Laboratory of Structural Biology

Structural biology and characterization of relationships between structure and function of medicinally relevant proteins

Group leader: RNDr. Cyril Bařinka, Ph.D.

The Laboratory of Structural Biology (LSB) was established in March 2010. We use methods of molecular, cell and structural biology to elucidate the structure-function relationship of several diagnostic/therapeutic targets. Ongoing projects include:
 
1. Identification and development of novel reagents, tools and techniques targeting human glutamate carboxypeptidase II (GCPII) and its paralogues/orthologues.
GCPII is a membrane-bound zinc metallopeptidase implicated in several physiological processes. Within the nervous system, GCPII exerts its peptidase activity by hydro- lysing N-acetyl-aspartyl-glutamate and the jejunal form of the enzyme acts as folate hydrolase, thus participating in the absorption of dietary folates (Fig. 1). GCPII-specific inhibitors have been reported to be neuroprotective in multiple preclinical models of neurodegeneration. Additionally, given the GCPII over-expression in prostate carcino- ma and within the neovasculature of solid tumours, the protein serves as an attractive target for imaging and therapy of a variety of cancers.

Our efforts are focused on addressing questions pertaining to the fundamental (patho) physiological roles of GCPII in healthy tissues as well as in cancer and neurodegene- ration. We solved the first high-resolution X-ray structure of human GCPII and these data, together with more than 40 reported structures of GCPII-inhibitor complexes, serve as a basis for our extensive programme aimed at the structure-assisted design of novel inhibitory compounds targeting GCPII (Fig. 2). In addition to projects involving small-molecule GCPII inhibitors we are actively pursuing the development of biolo- gics (Anticalin-based scaffolds) as novel agents for prostate cancer imaging. In our basic-oriented research we aimat unravelling putative non-enzymatic role(s) of human GCPII (receptor, chaperone) and defining functions of GCPII orthologues in several model organisms, including A. thaliana and C. elegans (Fig. 3).
 
2. Structure-function studies of histone deacetylases (HDACs)
Lysine acetylation is a major post-translational modification that plays a key role in many physiological processes and is believed to have a broad spectrumof modulatory functions within the cell. Acetylation levels of cellular targets are regulated by opposite activities of histone acyltransferease and histone deacetylases (HDACs). Eighteen hu- man HDACs identified so far can be divided into four major classes and our laboratory is specifically interested in class IIb (HDAC6, HDAC10) and class IV (HDAC11) pro- teins. To address basic questions related to the structure-function relationship of the- se hydrolases we employ a panel of molecular biology, biochemistry and enzymology techniques targeting the individual HDACs as well as their cognate substrates.

In addition to our own research, we actively participate in several collaborative projects that take advantage of aportfolio of techniques established in our lab. These techniques include heterologous protein expression in several systems including E. coli, K. lactis, insect S2 and Hi5 cells, and mammalian HEK293 cells, enzymatic and physicochemical characterization of purified proteins and X-ray structure determination, including robotic screening of crystallization conditions.
 
More information about projects and results of our lab can be found at the web lsb.avcr.cz.
Baranová Petra, Mgr.
Bařinka Cyril, RNDr., Ph.D.
Bašta Miroslav
Das Gargi
Havlínová Barbora, Ing.
Jelínková Iva
Komárek Jan, Mgr., Ph.D.
Kutil Zsófia, Ing., Ph.D.
Mikešová Jana, Mgr.
Motlová Lucia, Mgr.
Nedvědová Jana, Mgr.
Nováková Zora, RNDr., Ph.D.
Ondráková Markéta, Mgr.
Shukla Shivam

2020

  • Cardinale, J., Roscher, M., Schaefer, M., Geerlings, M., Benesova, M., Bauder-Wust, U., Remde, Y., Eder, M., Novakova, Z., Motlova, L., Barinka, C., Giesel, F. L., & Kopka, K. (2020). "Development of PSMA-1007 - Related Series of (18)F-Labeled Glu-ureido type PSMA inhibitors." J Med Chem.
  • Hapuarachchige, S., C. T. Huang, M. C. Donnelly, C. Barinka, S. E. Lupold, M. G. Pomper and D. Artemov (2020). "Cellular Delivery of Bioorthogonal Pretargeting Therapeutics in PSMA-Positive Prostate Cancer." Mol Pharm 17(1): 98-108.

  • Henrichs, V., Grycova, L., Barinka, C., Nahacka, Z., Neuzil, J., Diez, S., Rohlena, J., Braun, M., Lansky, Z. (2020). "Mitochondria-adaptor TRAK1 promotes kinesin-1 driven transport in crowded environments." Nature Communications, 11(1), 3123

  • Huang, C. T., Guo, X., Barinka, C., Lupold, S. E., Pomper, M. G., Gabrielson, K., Raman, V., Artemov, D., & Hapuarachchige, S. (2020). Development of 5D3-DM1: A Novel Anti-Prostate-Specific Membrane Antigen Antibody-Drug Conjugate for PSMA-Positive Prostate Cancer Therapy. Mol Pharm, 17(9), 3392-3402/p>

  • Kim, K., H. Kwon, C. Barinka, L. Motlova, S. Nam, D. Choi, H. Ha, H. Nam, S. H. Son, I. Minn, M. G. Pomper, X. Yang, Z. Kutil and Y. Byun (2020). "Novel beta- and gamma-Amino Acid-Derived Inhibitors of Prostate-Specific Membrane Antigen." J Med Chem.

  • Novakova, Z., Belousova, N., Foss, C. A., Havlinova, B., Gresova, M., Das, G., Lisok, A., Prada, A., Barinkova, M., Hubalek, M., Pomper, M. G., & Barinka, C. (2020). "Engineered Fragments of the PSMA-Specific 5D3 Antibody and Their Functional Characterization." Int J Mol Sci, 21(18).

  • Ptacek, J., Zhang, D., Qiu, L., Kruspe, S., Motlova, L., Kolenko, P., Novakova, Z., Shubham, S., Havlinova, B., Baranova, P., Chen, S. J., Zou, X., Giangrande, P., & Barinka, C. (2020). "Structural basis of prostate-specific membrane antigen recognition by the A9g RNA aptamer." Nucleic Acids Research, 48(19), 11130-11145.
  • Shen, S., M. Svoboda, G. Zhang, M. A. Cavasin, L. Motlova, T. A. McKinsey, J. H. Eubanks, C. Barinka and A. P. Kozikowski (2020). "Structural and in Vivo Characterization of Tubastatin A, a Widely Used Histone Deacetylase 6 Inhibitor." ACS Med Chem Lett 11(5): 706-712.

  • Ustinova, K., Z. Novakova, M. Saito, M. Meleshin, J. Mikesova, Z. Kutil, P. Baranova, B. Havlinova, M. Schutkowski, P. Matthias and C. Barinka (2020). "The disordered N-terminus of HDAC6 is a microtubule-binding domain critical for efficient tubulin deacetylation." J Biol Chem.

2019

  • Banerjee, S. R., V. Kumar, A. Lisok, D. Plyku, Z. Novakova, M. Brummet, B. Wharram, C. Barinka, R. Hobbs and M. G. Pomper (2019). "Evaluation of (111)In-DOTA-5D3, a Surrogate SPECT Imaging Agent for Radioimmunotherapy of Prostate-Specific Membrane Antigen." J Nucl Med 60(3): 400-406.

  • Barinka, C., Z. Novakova, N. Hin, D. Bim, D. V. Ferraris, B. Duvall, G. Kabarriti, R. Tsukamoto, M. Budesinsky, L. Motlova, C. Rojas, B. S. Slusher, T. A. Rokob, L. Rulisek and T. Tsukamoto (2019). "Structural and computational basis for potent inhibition of glutamate carboxypeptidase II by carbamate-based inhibitors." Bioorg Med Chem 27(2): 255-264.

  • Knox, T., E. Sahakian, D. Banik, M. Hadley, E. Palmer, S. Noonepalle, J. Kim, J. Powers, M. Gracia-Hernandez, V. Oliveira, F. Cheng, J. Chen, C. Barinka, J. Pinilla-Ibarz, N. H. Lee, A. Kozikowski and A. Villagra (2019). "Selective HDAC6 inhibitors improve anti-PD-1 immune checkpoint blockade therapy by decreasing the anti-inflammatory phenotype of macrophages and down-regulation of immunosuppressive proteins in tumor cells." Sci Rep 9(1): 6136.

  • Kozikowski, A. P., S. Shen, M. Pardo, M. T. Tavares, D. Szarics, V. Benoy, C. A. Zimprich, Z. Kutil, G. Zhang, C. Barinka, M. B. Robers, L. Van Den Bosch, J. H. Eubanks and R. S. Jope (2019). "Brain Penetrable Histone Deacetylase 6 Inhibitor SW-100 Ameliorates Memory and Learning Impairments in a Mouse Model of Fragile X Syndrome." ACS Chem Neurosci 10(3): 1679-1695.

  • Kutil, Z., J. Mikesova, M. Zessin, M. Meleshin, Z. Novakova, G. Alquicer, A. Kozikowski, W. Sippl, C. Barinka and M. Schutkowski (2019). "Continuous Activity Assay for HDAC11 Enabling Reevaluation of HDAC Inhibitors." ACS Omega 4(22): 19895-19904.

  • Kutil, Z., L. Skultetyova, D. Rauh, M. Meleshin, I. Snajdr, Z. Novakova, J. Mikesova, J. Pavlicek, M. Hadzima, P. Baranova, B. Havlinova, P. Majer, M. Schutkowski and C. Barinka (2019). "The unraveling of substrate specificity of histone deacetylase 6 domains using acetylome peptide microarrays and peptide libraries." FASEB J 33(3): 4035-4045.

  • Shen, S., M. Hadley, K. Ustinova, J. Pavlicek, T. Knox, S. Noonepalle, M. T. Tavares, C. A. Zimprich, G. Zhang, M. B. Robers, C. Barinka, A. P. Kozikowski and A. Villagra (2019). "Discovery of a New Isoxazole-3-hydroxamate-Based Histone Deacetylase 6 Inhibitor SS-208 with Antitumor Activity in Syngeneic Melanoma Mouse Models." J Med Chem 62(18): 8557-8577.

  • Tishchenko, G., Morganti, P., Stoller, M., Kelnar, I., Mikešová, J., Kovářová, J., Hašek, J., Kužel, R., Netopilík, M., Kaprálková, L., Špírková, M., Pavlova, E., Chánová, E., Brožová, L., Pekárek, M., Kobera, L., Sedláková, Z., Kubies, D. (2019). Chitin nanofibrils-chitosan composite films: characterization and properties. Bionanotechnology to save the environment. Plant and fishery's biomass as alternative to petrol. P. Morganti. Basel: 191-226.

  • Wu, H., K. Yang, Z. Zhang, E. D. Leisten, Z. Li, H. Xie, J. Liu, K. A. Smith, Z. Novakova, C. Barinka and W. Tang (2019). "Development of Multifunctional Histone Deacetylase 6 Degraders with Potent Antimyeloma Activity." J Med Chem 62(15): 7042-7057.

  • Zessin, M., Z. Kutil, M. Meleshin, Z. Novakova, E. Ghazy, D. Kalbas, M. Marek, C. Romier, W. Sippl, C. Barinka and M. Schutkowski (2019). "One-Atom Substitution Enables Direct and Continuous Monitoring of Histone Deacylase Activity." Biochemistry 58(48): 4777-4789.

2018

  • Kutil, Z., Z. Novakova, M. Meleshin, J. Mikesova, M. Schutkowski and C. Barinka (2018). "Histone Deacetylase 11 Is a Fatty-Acid Deacylase." ACS Chem Biol 13(3): 685-693.

  • Nakajima, R., Z. Novakova, W. Tueckmantel, L. Motlova, C. Barinka and A. P. Kozikowski (2018). "2-Aminoadipic Acid-C(O)-Glutamate Based Prostate-Specific Membrane Antigen Ligands for Potential Use as Theranostics." ACS Med Chem Lett 9(11): 1099-1104.

  • Porter, N. J., S. Shen, C. Barinka, A. P. Kozikowski and D. W. Christianson (2018). "Molecular Basis for the Selective Inhibition of Histone Deacetylase 6 by a Mercaptoacetamide Inhibitor." ACS Med Chem Lett 9(12): 1301-1305.

  • Ptacek, J., J. Nedvedova, M. Navratil, B. Havlinova, J. Konvalinka and C. Barinka (2018). "The calcium-binding site of human glutamate carboxypeptidase II is critical for dimerization, thermal stability, and enzymatic activity." Protein Sci 27(9): 1575-1584.

2017

  • Kopka, K., M. Benesova, C. Barinka, U. Haberkorn and J. Babich (2017). "Glu-Ureido-Based Inhibitors of Prostate-Specific Membrane Antigen: Lessons Learned During the Development of a Novel Class of Low-Molecular-Weight Theranostic Radiotracers." J Nucl Med 58(Suppl 2): 17S-26S.

  • Lv, W., G. Zhang, C. Barinka, J. H. Eubanks and A. P. Kozikowski (2017). "Design and Synthesis of Mercaptoacetamides as Potent, Selective, and Brain Permeable Histone Deacetylase 6 Inhibitors." ACS Med Chem Lett 8(5): 510-515.

  • Novakova, Z., C. A. Foss, B. T. Copeland, V. Morath, P. Baranova, B. Havlinova, A. Skerra, M. G. Pomper and C. Barinka (2017). "Novel Monoclonal Antibodies Recognizing Human Prostate-Specific Membrane Antigen (PSMA) as Research and Theranostic Tools." Prostate 77(7): 749-764.

  • Rais, R., J. Vavra, T. Tichy, R. P. Dash, A. J. Gadiano, L. Tenora, L. Monincova, C. Barinka, J. Alt, S. C. Zimmermann, C. E. Slusher, Y. Wu, K. Wozniak, P. Majer, T. Tsukamoto and B. S. Slusher (2017). "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 60(18): 7799-7809.

  • Skultetyova, L., K. Ustinova, Z. Kutil, Z. Novakova, J. Pavlicek, J. Mikesova, D. Trapl, P. Baranova, B. Havlinova, M. Hubalek, Z. Lansky and C. Barinka (2017). "Human histone deacetylase 6 shows strong preference for tubulin dimers over assembled microtubules." Sci Rep 7(1): 11547.

  • Tavares, M. T., S. Shen, T. Knox, M. Hadley, Z. Kutil, C. Barinka, A. Villagra and A. P. Kozikowski (2017). "Synthesis and Pharmacological Evaluation of Selective Histone Deacetylase 6 Inhibitors in Melanoma Models." ACS Med Chem Lett 8(10): 1031-1036.

2016

  • Barinka, C., J. Ptacek, A. Richter, Z. Novakova, V. Morath and A. Skerra (2016). "Selection and characterization of Anticalins targeting human prostate-specific membrane antigen (PSMA)." Protein Eng Des Sel 29(3): 105-115.

  • Bumba, L., J. Masin, P. Macek, T. Wald, L. Motlova, I. Bibova, N. Klimova, L. Bednarova, V. Veverka, M. Kachala, D. I. Svergun, C. Barinka and P. Sebo (2016). "Calcium-Driven Folding of RTX Domain beta-Rolls Ratchets Translocation of RTX Proteins through Type I Secretion Ducts." Mol Cell 62(1): 47-62.

  • Conway, R. E., C. Rojas, J. Alt, Z. Novakova, S. M. Richardson, T. C. Rodrick, J. L. Fuentes, N. H. Richardson, J. Attalla, S. Stewart, B. Fahmy, C. Barinka, M. Ghosh, L. H. Shapiro and B. S. Slusher (2016). "Prostate-specific membrane antigen (PSMA)-mediated laminin proteolysis generates a pro-angiogenic peptide." Angiogenesis 19(4): 487-500.

  • Dannoon, S., T. Ganguly, H. Cahaya, J. J. Geruntho, M. S. Galliher, S. K. Beyer, C. J. Choy, M. R. Hopkins, M. Regan, J. E. Blecha, L. Skultetyova, C. R. Drake, S. Jivan, C. Barinka, E. F. Jones, C. E. Berkman and H. F. VanBrocklin (2016). "Structure-Activity Relationship of (18)F-Labeled Phosphoramidate Peptidomimetic Prostate-Specific Membrane Antigen (PSMA)-Targeted Inhibitor Analogues for PET Imaging of Prostate Cancer." J Med Chem 59(12): 5684-5694.

  • Novakova, Z., K. Wozniak, A. Jancarik, R. Rais, Y. Wu, J. Pavlicek, D. Ferraris, B. Havlinova, J. Ptacek, J. Vavra, N. Hin, C. Rojas, P. Majer, B. S. Slusher, T. Tsukamoto and C. Barinka (2016). "Unprecedented Binding Mode of Hydroxamate-Based Inhibitors of Glutamate Carboxypeptidase II: Structural Characterization and Biological Activity." J Med Chem 59(10): 4539-4550.

  • Novakova, Z., J. Cerny, C. J. Choy, J. R. Nedrow, J. K. Choi, J. Lubkowski, C. E. Berkman and C. Barinka (2016). "Design of composite inhibitors targeting glutamate carboxypeptidase II: the importance of effector functionalities." FEBS J 283(1): 130-143.

2015

  • Ganguly, T., S. Dannoon, M. R. Hopkins, S. Murphy, H. Cahaya, J. E. Blecha, S. Jivan, C. R. Drake, C. Barinka, E. F. Jones, H. F. VanBrocklin and C. E. Berkman (2015). "A high-affinity [(18)F]-labeled phosphoramidate peptidomimetic PSMA-targeted inhibitor for PET imaging of prostate cancer." Nucl Med Biol 42(10): 780-787.

  • Tykvart, J., C. Barinka, M. Svoboda, V. Navratil, R. Soucek, M. Hubalek, M. Hradilek, P. Sacha, J. Lubkowski and J. Konvalinka (2015). "Structural and biochemical characterization of a novel aminopeptidase from human intestine." J Biol Chem 290(18): 11321-11336.

  • Youn, S., K. I. Kim, J. Ptacek, K. Ok, Z. Novakova, Y. Kim, J. Koo, C. Barinka and Y. Byun (2015). "Carborane-containing urea-based inhibitors of glutamate carboxypeptidase II: Synthesis and structural characterization." Bioorg Med Chem Lett 25(22): 5232-5236.

2014

  • Navratil, M., J. Ptacek, P. Sacha, J. Starkova, J. Lubkowski, C. Barinka and J. Konvalinka (2014). "Structural and biochemical characterization of the folyl-poly-gamma-l-glutamate hydrolyzing activity of human glutamate carboxypeptidase II." FEBS J 281(14): 3228-3242.

  • Pavlicek, J., J. Ptacek, J. Cerny, Y. Byun, L. Skultetyova, M. G. Pomper, J. Lubkowski and C. Barinka (2014). "Structural characterization of P1'-diversified urea-based inhibitors of glutamate carboxypeptidase II." Bioorg Med Chem Lett 24(10): 2340-2345.

  • Tykvart, J., V. Navratil, F. Sedlak, E. Corey, M. Colombatti, G. Fracasso, F. Koukolik, C. Barinka, P. Sacha and J. Konvalinka (2014). "Comparative analysis of monoclonal antibodies against prostate-specific membrane antigen (PSMA)." Prostate 74(16): 1674-1690.

2012

  • Alquicer, G., D. Sedlak, Y. Byun, J. Pavlicek, M. Stathis, C. Rojas, B. Slusher, M. G. Pomper, P. Bartunek and C. Barinka (2012). "Development of a high-throughput fluorescence polarization assay to identify novel ligands of glutamate carboxypeptidase II." J Biomol Screen 17(8): 1030-1040.

  • Barinka, C., C. Rojas, B. Slusher and M. Pomper (2012). "Glutamate carboxypeptidase II in diagnosis and treatment of neurologic disorders and prostate cancer." Curr Med Chem 19(6): 856-870.

  • Pavlicek, J., J. Ptacek and C. Barinka (2012). "Glutamate carboxypeptidase II: an overview of structural studies and their importance for structure-based drug design and deciphering the reaction mechanism of the enzyme." Curr Med Chem 19(9): 1300-1309.

  • Tykvart, J., P. Sacha, C. Barinka, T. Knedlik, J. Starkova, J. Lubkowski and J. Konvalinka (2012). "Efficient and versatile one-step affinity purification of in vivo biotinylated proteins: expression, characterization and structure analysis of recombinant human glutamate carboxypeptidase II." Protein Expr Purif 82(1): 106-115.

2011

  • Plechanovova, A., Y. Byun, G. Alquicer, L. Skultetyova, P. Mlcochova, A. Nemcova, H. J. Kim, M. Navratil, R. Mease, J. Lubkowski, M. Pomper, J. Konvalinka, L. Rulisek and C. Barinka (2011). "Novel substrate-based inhibitors of human glutamate carboxypeptidase II with enhanced lipophilicity." J Med Chem 54(21): 7535-7546.

2010

  • Zhang, A. X., R. P. Murelli, C. Barinka, J. Michel, A. Cocleaza, W. L. Jorgensen, J. Lubkowski and D. A. Spiegel (2010). "A remote arene-binding site on prostate specific membrane antigen revealed by antibody-recruiting small molecules." J Am Chem Soc 132(36): 12711-12716.

Patents

  • Skerra, A. R., A.; Morath, V.; Barinka, C.; Ptacek, J. ( 2019). US10406247. PSMA-Specific Binding Proteins., Technische Universität München (DE); Biotechnologický ústav AVCR, v.v.i. (CZ).

GACR: GJ19-22269Y, Kutil: Development of non-canonical inhibitors of glutamate carboxypeptidase II: structure-activity relationship studies and biological activity. 2019-2021.

MEYS: LTAUSA18196, Nováková: Recombinant antibody molecules for in vivo imaging and therapy of prostate cancer. 2019-2022.

GACR: GA18-04790S, Bařinka: Engineered antibodies as platforms for cancer imaging and therapy. 2018-2020.

GACR: GA14167S, Bařinka: Orthologs of glutamate carboxypeptidase 2 in model organisms - search for physiological roles and therapeutic potential of the enigmatic enzyme. 2018-2020.

AZV: NV17-32727A, Bařinka: Innovative strategies for personalized medicine: molecular approaches targeting the impact of inherited metabolic diseases caused by PPO mutations. 2017-2020.

Central Bohemia innovational vouchers: Providing knowledge to Apronex s.r.o. 2016.

GACR: GA15-19640S, Bařinka: Molecular basis of substrate recognition by histone deacetylase 6. 2015-2017.

MEYS: CZ.1.07/2.3.00/30.0045, Bařinka, Nováková, Kubista, Valihrach, Hejnalová: Biotechnological expert in structural biology and gene expression, OP Education for Competitiveness. 2012-2015.

GACR: GAP301/12/1513, Bařinka: Novel biologics for cancer imaging. 2012-2016.

EMBO: ASTF 56 – 2012, Bařinka: Short-Term Fellowship. 2012.

MEYS: ME10031, Bařinka: Structure-based design of ligands and inhibitors targeting glutamate carboxypeptidase II and its homologs. 2010-2012.

EMBO: 1978, Bařinka: EMBO Installation Grant. 2010-2015.

ASCR: Fellowship Jana Evangelisty Purkyně. 2010-2014.

FP7 IRG Reintegration Grant: 249220, Bařinka: Design and development of novel reagents, tools, and techniques targeting human glutamate carboxypeptidases II and III. 2010-2014.