The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Barbara S. Slusher - One of the best experts on this subject based on the ideXlab platform.
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Glutamate Carboxypeptidase II Inhibition Behaviorally and Physiologically Improves Pyridoxine-Induced Neuropathy in Rats
2016Co-Authors: Michelle C. Potter, Krystyna M Wozniak, Noëlle Callizot, Barbara S. SlusherAbstract:Pyridoxine is used as a supplement for treating conditions such as vitamin deficiency as well as neurological disorders such as depression, epilepsy and autism. A significant neurologic complication of pyridoxine therapy is peripheral neuropathy thought to be a result of long-term and high dose usage. Although pyridoxine-induced neuropathy is transient and can remit after its withdrawal, the process of complete recovery can be slow. Glutamate Carboxypeptidase II (GCP II) inhibition has been shown to improve symptoms of both chemotherapy- and diabetic-induced neuropathy. This study evaluated if GCP II inhibition could behaviorally and physiologically improve pyridoxine-induced neuropathy. In the current study, high doses of pyridoxine (400 mg/kg, twice a day for seven days) were used to induce neuropathy in rats. An orally bioavailable GCP II inhibitor, 2-(3-mercaptopropyl) pentanedioic acid (2-MPPA), was administered daily at a dose of 30 mg/kg starting from the onset of pyridoxine injections. Body weight, motor coordination, heat sensitivity, electromyographical (EMG) parameters and nerve morphological features were monitored. The results show beneficial effects of GCP II inhibition including normalization of hot plate reaction time, foot fault improvements and increased open field distance travelled. H wave frequency, amplitude and latency as well as sensory nerve conduction velocity (SNCV) were also significantly improved by 2-MPPA. Lastly, GCP II inhibition resulted in morphological protection in the spinal cord and sensory fibers in the lumbar region dorsal root ganglia (DRG). In conclusion, inhibition of GCP II may be beneficial against the periphera
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Glutamate Carboxypeptidase II Inhibition Behaviorally and Physiologically Improves Pyridoxine-Induced Neuropathy in Rats
PLoS ONE, 2014Co-Authors: Michelle C. Potter, Krystyna M Wozniak, Noëlle Callizot, Barbara S. SlusherAbstract:Pyridoxine is used as a supplement for treating conditions such as vitamin deficiency as well as neurological disorders such as depression, epilepsy and autism. A significant neurologic complication of pyridoxine therapy is peripheral neuropathy thought to be a result of long-term and high dose usage. Although pyridoxine-induced neuropathy is transient and can remit after its withdrawal, the process of complete recovery can be slow. Glutamate Carboxypeptidase II (GCP II) inhibition has been shown to improve symptoms of both chemotherapy- and diabetic-induced neuropathy. This study evaluated if GCP II inhibition could behaviorally and physiologically improve pyridoxine-induced neuropathy. In the current study, high doses of pyridoxine (400 mg/kg, twice a day for seven days) were used to induce neuropathy in rats. An orally bioavailable GCP II inhibitor, 2-(3-mercaptopropyl) pentanedioic acid (2-MPPA), was administered daily at a dose of 30 mg/kg starting from the onset of pyridoxine injections. Body weight, motor coordination, heat sensitivity, electromyographical (EMG) parameters and nerve morphological features were monitored. The results show beneficial effects of GCP II inhibition including normalization of hot plate reaction time, foot fault improvements and increased open field distance travelled. H wave frequency, amplitude and latency as well as sensory nerve conduction velocity (SNCV) were also significantly improved by 2-MPPA. Lastly, GCP II inhibition resulted in morphological protection in the spinal cord and sensory fibers in the lumbar region dorsal root ganglia (DRG). In conclusion, inhibition of GCP II may be beneficial against the peripheral sensory neuropathy caused by pyridoxine.
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δ-Thiolactones as Prodrugs of Thiol-Based Glutamate Carboxypeptidase II (GCPII) Inhibitors
Journal of Medicinal Chemistry, 2013Co-Authors: Dana Ferraris, Pavel Majer, Chiyou Ni, C. Ethan Slusher, Krystyna M Wozniak, Rana Rais, Ying Wu, Camilo Rojas, Barbara S. SlusherAbstract:δ-Thiolactones derived from thiol-based Glutamate Carboxypeptidase II (GCPII) inhibitors were evaluated as prodrugs. In rat liver microsomes, 2-(3-mercaptopropyl)pentanedioic acid (2-MPPA, 1) was gradually produced from 3-(2-oxotetrahydrothiopyran-3-yl)propionic acid (5), a thiolactone derived from 1. Compound 1 was detected in plasma at concentrations well above its IC50 for GCPII following oral administration of 5 in rats. Consistent with the oral plasma pharmacokinetics, thiolactone 5 exhibited efficacy in a rat model of neuropathic pain following oral administration.
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Glutamate Carboxypeptidase ii is not an amyloid peptide degrading enzyme
The FASEB Journal, 2013Co-Authors: Jesse Alt, Marigo Stathis, Camilo Rojas, Barbara S. SlusherAbstract:Glutamate Carboxypeptidase II (GCPII) is an exopeptidase that catalyzes the hydrolysis of N-acetylated aspartate-Glutamate (NAAG) to N-acetyl aspartate (NAA) and Glutamate. Consequently, GCPII inhibition has been of interest for the treatment of central and peripheral nervous system diseases associated with excess Glutamate. Recently, it was reported that GCPII can also serve as an endopeptidase cleaving amyloid β (Aβ) peptides and that its inhibition could increase the risk of Alzheimer's disease by increasing brain Aβ levels. This study aimed to corroborate and extend these new findings. We incubated Aβ peptides (20 μM) with human recombinant GCPII (300 ng/ml) and monitored the appearance of degradation products by mass spectrometry. Aβ peptides remained intact after 18 h incubation with GCPII. Under the same experimental conditions, Aβ1-40 (20 μM) was incubated with neprilysin (300 ng/ml), an endopeptidase known to hydrolyze Aβ1-40 and the expected cleavage products were observed. GCPII was confirmed active by catalyzing the complete hydrolysis of NAAG (100 μM). We also studied the hydrolysis of [(3)H]-NAAG (30 nM) catalyzed by GCPII (40 pM) in the presence of Aβ peptides (picomolar to micromolar range). The addition of Aβ peptides did not alter [(3)H]-NAAG hydrolysis. We conclude that GCPII is not an amyloid peptide-degrading enzyme.
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Glutamate Carboxypeptidase ii
Handbook of Proteolytic Enzymes, 2013Co-Authors: Barbara S. Slusher, Camilo Rojas, Joseph T CoyleAbstract:Publisher Summary This chapter examines the structural chemistry and the biological aspects of Glutamate Carboxypeptidase II. This enzyme was first characterized in the rat nervous system by its hydrolysis of the neuropeptide N-acetylaspartylGlutamate and was termed N-acetylated-alpha-linked acidic dipeptidase. The prostate cancer marker known as the prostate-specific membrane antigen also possesses NAALADase activity. Subcellular fractionation of brain reveals enrichment of the enzyme in neurosynaptosomal complexes. Electron microscopy localizes Glutamate Carboxypeptidase II immunoreactivity to the outer surface of the plasma membrane. The results indicate that the rat enzyme is expressed exclusively in astrocytic glial cells. Prostate-Specific Membrane (PSM) was characterized originally as the ligand of the monoclonal antibody 7E11.C5, which exhibited immunohistochemical staining restricted among human tissues to the prostate epithelium. The analyses have reported 7E11.C5 immunoreactivity and/or positive RNAase protection assays with PSM-derived probes in human salivary gland, brain, small intestine, seminal plasma and blood serum. Two gene loci closely related to PSM have been identified on human chromosome 11. The PSM form of Glutamate Carboxypeptidase II is expressed in normal and neoplastic prostate epithelial cells, and PSM has been examined for utility in the diagnosis, assessment and treatment of prostatic carcinoma.
Pavel Majer - One of the best experts on this subject based on the ideXlab platform.
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Discovery of Orally Available Prodrugs of the Glutamate Carboxypeptidase II (GCPII) Inhibitor 2-Phosphonomethylpentanedioic Acid (2-PMPA)
Journal of Medicinal Chemistry, 2016Co-Authors: Pavel Majer, Krystyna M Wozniak, Dana Ferraris, Andrej Jančařík, Marcela Krečmerová, Tomáš Tichý, Lukáš Tenora, Elie Pommier, Rana RaisAbstract:2-Phosphonomethylpentanedioic acid (1, 2-PMPA) is a potent inhibitor of Glutamate Carboxypeptidase II which has demonstrated robust neuroprotective efficacy in many neurological disease models. However, 1 is highly polar containing a phosphonate and two carboxylates, severely limiting its oral bioavailability. We strategized to mask the polar groups via a prodrug approach, increasing the likelihood of passive oral absorption. Our initial strategy was to cover the phosphonate with hydrophobic moieties such as pivaloyloxymethyl (POM) and isopropyloxycarbonyloxymethyl (POC) while keeping the α- and γ-carboxylates unsubstituted. This attempt was unsuccessful due to the chemical instability of the bis-POC/POM derivatives. Addition of α,γ-diesters and α-monoesters enhanced chemical stability and provided excellent oral exposure in mice, but these mixed esters were too stable in vivo, resulting in minimal release of 1. By introducing POC groups on both the phosphonate and α-carboxylate, we synthesized Tris-POC-2...
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Discovery of Orally Available Prodrugs of the Glutamate Carboxypeptidase II (GCPII) Inhibitor 2‑Phosphonomethylpentanedioic Acid (2-PMPA)
2016Co-Authors: Pavel Majer, Dana Ferraris, Andrej Jančařík, Krystyna Wozniak, Lukáš Tenora, Elie Pommier, Marcela Krečmerová, Tomáš Tichý, Rana RaisAbstract:2-Phosphonomethylpentanedioic acid (1, 2-PMPA) is a potent inhibitor of Glutamate Carboxypeptidase II which has demonstrated robust neuroprotective efficacy in many neurological disease models. However, 1 is highly polar containing a phosphonate and two carboxylates, severely limiting its oral bioavailability. We strategized to mask the polar groups via a prodrug approach, increasing the likelihood of passive oral absorption. Our initial strategy was to cover the phosphonate with hydrophobic moieties such as pivaloyloxymethyl (POM) and isopropyloxycarbonyloxymethyl (POC) while keeping the α- and γ-carboxylates unsubstituted. This attempt was unsuccessful due to the chemical instability of the bis-POC/POM derivatives. Addition of α,γ-diesters and α-monoesters enhanced chemical stability and provided excellent oral exposure in mice, but these mixed esters were too stable in vivo, resulting in minimal release of 1. By introducing POC groups on both the phosphonate and α-carboxylate, we synthesized Tris-POC-2-PMPA (21b), which afforded excellent release of 1 following oral administration in both mice and dog
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Design of highly potent urea-based, exosite-binding inhibitors selective for Glutamate Carboxypeptidase II.
Journal of Medicinal Chemistry, 2015Co-Authors: Jan Tykvart, Pavel Majer, J. Konvalinka, Jiří Schimer, Andrej Jančařík, Jitka Bařinková, Václav Navrátil, Jana Starková, Karolína Šrámková, Pavel SachaAbstract:We present here a structure-aided design of inhibitors targeting the active site as well as exosites of Glutamate Carboxypeptidase II (GCPII), a prostate cancer marker, preparing potent and selective inhibitors that are more than 1000-fold more active toward GCPII than its closest human homologue, Glutamate Carboxypeptidase III (GCPIII). Additionally, we demonstrate that the prepared inhibitor conjugate can be used for sensitive and selective imaging of GCPII in mammalian cells.
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Design of Highly Potent Urea-Based, Exosite-Binding Inhibitors Selective for Glutamate Carboxypeptidase II
2015Co-Authors: Jan Tykvart, Pavel Majer, J. Konvalinka, Jiří Schimer, Andrej Jančařík, Jitka Bařinková, Václav Navrátil, Jana Starková, Karolína Šrámková, Pavel ŠáchaAbstract:We present here a structure-aided design of inhibitors targeting the active site as well as exosites of Glutamate Carboxypeptidase II (GCPII), a prostate cancer marker, preparing potent and selective inhibitors that are more than 1000-fold more active toward GCPII than its closest human homologue, Glutamate Carboxypeptidase III (GCPIII). Additionally, we demonstrate that the prepared inhibitor conjugate can be used for sensitive and selective imaging of GCPII in mammalian cells
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Rational design of urea-based Glutamate Carboxypeptidase II (GCPII) inhibitors as versatile tools for specific drug targeting and delivery
Bioorganic & Medicinal Chemistry, 2014Co-Authors: Jan Tykvart, Pavel Majer, J. Konvalinka, Jiří Schimer, Jitka Bařinková, Petr Pachl, Lenka Poštová-slavětínská, Pavel SachaAbstract:Glutamate Carboxypeptidase II (GCPII), also known as prostate specific membrane antigen (PSMA), is an established prostate cancer marker and is considered a promising target for specific anticancer drug delivery. Low-molecular-weight inhibitors of GCPII are advantageous specific ligands for this purpose. However, they must be modified with a linker to enable connection of the ligand with an imaging molecule, anticancer drug, and/or nanocarrier. Here, we describe a structure-activity relationship (SAR) study of GCPII inhibitors with linkers suitable for imaging and drug delivery. Structure-assisted inhibitor design and targeting of a specific GCPII exosite resulted in a 7-fold improvement in Ki value compared to the parent structure. X-ray structural analysis of the inhibitor series led to the identification of several inhibitor binding modes. We also optimized the length of the inhibitor linker for effective attachment to a biotin-binding molecule and showed that the optimized inhibitor could be used to target nanoparticles to cells expressing GCPII.
J. Konvalinka - One of the best experts on this subject based on the ideXlab platform.
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the calcium binding site of human Glutamate Carboxypeptidase ii is critical for dimerization thermal stability and enzymatic activity
Protein Science, 2018Co-Authors: Jakub Ptacek, Barbora Havlinova, J. Konvalinka, Michal Navrátil, Jana Nedvedova, Cyril BarinkaAbstract: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.
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Design of highly potent urea-based, exosite-binding inhibitors selective for Glutamate Carboxypeptidase II.
Journal of Medicinal Chemistry, 2015Co-Authors: Jan Tykvart, Pavel Majer, J. Konvalinka, Jiří Schimer, Andrej Jančařík, Jitka Bařinková, Václav Navrátil, Jana Starková, Karolína Šrámková, Pavel SachaAbstract:We present here a structure-aided design of inhibitors targeting the active site as well as exosites of Glutamate Carboxypeptidase II (GCPII), a prostate cancer marker, preparing potent and selective inhibitors that are more than 1000-fold more active toward GCPII than its closest human homologue, Glutamate Carboxypeptidase III (GCPIII). Additionally, we demonstrate that the prepared inhibitor conjugate can be used for sensitive and selective imaging of GCPII in mammalian cells.
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Design of Highly Potent Urea-Based, Exosite-Binding Inhibitors Selective for Glutamate Carboxypeptidase II
2015Co-Authors: Jan Tykvart, Pavel Majer, J. Konvalinka, Jiří Schimer, Andrej Jančařík, Jitka Bařinková, Václav Navrátil, Jana Starková, Karolína Šrámková, Pavel ŠáchaAbstract:We present here a structure-aided design of inhibitors targeting the active site as well as exosites of Glutamate Carboxypeptidase II (GCPII), a prostate cancer marker, preparing potent and selective inhibitors that are more than 1000-fold more active toward GCPII than its closest human homologue, Glutamate Carboxypeptidase III (GCPIII). Additionally, we demonstrate that the prepared inhibitor conjugate can be used for sensitive and selective imaging of GCPII in mammalian cells
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Detection and quantitation of Glutamate Carboxypeptidase II in human blood
The Prostate, 2014Co-Authors: Tomáš Knedlík, Pavel Sacha, Václav Navrátil, Viktor Vik, Dalibor Pacík, J. KonvalinkaAbstract:BACKGROUND Glutamate Carboxypeptidase II (GCPII) is a transmembrane enzyme that cleaves N-acetyl-L-aspartyl-L-Glutamate (NAAG) in the brain. GCPII is highly expressed in the prostate and prostate cancer and might be associated with prostate cancer progression. Another exopeptidase, plasma Glutamate Carboxypeptidase (PGCP), was reported to be similar to GCPII and to share its NAAG-hydrolyzing activity. METHODS We performed a radioenzymatic assay with [H-3]NAAG as a substrate to detect and quantify the enzymatic activity of GCPII in plasma. Using a specific antibody raised against native GCPII (2G7), we immunoprecipitated GCPII from human plasma. We also cloned two PGCP constructs, expressed them in insect cells, and tested them for their NAAG-hydrolyzing activity. RESULTS We detected GCPII protein in human plasma and found that its concentration ranges between 1.3 and 17.2 ng/ml in volunteers not diagnosed with prostate cancer. Recombinant PGCP was enzymatically active but exhibited no NAAG-hydrolyzing activity. CONCLUSION GCPII is present in human blood, and its concentration within a healthy population varies. Recombinant PGCP does not hydrolyze NAAG, suggesting that GCPII alone is responsible for the NAAG-hydrolyzing activity observed in human blood. The potential correlation between GCPII serum levels and the disease status of prostate cancer patients will be further investigated.
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Rational design of urea-based Glutamate Carboxypeptidase II (GCPII) inhibitors as versatile tools for specific drug targeting and delivery
Bioorganic & Medicinal Chemistry, 2014Co-Authors: Jan Tykvart, Pavel Majer, J. Konvalinka, Jiří Schimer, Jitka Bařinková, Petr Pachl, Lenka Poštová-slavětínská, Pavel SachaAbstract:Glutamate Carboxypeptidase II (GCPII), also known as prostate specific membrane antigen (PSMA), is an established prostate cancer marker and is considered a promising target for specific anticancer drug delivery. Low-molecular-weight inhibitors of GCPII are advantageous specific ligands for this purpose. However, they must be modified with a linker to enable connection of the ligand with an imaging molecule, anticancer drug, and/or nanocarrier. Here, we describe a structure-activity relationship (SAR) study of GCPII inhibitors with linkers suitable for imaging and drug delivery. Structure-assisted inhibitor design and targeting of a specific GCPII exosite resulted in a 7-fold improvement in Ki value compared to the parent structure. X-ray structural analysis of the inhibitor series led to the identification of several inhibitor binding modes. We also optimized the length of the inhibitor linker for effective attachment to a biotin-binding molecule and showed that the optimized inhibitor could be used to target nanoparticles to cells expressing GCPII.
Takashi Tsukamoto - One of the best experts on this subject based on the ideXlab platform.
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Structure-activity relationships of Glutamate Carboxypeptidase II (GCPII) inhibitors.
Current Medicinal Chemistry, 2012Co-Authors: Dana Ferraris, Krupa Shukla, Takashi TsukamotoAbstract:Glutamate Carboxypeptidase II (GCPII, EC 3.4.17.21) is a zinc metallopeptidase that hydrolyzes N-acetylaspartylGlutamate (NAAG) into N-acetylaspartate (NAA) and Glutamate in the nervous system. Inhibition of GCPII has the potential to reduce extracellular Glutamate and represents an opportune target for treating neurological disorders in which excess Glutamate is considered pathogenic. Furthermore, GCPII was found to be identical to a tumor marker, prostate-specific membrane antigen (PSMA), and has drawn significant interest as a diagnostic and/or therapeutic target in oncology. Over the past 15 years, tremendous efforts have been made in the discovery of potent GCPII inhibitors, particularly those with phosphorus-, urea- and thiol-based zinc binding groups. In addition, significant progress has been made in understanding the three-dimensional structural characteristics of GCPII in complex with various ligands. The purpose of this review article is to analyze the structure-activity relationships (SAR) of GCPII inhibitors reported to date, which are classified on the basis of their zinc-binding group. SAR and crystallographic data are evaluated in detail for each of these series to highlight the future challenges and opportunities to identify clinically viable GCPII inhibitors.
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orally active Glutamate Carboxypeptidase ii inhibitor 2 mppa attenuates dizocilpine induced prepulse inhibition deficits in mice
Brain Research, 2011Co-Authors: Yuto Takatsu, Barbara S. Slusher, Takashi Tsukamoto, Yuko Fujita, Kenji HashimotoAbstract:Abstract Glutamate Carboxypeptidase II (GCP II) is a glial enzyme responsible for the hydrolysis of N-acetylaspartylGlutamate (NAAG) into Glutamate and N-acetylaspartate (NAA). Abnormalities in Glutamate neurotransmission are implicated in the pathophysiology of schizophrenia. In this study, we examined the effects of a novel, orally active GCP II inhibitor, 2-(3-mercaptopropyl)pentanedioic acid (2-MPPA), on the prepulse inhibition (PPI) deficits after administration of the N-methyl- d -aspartate (NMDA) receptor antagonist dizocilpine. Oral administration of 2-MPPA (10, 30 or 100 mg/kg) significantly attenuated dizocilpine (0.1 mg/kg)-induced PPI deficits in mice, in a dose dependent manner. Furthermore, the efficacy of 2-MPPA on dizocilpine-induced PPI deficits was significantly antagonized by pretreatment with the selective group II metabotropic Glutamate receptor (mGluR) antagonist LY341495 (1.0 mg/kg). In the same model, however, the selective group II mGluR agonist LY354740 (3, 10 or 30 mg/kg) significantly attenuated dizocilpine-induced PPI deficits at only one dose and prepulse intensity. Our findings suggest that GCP II inhibition may be useful therapeutic strategy for schizophrenia. From a mechanistic perspective, while increased NAAG and activation of group II mGluRs may contribute to the therapeutic efficacy of 2-MPPA, it is likely that additional pharmacological activities are also involved.
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Structural basis of interactions between human Glutamate Carboxypeptidase II and its substrate analogs
Journal of Molecular Biology, 2008Co-Authors: Cyril Barinka, Pavel Majer, Barbara S. Slusher, Takashi Tsukamoto, Miroslava Rovenska, Klára Hlouchová, Niyada Hin, Miroslawa Dauter, J. KonvalinkaAbstract:Human Glutamate Carboxypeptidase II (GCPII) is involved in neuronal signal transduction and intestinal folate absorption by means of the hydrolysis of its two natural substrates, N-acetyl-aspartyl-Glutamate (NAAG) and folyl-poly-γ-Glutamates, respectively. During the past years, tremendous efforts have been made towards the structural analysis of GCPII. Crystal structures of GCPII in complex with various ligands have provided insight into the binding of these ligands, particularly to the S1′ site of the enzyme. In this paper, we have extended structural characterization of GCPII to its S1 site by using dipeptide-based inhibitors that interact with both S1 and S1′ sites of the enzyme. To this end, we have determined crystal structures of human GCPII in complex with phosphapeptide analogs of folyl-γ-Glutamate, aspartyl-Glutamate and γ-glutamyl-Glutamate, reined at resolution of 1.50 A, 1.60 A and 1.67 A, respectively. The S1 pocket of GCPII could be accurately defined and analyzed for the first time, and the data indicate the importance of Asn519, Arg463, Arg534, and Arg536 for recognition of the penultimate (i.e., P1) substrate residues. Direct interactions between the positively charged guanidinium groups of Arg534 and Arg536 and a P1 moiety of a substrate/inhibitor provide mechanistic explanation of GCPII preference for acidic dipeptides. Additionally, observed conformational flexibility of the Arg463 and Arg536 side chains likely regulates GCPII affinity towards different inhibitors and modulates GCPII substrate specificity. The biochemical experiments assessing the hydrolysis of several GCPII substrate derivatives modified at the P1 position, also included in this report, further complement and extend conclusions derived from the structural analysis. The data described here form an excellent foundation for the structurally aided design of novel low-molecular weight GCPII inhibitors and imaging agents.
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Progress in the discovery and development of Glutamate Carboxypeptidase II inhibitors
Drug Discovery Today, 2007Co-Authors: Takashi Tsukamoto, Krystyna M Wozniak, Barbara S. SlusherAbstract:During the past 10 years, substantial progress has been made in the discovery and development of small molecule Glutamate Carboxypeptidase II (GCP II) inhibitors. These inhibitors have provided the necessary tools to investigate the physiological role of GCP II as well as the potential therapeutic benefits of its inhibition in neurological disorders of Glutamatergic dysregulation. This review article details key GCP II inhibitors discovered in the last decade and important findings from preclinical and clinical studies.
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Structural insight into the pharmacophore pocket of human Glutamate Carboxypeptidase II.
Journal of Medicinal Chemistry, 2007Co-Authors: Cyril Barinka, Pavel Majer, Barbara S. Slusher, J. Konvalinka, Takashi Tsukamoto, Miroslava Rovenska, Petra Mlčochová, Klára Hlouchová, Anna Plechanovová, Jacek LubkowskiAbstract:Inhibition of Glutamate Carboxypeptidase II (GCPII) has been shown to be neuroprotective in multiple preclinical models in which dysregulated Glutamatergic transmission is implicated. Herein, we report crystal structures of the human GCPII complexed with three Glutamate mimetics/derivatives, 2-(phosphonomethyl)pentanedioic acid (2-PMPA), quisqualic acid (QA), and L-serine O-sulfate (L-SOS), at 1.72, 1.62, and 2.10 A resolution, respectively. Despite the structural differences between the distal parts of the inhibitors, all three compounds share similar binding modes in the pharmacophore (i.e., S1') pocket of GCPII, where they are stabilized by a combination of polar and van der Waals interactions. The structural diversity of the distal parts of the inhibitors leads to rearrangements of the S1' site that are necessary for efficient interactions between the enzyme and an inhibitor. The set of structures presented here, in conjunction with the available biochemical data, illustrates a flexibility of the GCPII pharmacophore pocket and highlights the structural features required for potent GCPII inhibition. These findings could facilitate the rational structure-based drug design of new GCPII inhibitors in the future.
Camilo Rojas - One of the best experts on this subject based on the ideXlab platform.
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δ-Thiolactones as Prodrugs of Thiol-Based Glutamate Carboxypeptidase II (GCPII) Inhibitors
Journal of Medicinal Chemistry, 2013Co-Authors: Dana Ferraris, Pavel Majer, Chiyou Ni, C. Ethan Slusher, Krystyna M Wozniak, Rana Rais, Ying Wu, Camilo Rojas, Barbara S. SlusherAbstract:δ-Thiolactones derived from thiol-based Glutamate Carboxypeptidase II (GCPII) inhibitors were evaluated as prodrugs. In rat liver microsomes, 2-(3-mercaptopropyl)pentanedioic acid (2-MPPA, 1) was gradually produced from 3-(2-oxotetrahydrothiopyran-3-yl)propionic acid (5), a thiolactone derived from 1. Compound 1 was detected in plasma at concentrations well above its IC50 for GCPII following oral administration of 5 in rats. Consistent with the oral plasma pharmacokinetics, thiolactone 5 exhibited efficacy in a rat model of neuropathic pain following oral administration.
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Glutamate Carboxypeptidase ii is not an amyloid peptide degrading enzyme
The FASEB Journal, 2013Co-Authors: Jesse Alt, Marigo Stathis, Camilo Rojas, Barbara S. SlusherAbstract:Glutamate Carboxypeptidase II (GCPII) is an exopeptidase that catalyzes the hydrolysis of N-acetylated aspartate-Glutamate (NAAG) to N-acetyl aspartate (NAA) and Glutamate. Consequently, GCPII inhibition has been of interest for the treatment of central and peripheral nervous system diseases associated with excess Glutamate. Recently, it was reported that GCPII can also serve as an endopeptidase cleaving amyloid β (Aβ) peptides and that its inhibition could increase the risk of Alzheimer's disease by increasing brain Aβ levels. This study aimed to corroborate and extend these new findings. We incubated Aβ peptides (20 μM) with human recombinant GCPII (300 ng/ml) and monitored the appearance of degradation products by mass spectrometry. Aβ peptides remained intact after 18 h incubation with GCPII. Under the same experimental conditions, Aβ1-40 (20 μM) was incubated with neprilysin (300 ng/ml), an endopeptidase known to hydrolyze Aβ1-40 and the expected cleavage products were observed. GCPII was confirmed active by catalyzing the complete hydrolysis of NAAG (100 μM). We also studied the hydrolysis of [(3)H]-NAAG (30 nM) catalyzed by GCPII (40 pM) in the presence of Aβ peptides (picomolar to micromolar range). The addition of Aβ peptides did not alter [(3)H]-NAAG hydrolysis. We conclude that GCPII is not an amyloid peptide-degrading enzyme.
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Glutamate Carboxypeptidase ii
Handbook of Proteolytic Enzymes, 2013Co-Authors: Barbara S. Slusher, Camilo Rojas, Joseph T CoyleAbstract:Publisher Summary This chapter examines the structural chemistry and the biological aspects of Glutamate Carboxypeptidase II. This enzyme was first characterized in the rat nervous system by its hydrolysis of the neuropeptide N-acetylaspartylGlutamate and was termed N-acetylated-alpha-linked acidic dipeptidase. The prostate cancer marker known as the prostate-specific membrane antigen also possesses NAALADase activity. Subcellular fractionation of brain reveals enrichment of the enzyme in neurosynaptosomal complexes. Electron microscopy localizes Glutamate Carboxypeptidase II immunoreactivity to the outer surface of the plasma membrane. The results indicate that the rat enzyme is expressed exclusively in astrocytic glial cells. Prostate-Specific Membrane (PSM) was characterized originally as the ligand of the monoclonal antibody 7E11.C5, which exhibited immunohistochemical staining restricted among human tissues to the prostate epithelium. The analyses have reported 7E11.C5 immunoreactivity and/or positive RNAase protection assays with PSM-derived probes in human salivary gland, brain, small intestine, seminal plasma and blood serum. Two gene loci closely related to PSM have been identified on human chromosome 11. The PSM form of Glutamate Carboxypeptidase II is expressed in normal and neoplastic prostate epithelial cells, and PSM has been examined for utility in the diagnosis, assessment and treatment of prostatic carcinoma.
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inhibition of Glutamate Carboxypeptidase ii gcpii activity as a treatment for cognitive impairment in multiple sclerosis
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Kristen A Rahn, Rana Rais, Martin G Pomper, Camilo Rojas, Marigo Stathis, Crystal C Watkins, Inna V Grishkan, Ciprian M Crainiceau, Mikhail V Pletnikov, Peter A CalabresiAbstract:Half of all patients with multiple sclerosis (MS) experience cognitive impairment, for which there is no pharmacological treatment. Using magnetic resonance spectroscopy (MRS), we examined metabolic changes in the hippocampi of MS patients, compared the findings to performance on a neurocognitive test battery, and found that N-acetylaspartylGlutamate (NAAG) concentration correlated with cognitive functioning. Specifically, MS patients with cognitive impairment had low hippocampal NAAG levels, whereas those with normal cognition demonstrated higher levels. We then evaluated Glutamate Carboxypeptidase II (GCPII) inhibitors, known to increase brain NAAG levels, on cognition in the experimental autoimmune encephalomyelitis (EAE) model of MS. Whereas GCPII inhibitor administration did not affect physical disabilities, it increased brain NAAG levels and dramatically improved learning and memory test performance compared with vehicle-treated EAE mice. These data suggest that NAAG is a unique biomarker for cognitive function in MS and that inhibition of GCPII might be a unique therapeutic strategy for recovery of cognitive function.
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Development of a high-throughput fluorescence polarization assay to identify novel ligands of Glutamate Carboxypeptidase II.
Journal of Biomolecular Screening, 2012Co-Authors: Glenda Alquicer, Jan Pavlíček, Youngjoo Byun, Martin G Pomper, Barbara S. Slusher, Camilo Rojas, Marigo Stathis, David Sedlák, Petr Bartůněk, Cyril BařinkaAbstract: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.