCPII-specific inhibitors exist, the development of novel active scaffolds with different pharmacological profiles remains a challenge. Virtual screening followed by in vitro testing is an effective means for the discovery of novel active compounds. Structure- and ligand-based pharmacophore models were created based on a dataset of known GCPII-selective ligands. These models were used in a virtual screening of the SPECS compound library (∼209.000 compounds). Fifty top-scoring virtual hits were further experimentally tested for their ability to inhibit GCPII enzymatic activity in vitro. Six hits were found to have moderate to high inhibitory potency with the best virtual hit, a modified xanthene, inhibiting GCPII with an IC50 value of 353 ± 24 nM. The identification of this novel inhibitory scaffold illustrates the applicability of pharmacophore-based modeling for the discovery of GCPII-specific inhibitors.

• MeSH
• Glutamate Carboxypeptidase II * MeSH
• Humans MeSH
• Ligands MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Glutamate Carboxypeptidase II * MeSH
• Ligands 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

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

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