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.

Department of Pharmaceutical and Medicinal Chemistry Institute of Pharmacy Paracelsus Medical University Strubergasse 21 5020 Salzburg Austria

Laboratory of Structural Biology Institute of Biotechnology of the Czech Academy of Sciences BIOCEV Prumyslova 595 252 50 Vestec Czech Republic

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Tsukamoto T.; Wozniak K. M.; Slusher B. S. Progress in the Discovery and Development of Glutamate Carboxypeptidase II Inhibitors. Drug Discovery Today 2007, 12, 767–776. 10.1016/j.drudis.2007.07.010. PubMed DOI

Kamiya H.; Shinozaki H.; Yamamoto C. Activation of Metabotropic Glutamate Receptor Type 2/3 Suppresses Transmission at Rat Hippocampal Mossy Fibre Synapses. J. Physiol. 1996, 493, 447–455. 10.1113/jphysiol.1996.sp021395. PubMed DOI PMC

Mesters J. R.; Barinka C.; Li W.; Tsukamoto T.; Majer P.; Slusher B. S.; Konvalinka J.; Hilgenfeld R. Structure of Glutamate Carboxypeptidase II, a Drug Target in Neuronal Damage and Prostate Cancer. EMBO J. 2006, 25, 1375–1384. 10.1038/sj.emboj.7600969. PubMed DOI PMC

Barinka C.; Starkova J.; Konvalinka J.; Lubkowski J. A High-Resolution Structure of Ligand-Free Human Glutamate Carboxypeptidase II. Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 2007, 63, 150–153. 10.1107/S174430910700379X. PubMed DOI PMC

Bařinka C.; Rojas C.; Slusher B.; Pomper M. Glutamate Carboxypeptidase II in Diagnosis and Treatment of Neurologic Disorders and Prostate Cancer. Curr. Med. Chem. 2012, 19, 856–870. 10.2174/092986712799034888. PubMed DOI PMC

Barinka C.; Byun Y.; Dusich C. L.; Banerjee S. R.; Chen Y.; Castanares M.; Kozikowski A. P.; Mease R. C.; Pomper M. G.; Lubkowski J. Interactions between Human Glutamate Carboxypeptidase II and Urea-Based Inhibitors: Structural Characterization. J. Med. Chem. 2008, 51, 7737–7743. 10.1021/jm800765e. PubMed DOI PMC

Nakajima R.; Nováková Z.; Tueckmantel W.; Motlová L.; Bařinka C.; Kozikowski A. P. 2-Aminoadipic Acid-C(O)-Glutamate Based Prostate-Specific Membrane Antigen Ligands for Potential Use as Theranostics. ACS Med. Chem. Lett. 2018, 9, 1099–1104. 10.1021/acsmedchemlett.8b00318. PubMed DOI PMC

Barinka C.; Hlouchova K.; Rovenska M.; Majer P.; Dauter M.; Hin N.; Ko Y.-S.; Tsukamoto T.; Slusher B. S.; Konvalinka J.; Lubkowski J. Structural Basis of Interactions between Human Glutamate Carboxypeptidase II and Its Substrate Analogs. J. Mol. Biol. 2008, 376, 1438–1450. 10.1016/j.jmb.2007.12.066. PubMed DOI PMC

Zhang A. X.; Murelli R. P.; Barinka C.; Michel J.; Cocleaza A.; Jorgensen W. L.; Lubkowski J.; Spiegel D. A. A Remote Arene-Binding Site on Prostate Specific Membrane Antigen Revealed by Antibody-Recruiting Small Molecules. J. Am. Chem. Soc. 2010, 132, 12711–12716. 10.1021/ja104591m. PubMed DOI PMC

Tykvart J.; Schimer J.; Bařinková J.; Pachl P.; Poštová-Slavětínská L.; Majer P.; Konvalinka J.; Šácha P. Rational Design of Urea-Based Glutamate Carboxypeptidase II (GCPII) Inhibitors as Versatile Tools for Specific Drug Targeting and Delivery. Bioorg. Med. Chem. 2014, 22, 4099–4108. 10.1016/J.BMC.2014.05.061. PubMed DOI

Barinka C.; Novakova Z.; Hin N.; Bím D.; Ferraris D. V.; Duvall B.; Kabarriti G.; Tsukamoto R.; Budesinsky M.; Motlova L.; Rojas C.; Slusher B. S.; Rokob T. A.; Rulíšek L.; Tsukamoto T. Structural and Computational Basis for Potent Inhibition of Glutamate Carboxypeptidase II by Carbamate-Based Inhibitors. Bioorg. Med. Chem. 2019, 27, 255–264. 10.1016/j.bmc.2018.11.022. PubMed DOI PMC

Majer P.; Jackson P. F.; Delahanty G.; Grella B. S.; Ko Y. Sen.; Li W.; Liu Q.; Maclin K. M.; Poláková J.; Shaffer K. A.; Stoermer D.; Vitharana D.; Yanjun Wang E.; Zakrzewski A.; Rojas C.; Slusher B. S.; Wozniak K. M.; Burak E.; Limsakun T.; Tsukamoto T. Synthesis and Biological Evaluation of Thiol-Based Inhibitors of Glutamate Carboxypeptidase II: Discovery of an Orally Active GCP II Inhibitor. J. Med. Chem. 2003, 46, 1989–1996. 10.1021/jm020515w. PubMed DOI

Liu T.; Wu L. Y.; Hopkins M. R.; Choi J. K.; Berkman C. E. A Targeted Low Molecular Weight Near-Infrared Fluorescent Probe for Prostate Cancer. Bioorg. Med. Chem. Lett. 2010, 20, 7124–7126. 10.1016/J.BMCL.2010.09.057. PubMed DOI PMC

Wu L. Y.; Anderson M. O.; Toriyabe Y.; Maung J.; Campbell T. Y.; Tajon C.; Kazak M.; Moser J.; Berkman C. E. The Molecular Pruning of a Phosphoramidate Peptidomimetic Inhibitor of Prostate-Specific Membrane Antigen. Bioorg. Med. Chem. 2007, 15, 7434–7443. 10.1016/J.BMC.2007.07.028. PubMed DOI PMC

Liu T.; Nedrow-Byers J. R.; Hopkins M. R.; Berkman C. E. Spacer Length Effects on in Vitro Imaging and Surface Accessibility of Fluorescent Inhibitors of Prostate Specific Membrane Antigen. Bioorg. Med. Chem. Lett. 2011, 21, 7013–7016. 10.1016/J.BMCL.2011.09.115. PubMed DOI PMC

Mesters J. R.; Henning K.; Hilgenfeld R. Human Glutamate Carboxypeptidase II Inhibition: Structures of GCPII in Complex with Two Potent Inhibitors, Quisqualate and 2-PMPA. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2007, 63, 508–513. 10.1107/S090744490700902X. PubMed DOI

Slusher B. S.; Vornov J. J.; Thomas A. G.; Hurn P. D.; Harukuni I.; Bhardwaj A.; Traystman R. J.; Robinson M. B.; Britton P.; Lu X. C. M.; Tortella F. C.; Wozniak K. M.; Yudkoff M.; Potter B. M.; Jackson P. F. Selective Inhibition of NAALADase, Which Converts NAAG to Glutamate, Reduces Ischemic Brain Injury. Nat. Med. 1999, 5, 1396–1402. 10.1038/70971. PubMed DOI

Vornov J. J.; Hollinger K. R.; Jackson P. F.; Wozniak K. M.; Farah M. H.; Majer P.; Rais R.; Slusher B. S. Still NAAG’ing After All These Years: The Continuing Pursuit of GCPII Inhibitors. Adv. Pharmacol. 2016, 76, 215–255. 10.1016/BS.APHA.2016.01.007. PubMed DOI

Van Der Post J. P.; De Visser S. J.; De Kam M. L.; Woelfler M.; Hilt D. C.; Vornov J.; Burak E. S.; Bortey E.; Slusher B. S.; Limsakun T.; Cohen A. F.; Van Gerven J. M. A. The Central Nervous System Effects, Pharmacokinetics and Safety of the NAALADase-Inhibitor GPI 5693. Br. J. Clin. Pharmacol. 2005, 60, 128–136. 10.1111/J.1365-2125.2005.02396.X. PubMed DOI PMC

Dash R. P.; Tichý T.; Veeravalli V.; Lam J.; Alt J.; Wu Y.; Tenora L.; Majer P.; Slusher B. S.; Rais R. Enhanced Oral Bioavailability of 2-(Phosphonomethyl)-Pentanedioic Acid (2-PMPA) from Its (5-Methyl-2-Oxo-1,3-Dioxol-4-Yl)Methyl (ODOL)-Based Prodrugs. Mol. Pharmaceutics 2019, 16, 4292–4301. 10.1021/acs.molpharmaceut.9b00637. PubMed DOI PMC

Cardinale J.; Roscher M.; Schäfer M.; Geerlings M.; Benešová M.; Bauder-Wüst U.; Remde Y.; Eder M.; Nováková Z.; Motlová L.; Barinka C.; Giesel F. L.; Kopka K. Development of PSMA-1007-Related Series of 18F-Labeled Glu-Ureido-Type PSMA Inhibitors. J. Med. Chem. 2020, 63, 10897–10907. 10.1021/acs.jmedchem.9b01479. PubMed DOI

Voet A.; Qing X.; Lee X. Y.; De Raeymaecker J.; Tame J.; Zhang K.; De Maeyer M. Pharmacophore Modeling: Advances, Limitations, and Current Utility in Drug Discovery. J. Recept., Ligand Channel Res. 2014, 7, 81.10.2147/JRLCR.S46843. DOI

Kaserer T.; Beck K. R.; Akram M.; Odermatt A.; Schuster D.; Willett P. Pharmacophore Models and Pharmacophore-Based Virtual Screening: Concepts and Applications Exemplified on Hydroxysteroid Dehydrogenases. Molecules 2015, 20, 22799.10.3390/MOLECULES201219880. PubMed DOI PMC

Seidel T.; Wieder O.; Garon A.; Langer T. Applications of the Pharmacophore Concept in Natural Product Inspired Drug Design. Mol. Inf. 2020, 39, 200005910.1002/minf.202000059. PubMed DOI PMC

Wolber G.; Seidel T.; Bendix F.; Langer T. Molecule-Pharmacophore Superpositioning and Pattern Matching in Computational Drug Design. Drug Discovery Today 2008, 13, 23–29. 10.1016/j.drudis.2007.09.007. PubMed DOI

Vuorinen A.; Schuster D. Methods for Generating and Applying Pharmacophore Models as Virtual Screening Filters and for Bioactivity Profiling. Methods 2015, 71, 113–134. 10.1016/j.ymeth.2014.10.013. PubMed DOI

Berman H. M.; Westbrook J.; Feng Z.; Gilliland G.; Bhat T. N.; Weissig H.; Shindyalov I. N.; Bourne P. E. The Protein Data Bank. Nucleic Acids Res. 2000, 28, 235–242. 10.1093/nar/28.1.235. PubMed DOI PMC

Gaulton A.; Hersey A.; Nowotka M. L.; Patricia Bento A.; Chambers J.; Mendez D.; Mutowo P.; Atkinson F.; Bellis L. J.; Cibrian-Uhalte E.; Davies M.; Dedman N.; Karlsson A.; Magarinos M. P.; Overington J. P.; Papadatos G.; Smit I.; Leach A. R. The ChEMBL Database in 2017. Nucleic Acids Res. 2017, 45, D945–D954. 10.1093/NAR/GKW1074. PubMed DOI PMC

Wolber G.; Langer T. LigandScout: 3-D Pharmacophores Derived from Protein-Bound Ligands and Their Use as Virtual Screening Filters. J. Chem. Inf. Model. 2005, 45, 160–169. 10.1021/ci049885e. PubMed DOI

Wolber G.; Dornhofer A. A.; Langer T. Efficient Overlay of Small Organic Molecules Using 3D Pharmacophores. J. Comput.-Aided Mol. Des. 2007, 20, 773–788. 10.1007/s10822-006-9078-7. PubMed DOI

Hawkins P. C. D.; Skillman A. G.; Warren G. L.; Ellingson B. A.; Stahl M. T. Conformer Generation with OMEGA: Algorithm and Validation Using High Quality Structures from the Protein Databank and Cambridge Structural Database. J. Chem. Inf. Model. 2010, 50, 572–584. 10.1021/ci100031x. PubMed DOI PMC

Omega 4.1.2.0. OpenEye Scientific Software, 2022. http://www.eyesopen.com.

Güner O. F.; Bowen J. P. Setting the Record Straight: The Origin of the Pharmacophore Concept. J. Chem. Inf. Model. 2014, 54, 1269–1283. 10.1021/ci5000533. PubMed DOI

Vuorinen A.; Nashev L. G.; Odermatt A.; Rollinger J. M.; Schuster D. Pharmacophore Model Refinement for 11β-Hydroxysteroid Dehydrogenase Inhibitors: Search for Modulators of Intracellular Glucocorticoid Concentrations. Mol. Inf. 2014, 33, 15–25. 10.1002/minf.201300063. PubMed DOI

Triballeau N.; Acher F.; Brabet I.; Pin J.-P.; Bertrand H.-O. Virtual Screening Workflow Development Guided by the “Receiver Operating Characteristic” Curve Approach. Application to High-Throughput Docking on Metabotropic Glutamate Receptor Subtype 4. J. Med. Chem. 2005, 48, 2534–2547. 10.1021/jm049092j. PubMed DOI

Güner F.; Henry R.. Metric for Analyzing Hit-Lists and Pharmacophores. In Pharmacophore Perception, Development, and Use in Drug Design, Osman F. G., Ed.; International University Line: La Jolla, 2000; pp 193–212.

Jacobsson M.; Lidén P.; Stjernschantz E.; Boström H.; Norinder U. Improving Structure-Based Virtual Screening by Multivariate Analysis of Scoring Data. J. Med. Chem. 2003, 46, 5781–5789. 10.1021/jm030896t. PubMed DOI

Zweig M. H.; Campbell G. Receiver-Operating Characteristic (ROC) Plots: A Fundamental Evaluation Tool in Clinical Medicine. Clin. Chem. 1993, 39, 561–577. 10.1093/clinchem/39.4.561. PubMed DOI

Temml V.; Kutil Z. Structure-Based Molecular Modeling in SAR Analysis and Lead Optimization. Comput. Struct. Biotechnol. J. 2021, 19, 1431–1444. 10.1016/j.csbj.2021.02.018. PubMed DOI PMC

Tykvart J.; Sácha P.; Bařinka C.; Knedlík T.; Starková J.; Lubkowski J.; Konvalinka J. Efficient and Versatile One-Step Affinity Purification of in Vivo Biotinylated Proteins: Expression, Characterization and Structure Analysis of Recombinant Human Glutamate Carboxypeptidase II. Protein Expression Purif. 2012, 82, 106–115. 10.1016/j.pep.2011.11.016. PubMed DOI PMC

Barinka C.; Ptacek J.; Richter A.; Novakova Z.; Morath V.; Skerra A. Selection and Characterization of Anticalins Targeting Human Prostate-Specific Membrane Antigen (PSMA). Protein Eng., Des. Sel. 2016, 29, 105–115. 10.1093/protein/gzv065. PubMed DOI

Barinka C.; Rinnová M.; Šácha P.; Rojas C.; Majer P.; Slusher B. S.; Konvalinka J. Substrate Specificity, Inhibition and Enzymological Analysis of Recombinant Human Glutamate Carboxypeptidase II. J. Neurochem. 2002, 80, 477–487. 10.1046/J.0022-3042.2001.00715.X. PubMed DOI

Hai Y.; Shinsky S. A.; Porter N. J.; Christianson D. W. Histone Deacetylase 10 Structure and Molecular Function as a Polyamine Deacetylase. Nat. Commun. 2017, 8, 1536810.1038/ncomms15368. PubMed DOI PMC

GraphPad Prism 8.4.0 for Windows; GraphPad Software: San Diego, California, USA, 2022. www.graphpad.com.

Novakova Z.; Wozniak K.; Jancarik A.; Rais R.; Wu Y.; Pavlicek J.; Ferraris D.; Havlinova B.; Ptacek J.; Vavra J.; Hin N.; Rojas C.; Majer P.; Slusher B. S.; Tsukamoto T.; Barinka C. Unprecedented Binding Mode of Hydroxamate-Based Inhibitors of Glutamate Carboxypeptidase II: Structural Characterization and Biological Activity. J. Med. Chem. 2016, 59, 4539–4550. 10.1021/acs.jmedchem.5b01806. PubMed DOI

Plechanovová A.; Byun Y.; Alquicer G.; Škultétyová L.; Mlčochová P.; Němcová A.; Kim H.-J.; Navrátil M.; Mease R.; Lubkowski J.; Pomper M.; Konvalinka J.; Rulíšek L.; Bařinka C. Novel Substrate-Based Inhibitors of Human Glutamate Carboxypeptidase II with Enhanced Lipophilicity. J. Med. Chem. 2011, 54, 7535–7546. 10.1021/jm200807m. PubMed DOI PMC

Limongelli V.; Bonomi M.; Marinelli L.; Gervasio F. L.; Cavalli A.; Novellino E.; Parrinello M. Molecular Basis of Cyclooxygenase Enzymes (COXs) Selective Inhibition. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 5411.10.1073/PNAS.0913377107. PubMed DOI PMC

Baell J. B.; Holloway G. A. New Substructure Filters for Removal of Pan Assay Interference Compounds (PAINS) from Screening Libraries and for Their Exclusion in Bioassays. J. Med. Chem. 2010, 53, 2719–2740. 10.1021/jm901137j. PubMed DOI

Huth J. R.; Mendoza R.; Olejniczak E. T.; Johnson R. W.; Cothron D. A.; Liu Y.; Lerner C. G.; Chen J.; Hajduk P. J. ALARM NMR: A Rapid and Robust Experimental Method to Detect Reactive False Positives in Biochemical Screens. J. Am. Chem. Soc. 2005, 127, 217–224. 10.1021/ja0455547. PubMed DOI

Morgan R. E.; Ahn S.; Nzimiro S.; Fotie J.; Phelps M. A.; Cotrill J.; Yakovich A. J.; Sackett D. L.; Dalton J. T.; Werbovetz K. A. Inhibitors of Tubulin Assembly Identified through Screening a Compound Library. Chem. Biol. Drug Des. 2008, 72, 513–524. 10.1111/j.1747-0285.2008.00729.x. PubMed DOI PMC

Patch R. J.; Baumann C. A.; Liu J.; Gibbs A. C.; Ott H.; Lattanze J.; Player M. R. Identification of 2-Acylaminothiophene-3-Carboxamides as Potent Inhibitors of FLT3. Bioorg. Med. Chem. Lett. 2006, 16, 3282–3286. 10.1016/J.BMCL.2006.03.032. PubMed DOI

Louise-May S.; Yang W.; Nie X.; Liu D.; Deshpande M. S.; Phadke A. S.; Huang M.; Agarwal A. Discovery of Novel Dialkyl Substituted Thiophene Inhibitors of HCV by in Silico Screening of the NS5B RdRp. Bioorg. Med. Chem. Lett. 2007, 17, 3905–3909. 10.1016/J.BMCL.2007.04.103. PubMed DOI

Tanimoto T. T.An Elementary Mathematical Theory of Classification and Prediction|National Library of Australia; International Business Machines Corporation, 1958; pp 1–10.

Rogers D.; Brown R. D.; Hahn M. Using Extended-Connectivity Fingerprints with Laplacian-Modified Bayesian Analysis in High-Throughput Screening Follow-Up. J. Biomol. Screen. 2005, 10, 682–686. 10.1177/1087057105281365. PubMed DOI

Rogers D.; Hahn M. Extended-Connectivity Fingerprints. J. Chem. Inf. Model. 2010, 50, 742–754. 10.1021/ci100050t. PubMed DOI

Rais R.; Vávra J.; Tichý T.; Dash R. P.; Gadiano A. J.; Tenora L.; Monincová L.; Bařinka C.; Alt J.; Zimmermann S. C.; Slusher C. E.; Wu Y.; Wozniak K.; Majer P.; Tsukamoto T.; Slusher B. S. Discovery of a Para-Acetoxy-Benzyl Ester Prodrug of a Hydroxamate-Based Glutamate Carboxypeptidase II Inhibitor as Oral Therapy for Neuropathic Pain. J. Med. Chem. 2017, 60, 7799–7809. 10.1021/ACS.JMEDCHEM.7B00825. PubMed DOI PMC