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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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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.
Barbara S. Slusher - One of the best experts on this subject based on the ideXlab platform.
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structural and computational basis for potent inhibition of Glutamate Carboxypeptidase II by carbamate based inhibitors
Bioorganic & Medicinal Chemistry, 2019Co-Authors: Cyril Barinka, Bridget Duvall, Gabriel Kabarriti, Reiji Tsukamoto, Milos Budesinsky, L Motlova, Zora Novakova, Dana Ferraris, Camilo Rojas, Barbara S. SlusherAbstract:Abstract 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.
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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, Camilo Rojas, Marigo Stathis, 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 a...
Cyril Barinka - One of the best experts on this subject based on the ideXlab platform.
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structural and computational basis for potent inhibition of Glutamate Carboxypeptidase II by carbamate based inhibitors
Bioorganic & Medicinal Chemistry, 2019Co-Authors: Cyril Barinka, Bridget Duvall, Gabriel Kabarriti, Reiji Tsukamoto, Milos Budesinsky, L Motlova, Zora Novakova, Dana Ferraris, Camilo Rojas, Barbara S. SlusherAbstract:Abstract 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.
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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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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 composite inhibitors targeting Glutamate Carboxypeptidase II: the importance of effector functionalities.
FEBS Journal, 2015Co-Authors: Zora Novakova, Jacek Lubkowski, Cindy J. Choy, Clifford E. Berkman, Jiri Cerny, Jessie R. Nedrow, Joeseph K. Choi, Cyril BarinkaAbstract: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).
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Carborane-containing urea-based inhibitors of Glutamate Carboxypeptidase II: Synthesis and structural characterization.
Bioorganic & Medicinal Chemistry Letters, 2015Co-Authors: Sihyun Youn, Zora Novakova, Jakub Ptacek, Cyril Barinka, Kyung Im Kim, Yunhye Kim, Jaehyung Koo, Youngjoo ByunAbstract:Abstract Glutamate Carboxypeptidase II (GCPII) is a zinc metalloprotease on the surface of astrocytes which cleaves N-acetylaspartylGlutamate to release N-acetylaspartate and Glutamate. GCPII inhibitors can decrease Glutamate concentration and play a protective role against apoptosis or degradation of brain neurons. Herein, we report the synthesis and structural analysis of novel carborane-based GCPII inhibitors. We determined the X-ray crystal structure of GCPII in complex with a carborane-containing inhibitor at 1.79 A resolution. The X-ray analysis revealed that the bulky closo-carborane cluster is located in the spacious entrance funnel region of GCPII, indicating that the carborane cluster can be further structurally modified to identify promising lead structures of novel GCPII inhibitors.
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.
Camilo Rojas - One of the best experts on this subject based on the ideXlab platform.
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structural and computational basis for potent inhibition of Glutamate Carboxypeptidase II by carbamate based inhibitors
Bioorganic & Medicinal Chemistry, 2019Co-Authors: Cyril Barinka, Bridget Duvall, Gabriel Kabarriti, Reiji Tsukamoto, Milos Budesinsky, L Motlova, Zora Novakova, Dana Ferraris, Camilo Rojas, Barbara S. SlusherAbstract:Abstract 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.
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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, Camilo Rojas, Marigo Stathis, 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 a...
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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.