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
• glutamátkarboxypeptidasa II * MeSH
• lidé MeSH
• ligandy MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• glutamátkarboxypeptidasa II * MeSH
• ligandy 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.

• Klíčová slova
• Fasciola hepatica, Folate hydrolase, Immunohistochemistry, M28B metalloproteases, NAALADase, Platyhelminth, Prostate specific-membrane antigen, RNA in situ hybridization, Schistosoma mansoni,
• MeSH
• Fasciola hepatica * genetika   MeSH
• myši MeSH
• proteasy MeSH
• savci MeSH
• Schistosoma mansoni MeSH
• Trematoda * genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• glutamate carboxypeptidase MeSH Prohlížeč
• proteasy MeSH

Peptide display methods are a powerful tool for discovering new ligands of pharmacologically relevant targets. However, the selected ligands often suffer from low affinity. Using phage display, we identified a new bicyclic peptide binder of prostate-specific membrane antigen (PSMA), a metalloprotease frequently overexpressed in prostate cancer. We show that linking multiple copies of a selected low-affinity peptide to a biocompatible water-soluble N-(2-hydroxypropyl)methacrylamide copolymer carrier (iBody) improved binding of the conjugate by several orders of magnitude. Furthermore, using ELISA, enzyme kinetics, confocal microscopy, and other approaches, we demonstrate that the resulting iBody can distinguish between different conformations of the target protein. The possibility to develop stable, fully synthetic, conformation-selective antibody mimetics has potential applications for molecular recognition, diagnosis and treatment of many pathologies. This strategy could significantly contribute to more effective drug discovery and design.

• Klíčová slova
• HPMA copolymer, PSMA, antibody mimetics, bicyclic phage display, chemical biology, cyclic peptide, molecular recognition, nanotechnology, phage display, protein targeting,
• MeSH
• biomimetické materiály chemie   MeSH
• kalikreiny chemie   MeSH
• lidé MeSH
• nosiče léků chemie   MeSH
• peptidová knihovna * MeSH
• prostatický specifický antigen chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kalikreiny MeSH
• KLK3 protein, human MeSH Prohlížeč
• nosiče léků MeSH
• peptidová knihovna * MeSH
• prostatický specifický antigen 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.

• Klíčová slova
• glutamate carboxypeptidase II, mouse animal model, neuronal disorders, prostate cancer, prostate‐specific membrane antigen,
• Publikační typ
• časopisecké články 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
• biotest * MeSH
• DNA * MeSH
• enzymy metabolismus   MeSH
• inhibitory enzymů farmakologie   MeSH
• lidé MeSH
• objevování léků * MeSH
• reprodukovatelnost výsledků MeSH
• senzitivita a specificita MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA * MeSH
• enzymy MeSH
• inhibitory enzymů MeSH

Antibodies are indispensable tools for biomedicine and anticancer therapy. Nevertheless, their use is compromised by high production costs, limited stability, and difficulty of chemical modification. The design and preparation of synthetic polymer conjugates capable of replacing antibodies in biomedical applications such as ELISA, flow cytometry, immunocytochemistry, and immunoprecipitation is reported. The conjugates, named "iBodies", consist of an HPMA copolymer decorated with low-molecular-weight compounds that function as targeting ligands, affinity anchors, and imaging probes. We prepared specific conjugates targeting several proteins with known ligands and used these iBodies for enzyme inhibition, protein isolation, immobilization, quantification, and live-cell imaging. Our data indicate that this highly modular and versatile polymer system can be used to produce inexpensive and stable antibody substitutes directed toward virtually any protein of interest with a known ligand.

• Klíčová slova
• HPMA, antibody mimetics, molecular recognition, polymer conjugates, protein targeting,
• MeSH
• hydrofobní a hydrofilní interakce MeSH
• lidé MeSH
• molekulární mimikry * MeSH
• nádorové buněčné linie MeSH
• polymery chemie   MeSH
• protilátky chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• polymery MeSH
• protilátky MeSH