DC10 - Dissecting glycan-protein interactions with cell-based assays

Cathal Patrick Forkan

Biography

I am Cathal Forkan, originally from Galway, Ireland. I completed my Bsc. in Biotechnology at the University of Galway in 2022. I proceeded to study an EMJMD master’s in Precision Medicine at the Université Grenoble-Alpes, France, graduating in 2024. My master’s thesis research on extracellular-vesicle based therapy for type-1 diabetes was conducted at the Steno Diabetes Centre in Copenhagen. My background of experience is in cellular and molecular biology, strongly driven by a curiosity and passion for science, and the desire to play a part in meaningful advances in science and ultimately, clinical therapies.

Research project objectives

In my project I aim to characterise isoenzyme-specific inhibitors of glycosyltransferases. Targeted glycosyltransferases have been linked to the expression of cancer-associated glycan motifs. Glycosyltransferase inhibitors developed by the DN will be tested in custom-engineered cell-based glycan arrays, to test for their specificity and efficacy as inhibitors of glycosylation, with a key focus on inhibitor specificity for individual isoenzymes. Key targets will be the STn-associated ST6GALNAC1 and the SLeX associated FUT6 and FUT7.

Graduate Program for Cellular & Genetic Medicine (CeGem), University of Copenhagen

GlycoDisplay is a spin-out from the Copenhagen Center for Glycomics (CCG), Copenhagen University, a world leader in the field of glyco-engineering and glycomics. GlycoDisplay develops and utilizes cell-based glycan arrays and recombinant expressed reporters for screening of biological functions of glycans and glycan binding proteins. GlycoDisplay is located at CCG and shares its state-of-the-art facilities for mass spectrometry, flow cytometry, cell engineering, and glycan arrays.

Selected papers (10):

  1. Sørensen DM, Büll C, Madsen TD, Lira-Navarrete E, Clausen TM, Clark AE, Garretson AF, Karlsson R, Pijnenborg JFA, Yin X, Miller RL, Chanda SK, Boltje TJ, Schjoldager KT, Vakhrushev SY, Halim A, Esko JD, Carlin AF, Hurtado-Guerrero R, Weigert R, Clausen H, Narimatsu Y (2023): Identification of global inhibitors of cellular glycosylation. Nat Commun. 20;14(1):948. doi: 10.1038/s41467-023-36598-7.
  2. Chen Y-H, Tian W, Yasuda M, Ye Z, Song M, Mandel U, Kristensen C, Povolo L, Marques A R, Caval T, Heck A J R, Sampaio J L, Johannes L, Tsukimura T, Desnick R, Vakhrushev S Y, Yang Z, Clausen H (2023): A universal GlycoDesign for lysosomal replacement enzymes to improve circulation time and biodistribution. Front. Bioeng. Biotechnool, 11:1128371. DOI: 10.3389/fbioe.2023.1128371
  3. Nason R, Bull C, Konstantinidi A, Sun L, Ye Z, Halim A, Du W, Sørensen DM, Durbesson F, Furukawa S, Mandel U, Joshi HJ, Dworkin LA, Hansen L, David L, Iverson TM, Bensing BA, Sullam PM, Varki A, Vries E, Haan C, Vincentelli R, Henrissat B, Vakhrushev S, Clausen H, Narimatsu Y (2021): Display of the human mucinome with defined O-glycans by gene engineered cells. Nat Commun1;12(1):4070. DOI: 10.1038/s41467-021-24366-4
  4. Narimatsu Y, Joshi H, Nason R, Van Coillie J, Karlsson R, Sun L, Ye Z, Chen Y-H, Schjoldager KT, Steentoft C, Furukawa S, Bensing BA, Sullam PM, Thompson AJ, Paulson JC, Bull C, Adema GJ, Mandel U, Hansen L, Bennett EP, Varki A, Vakhrushev SY, Yang Z, Clausen H (2019): An Atlas of Human Glycosylation Pathways Enables Display of the Human Glycome by Gene Engineered Cells. Mol Cell75:1-14. DOI: 10.1016/j.molcel.2019.05.017
  5. Tian W, Ye Z, Wang S, Schulz MA, Van Coillie J, Sun L, Chen Y-H, Narimatsu Y, Hansen L, Kristensen C, Mandel U, Bennett EP, Jabbarzadeh-Tabrizi S, Schiffmann R, Shen J-S, Vakhrushev SY, Clausen H, Yang Z. (2019): The Glycosylation Design Space for Recombinant Lysosomal Replacement Enzymes Produced in CHO Cells. Nat Commun. 10:1785. DOI: 10.1038/s41467-019-09809-3
  6. Chen Y-H, Narimatsu Y, Clausen TM, Gomes C, Karlsson R, Steentoft C, Spliid CB, Gustavsson T, Salanti A, Persson A, Malmström A, Willén W, Ellervik U, Bennett EP, Mao Y, Clausen H & Yang Z (2018): The GAGOme: a cell-based library of displayed glycosaminoglycans. Nat Methods15(11):881-888. DOI: 10.1038/s41592-018-0086-z
  7. Schulz MA, Tian W, Mao Y, van Coillie J, Sun L, Larsen JS, Chen YH, Kristensen C, Vakhrushev SY, Clausen H, Yang Z. (2018): Glycoengineering design options for IgG1 in CHO cells using precise gene editing. Glycobiology 1;28(7):542-549. DOI: 10.1093/glycob/cwy022
  8. Narimatsu Y, Joshi HJ, Zhang Y, Gomes C, Chen Y-H, Lorenzetti F, Furukawa S, Schjoldager K, Hansen L, Clausen H, Bennett EP, Wandall HH (2018): A validated gRNA library for CRISPR/Cas9 targeting of the human glycosyltransferase genome. Glycobiology1;28(5):295-305. DOI: 10.1093/glycob/cwx101
  9. Lonowski LA, Narimatsu Y, Riaz A, Delay CE, Yang Z, Niola F, Duda K, Ober EA, Clausen H, Wandall HH, Hansen SH, Bennett EP, Frödin M (2017): Genome editing using FACS enrichment of nuclease-expressing cells and idel detection by amplicon analysis. Nat Protocols12(3):581-603. DOI: 10.1038/nprot.2016.165
  10. Yang Z, Wang S, Halim A, Schulz MA, Frodin M, Rahman SH, Vester-Christensen MB, Behrens C, Kristensen C, Vakhrushev SY, Bennett EP, Wandall HH, Clausen H. (2015): Engineered CHO cells for production of diverse, homogeneous glycoproteins. Nat Biotechnol33:842-4. DOI: 10.1038/nbt.3280

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DC6 - Identification of small molecule inhibitors of sialyl and fucosyltransferases

George Lasisi

Biography

I chose to study organic chemistry to use my passion for chemistry to help those suffering from autoimmune diseases, which have also affected people close to me. After achieving my BSc degree in Chemistry at the HAN University of Applied Sciences in the Netherlands in 2018, I completed an MSc in Chemistry at Lund University in Sweden in 2020. While there are many ways to support people worldwide, using my affinity for chemistry allows me to help others while studying fields I genuinely enjoy every day.

Research project objectives

This DC will focus on the development of fluorescence polarization (FP) and high-throughput screening (HTS) assays for the identification and potency determination of novel inhibitors of glycosyltransferases. While working towards this objective, the DC will implement a method for the HTS assay that will dramatically reduce the amounts of consumables required. This will enable the DC to efficiently screen up to 50,000 molecules from a proprietary collection for the identification of potential new inhibitors of glycosyltransferases while minimizing costs.

Technical University of Denmark (DTU), Department of Biotechnology and Biomedicine

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DC2- Synthetic approaches for targeting cancer glycosylation

Georgia-Myrto Prifti

Biography

I am Georgia-Myrto Prifti, originally from Greece and born on December 18th, 1998. I earned my MPharm in Pharmacy from the National and Kapodistrian University of Athens in 2021, followed by an MPhil in Medicinal Chemistry from the same institution in 2023. Transitioning to Copenhagen, I embarked on a six-month Erasmus+ Internship in Medicinal Chemistry at the Technical University of Denmark. So far, my focus has been on the design and synthesis of antiviral compounds targeting Hepatitis B and SARS-CoV-2, with a minor involvement in anticancer compound research. Joining the GlyCanDrug network presents an exciting opportunity for me to contribute to this exceptional project.

Research project objectives

The research project aims to develop synthetic approaches for precisely targeting cancer glycosylation. Specifically, it focuses on delivering therapeutics to cancer cells with precision. This involves conjugating cell organelles-targeting molecules with selected synthetic glycosyltransferase inhibitors to enhance their efficacy in cancer cell targeting. Additionally, nanoparticles decorated with Tn/STn-specific scFv antibodies will be prepared for selective targeting of cancer cells, ensuring high specificity and minimizing off-target effects. The project will also involve evaluating the intracellular fate of synthetic constructs within cancer cells to understand their mechanisms of action and optimize therapeutic outcomes. Furthermore, glycoengineering studies will be conducted to analyze the glycoprofile of engineered cancer cells treated with synthetic constructs, providing insights into changes in glycosylation patterns and their implications for therapeutic response.

PhD School in Chemical Science XXXIX Cycle (University of Florence, Department of Chemistry ‘Ugo Schiff’, UNIFI-DICUS)

The host laboratories (DICUS–UNIFI) are equipped with modern equipment for organic synthesis and nanomaterials, including 400MHz NMR spectroscopy facilities, a state of the art cryo-electron microscope (cryo-EM), polarimeter, DLS, microwave reactors, HPLCs, FT-IR, and mass (GC/MS, ESI/MS) spectrometers, UV-Vis spectrophotometers, freeze dryers, and centrifuges. Full access to the literature databases (Reaxys, Web of Science, ScFinder) and on-line access to the primary lit. https://www.chim.unifi.it/vp-363-strumentazione-dipartimentale.html

Selected papers (10):

  1. Biagiotti G.; G. Toniolo G.; Albino M.; Severi M.; Andreozzi P.; Marelli M.; Kokot H.; Tria G.; Guerri A.; Sangregorio C.; Rojo J.; Berti D.; Marradi M.; Cicchi S.; Urbancˇicˇ I.; van Kooyk Y.; Chiodo F.; Richichi B. Simple engineering of hybrid cellulose nanocrystal–gold nanoparticles results in a functional glyconanomaterial with biomolecular recognition properties. Nanoscale Horizons, 2023, DOI: 10.1039/d3nh00063j
  2. Tricomi, J.; Cacaci, M.; Biagiotti, G.; Caselli, L.; Niccoli, F.; Torelli, R. Gabbani, A.; Di Vito, M.; Pineider, F.; Severi, M.; Sanguinetti, M.; Menna, E.; Lelli, M.; Berti, D.; Cicchi, S.; Bugli, F.; Richichi, B. Ball Milled Glyco-graphene oxide conjugates markedly disrupted Pseudomonas aeruginosa biofilm, Nanoscale, 2022, DOI: 10.1039/D2NR02027K.
  3. Biagiotti, G.; Legnani, L.; Aresta, G.; Chiacchio, M.A.; Richichi, B.  Benzo[c][1,2]thiazine-Based Analogs in the Inverse Electron Demand [4+2] Hetero Diels-Alder Reaction with Glycals: Access to Tetracyclic Fused Galactose and Fucose Derivatives. Eur. J. Org. Chem., 2022, e202200769. doi: 10.1002/ejoc.202200769.
  4. Anderluh, M.; Berti, F.; Bzducha‐Wróbel, A.; Chiodo, F.; Colombo, C.; Compostella, F.; Durlik, K.; Ferhati, X.; Holmdahl, R.; Jovanovic, D.; Kaca, W.; Lay, L.; Marinovic‐Cincovic, M.; Marradi, M.; Ozil, M.; Polito, L.; Reina, J.J.; Reis, C.A.; Sackstein, R.; Silipo, A.; Švajger, U.; Vaněk, O.; Yamamoto, F.; Richichi, B.; S. J. van Vliet, Recent advances on smart glycoconjugate vaccines in infections and cancer. FEBS J., 2021, doi: 10.1111/febs.15909
  5. Martin, K.C.; Tricomi, J.; Corzana, F.; García-García, A.; Ceballos-Laita, L.; Hicks, T.; Monaco, S.; Angulo, J.; Hurtado-Guerrero, R.; Richichi, B.; Sackstein, R. Fucosyltransferase-specific inhibition via next generation of fucose mimetics. ChemCommun, 2021, 57, 1145-1148.
  6. Biagiotti G.; Purić E.; Urbančič I.; Krišelj A.; Weiss M.; Mravljak J.; Gellini C.; Lay L.; Chiodo F.; Anderluh M.; Cicchi S.; Richichi B. Combining cross-coupling reaction and Knoevenagel condensation in the synthesis of glyco-BODIPY probes for DC-SIGN super-resolution bioimaging. BioorgChem, 2021, 109, 104730.
  7. Andreozzi P.; Simó C.; Moretti P.; Martinez Porcel J.; Ursula Lüdtke T.; de los Angeles Ramirez M.; Tamberi L.; Marradi M.; Amenitsch H.; Llop J.; Grazia Ortore M.; Moya S.E. Novel Core–Shell Polyamine Phosphate Nanoparticles Self-Assembled from PEGylated Poly (allylamine hydrochloride) with low toxicity and increased in vivo circulation time, Small 2021, 17, 2102211. DOI: 10.1002/smll.202102211.
  8. Vetro M.; Safari, D.; Fallarini, S.; Salsabila K.; Lahmann M.; Penadés, S.; Lay L.; Marradi M.; Compostella F. Preparation and immunogenicity of gold glyco-nanoparticles as antipneumococcal vaccine model Nanomedicine 2017, 12(1), 13–23. DOI: 10.2217/nnm-2016-0306
  9. Chiodo F.; Marradi M.; Park J.; Ram A.F.J.; Penadés S.; van Die I.; Tefsen B.Galactofuranose-Coated Gold Nanoparticles Elicit a Proinflammatory Response in Human Monocyte-Derived Dendritic Cells and Are Recognized by DC-SIGN. ACS Chem. Biol. 2014, 9, 383−389. DOI: 10.1021/cb4008265
  10. Terán-Navarro H.; Zeoli A.; Salines-Cuevas D.; Marradi M.; Montoya N.; Gonzalez-Lopez E.; Ocejo-Vinyals J.G.; Dominguez-Esteban M.; Gutierrez-Baños J.L.; Campos-Juanatey F.; Yañez-Diaz S.; Garcia-Castaño A.; Rivera F.; Duran I.; Alvarez-Dominguez C. Gold Glyconanoparticles Combined with 91–99 Peptide of the Bacterial Toxin, Listeriolysin O, Are Efficient Immunotherapies in Experimental Bladder Tumors. Cancers 2022, 14, 2413. DOI: 10.3390/cancers14102413

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DC5 - Combotope-specific single chain fragments (scFv) antibodies by Phage Display Technologies

Edward Meier

Biography

Edward Meier earned his MSc from the University of Victoria in 2021, specializing in carbohydrate processing of pathogens. Following his postgraduate studies, he dedicated three years to the biopharmaceutical industry, contributing significantly to the advancement of multi-specific T-cell engager, ADC, and radio-conjugate antibody formats for the treatment of diverse cancers and immunological disorders. His scientific pursuits are fueled by a passion for chemistry, biochemistry, and structural biology, which he applies adeptly to address challenges in health and disease. Beyond the confines of the laboratory, Edward finds solace and joy in outdoor activities, such as hiking, biking, traveling, and rock climbing. Additionally, he indulges his creative side through painting, playing guitar and brewing beer, among other hobbies.

Research project objectives

The project focuses on the development of cutting-edge anti-combotope scFv antibodies as part of the GlyCanDrug program. The main objectives include preparation of synthetic combotope peptides, development of the iDEAL phage display antibody discovery platform, molecular-level study of combotope/scFv interactions, specificity assessment and target validation using various engineered cancer cell models and human patient tissue samples. This training will involve understanding the implications of scFv antibodies as potential chimeric antigen receptors to enhance NK cells’ ability to kill cancer cells. This work will contribute to the ambitious studies of the GlyCanDrug program and lay the groundwork for future projects.

Technical University of Denmark (DTU)

Selected papers:

  1. Sørensen CV, […], Laustsen AH*. Antibody-dependent enhancement of toxicity of myotoxin II from Bothrops asper. Nature Communications 2023, in press.
  2. Tulika T, […], Laustsen AH*. Phage display assisted discovery of a pH-dependent anti-α-cobratoxin antibody from a natural variable domain library. Protein Science 2023, 32, p1-17.
  3. Ledsgaard L, […], Laustsen AH*, Karatt-Vellatt A. Discovery and optimization of a broadly-neutralizing human monoclonal antibody against long-chain α-neurotoxins from snakes. Nature Communications 2023, 14, p1-14.
  4. Laustsen AH*, […], McCafferty J. In vivo neutralization of dendrotoxin-mediated neurotoxicity of black mamba venom by mixtures of human IgG monoclonal antibodies. Nature Communications 2018, 9: p1-9.
  5. Persson N, […] Blixt O. Epitope mapping of a new anti-Tn antibody detecting gastric cancer cells. Glycobiology 2017, 27, 635-645. DOI: 10.1093/glycob/cwx033.
  6. Persson N, […] Blixt O. A Combinatory Antibody-Antigen Microarray Assay for High-Content Screening of Single-Chain Fragment Variable Clones from Recombinant Libraries. PLoS One 2016, 11:e0168761. DOI: 10.1371/journal.pone.0168761.
  7. Blixt O, […] Filatov AV. Analysis of Tn-antigenicity with a panel of new IgM and IgG1 monoclonal antibodies raised against leukemic cells. Glycobiology. 2012, 22, 529-42.
  8. Gong Y, Klein Wolterink RGJ, Gulaia V, Cloosen S, Ehlers FAI, Wieten L, Graus YF, Bos GMJ, Germeraad WTV. Defucosylation of tumor-specific humanized anti-MUC1 monoclonal antibody enhances NK cell-mediated anti-tumor cell Cytotoxicity. Cancers, May 2021, 13, 2579.
  9. Gong Y, Klein Wolterink RJG, Wang JX, Bos GMJ, Germeraad WTV. Generation chimeric antigen receptor Natural Killer cells for tumor immunotherapy. J Hematol Oncol. 2021. 14:73.
  10. Huijskens MJAJ, Walczak M, Sarkar S, Atrafi F, Senden-Gijsbers BLMG, Bos GMJ, Wieten L, Germeraad WTV. Ascorbic acid promotes proliferation of NK cell populations in culture systems applicable for NK cell therapy. Cytotherapy. 2015 May;17(5):613-20.
  11. Sarkar S, Germeraad WTV, Rouschop KM, Steeghs EM, van Gelder M, Bos GMJ, Wieten L. Hypoxia induced impairment of NK cell cytotoxicity against multiple myeloma can be overcome by IL-2 activation of the NK cells. PLoS One. 2013 May 28;8(5):e64835.
  12. Cloosen S, Arnold J, Thio M, Bos GMJ, Kyewski B, and Germeraad WTV. Expression of tumor-associated differentiation antigens CEA and MUC1 glycoforms in human thymic epithelial cells: 2implications for self-tolerance and tumor therapy. Cancer Res. 67(8): 3919-3926, 2007.

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DC7 - Screening and hit development towards novel non-carbohydrate inhibitors of glycosyltransferases

Natan Koraj

Biography

Originally from a small village in the north of Croatia, I moved to a big city to study pharmacy at the Faculty of Pharmacy and Biochemistry, University of Zagreb. I discovered my love for science through bioanalytical method development at the faculty, which resulted in the Rector’s award and a publication. The next big challenge was my master’s thesis in the field of medicinal chemistry, where I developed potential novel antimalarials. My student researcher days concluded with exploration of glycobiology. After graduating, I explored working as a pharmacist and as a pharmaceutical representative before finally returning to my dear science.

Research project objectives

DC7 will perform virtual screening and hit analysis to identify FTs and STs inhibitors. Starting hits will be alternatively discovered by using DNA-encoded libraries screening of compounds vs FTs and STs by using DELopen technology to minimize the risk of hit identification failure. Hits will be synthesized and further optimized in terms of potency, target selectivity and cell permeability. Co-crystallization experiments of enzyme-inhibitor complexes will be performed to elucidate interactions X-ray crystallographic experiments or alternatively by NMR. These data will in turn allow structure-based design and new iterations towards lead compounds to be assayed in cell-based assays.

University of Ljubljana, Scientific field of Pharmacy

The host laboratories (Anderluh, UL FFA, Department of Pharmaceutical Chemistry) are equipped with modern equipment for molecular modelling (supermicro servers for high-performance computing equipped with modern scientific software packages – OpenEye, GOLD, MOE, BiosolveIT, KNIME, OpenBabel, Modeller, VMD/NAMD, GROMACS, BLAST, PyMol), and organic synthesis and nanomaterials, including 400-800MHz NMR spectroscopy facilities, state of the art LC/MS/MS systems), polarimeter, AutoChem system, microwave reactors, bench top reactor Parr 4560 HPLCs, flash purification systems (Biotage Isolera), FT-IR, and mass (GC/MS, ESI/MS) spectrometers, UV-Vis spectrophotometers, freeze dryers, and centrifuges. Furthermore, a dedicated laboratory is available for biochemical studies with several microplate readers (TECAN Safire II and BioTek Synergy H4, microplate sample processor (robotic pipetor) BioTek Precision XS), biolayer interferometry instrument (Sartorius Octet® R4 Protein Analysis System), ITC system for microcalorimetry (TA Instruments Nano), flow cytometers (Attune NxT flow cytometer with automated plate reader), gel and membrane imager (UVItec Alliance 9.7), centrifuges (Hereus Megafuge 16R, Thermo Scientific and StatSpin Cytofuge), ultracentrifuge (Thermo Scientific Sorvall WX), incubator (WTB BINDER). Full access to the literature databases (Reaxys, Web of Science, ScFinder) and on-line access to the primary literature.

Selected papers (10):

  1. Kokot M, Weiss M, Zdovc I, Senerovic L, Radakovic N, Anderluh M, Minovski N, Hrast M. Amide containing NBTI antibacterials with reduced hERG inhibition, retained antimicrobial activity against gram-positive bacteria and in vivo efficacy. Eur J Med Chem. 2023, 250, 115160. doi: 10.1016/j.ejmech.2023.115160.
  2. van Klaveren S, Dernovšek J, Jakopin Ž, Anderluh M, Leffler H, Nilsson UJ, Tomašič T. Design and synthesis of novel 3-triazolyl-1-thiogalactosides as galectin-1, -3 and -8 inhibitors. RSC Adv. 2022,12(29),18973-18984. doi: 10.1039/d2ra03163a.
  3. Girardi B, Manna M, Van Klaveren S, Tomašič T, Jakopin Ž, Leffler H, Nilsson UJ, Ricklin D, Mravljak J, Schwardt O, Anderluh M. Selective Monovalent Galectin-8 Ligands Based on 3-Lactoylgalactoside. ChemMedChem. 2022,17(3):e202100514. doi: 10.1002/cmdc.202100514.
  4. Hassan M, Baussière F, Guzelj S, Sundin AP, Håkansson M, Kovačič R, Leffler H, Tomašič T, Anderluh M, Jakopin Ž, Nilsson UJ. Structure-Guided Design of d-Galactal Derivatives with High Affinity and Selectivity for the Galectin-8 N-Terminal Domain. ACS Med Chem Lett. 2021, 12(11),1745-1752. doi: 10.1021/acsmedchemlett.1c00371.
  5. Anderluh, M.; Berti, F.; Bzducha‐Wróbel, A.; Chiodo, F.; Colombo, C.; Compostella, F.; Durlik, K.; Ferhati, X.; Holmdahl, R.; Jovanovic, D.; Kaca, W.; Lay, L.; Marinovic‐Cincovic, M.; Marradi, M.; Ozil, M.; Polito, L.; Reina, J.J.; Reis, C.A.; Sackstein, R.; Silipo, A.; Švajger, U.; Vaněk, O.; Yamamoto, F.; Richichi, B.; S. J. van Vliet, Recent advances on smart glycoconjugate vaccines in infections and cancer. FEBS J., 2021, doi: 10.1111/febs.15909
  6. Kokot M, Weiss M, Zdovc I, Hrast M, Anderluh M, Minovski N. Structurally Optimized Potent Dual-Targeting NBTI Antibacterials with an Enhanced Bifurcated Halogen-Bonding Propensity. ACS Med Chem Lett. 2021, 12(9), 1478-1485. doi: 10.1021/acsmedchemlett.1c00345.
  7. Hassan M, van Klaveren S, Håkansson M, Diehl C, Kovačič R, Baussière F, Sundin AP, Dernovšek J, Walse B, Zetterberg F, Leffler H, Anderluh M, Tomašič T, Jakopin Ž, Nilsson UJ. Benzimidazole-galactosides bind selectively to the Galectin-8 N-Terminal domain: Structure-based design and optimisation. Eur J Med Chem. 2021, 223, 113664. doi: 10.1016/j.ejmech.2021.113664.
  8. Weiss M, Loi EM, Sterle M, Balsollier C, Tomašič T, Pieters RJ, Gobec M, Anderluh M. New Quinolinone O-GlcNAc Transferase Inhibitors Based on Fragment Growth. Front Chem. 2021, 9., 666122. doi: 10.3389/fchem.2021.666122.
  9. Anderluh M, Berti F, Bzducha-Wróbel A, Chiodo F, Colombo C, Compostella F, Durlik K, Ferhati X, Holmdahl R, Jovanovic D, Kaca W, Lay L, Marinovic-Cincovic M, Marradi M, Ozil M, Polito L, Reina-Martin JJ, Reis CA, Sackstein R, Silipo A, Švajger U, Vaněk O, Yamamoto F, Richichi B, van Vliet SJ. Emerging glyco-based strategies to steer immune responses. FEBS J. 2021, 288(16), 4746-4772. doi: 10.1111/febs.15830.
  10. Kolarič A, Germe T, Hrast M, Stevenson CEM, Lawson DM, Burton NP, Vörös J, Maxwell A, Minovski N, Anderluh M. Potent DNA gyrase inhibitors bind asymmetrically to their target using symmetrical bifurcated halogen bonds. Nat Commun. 2021, 12(1), 150. doi: 10.1038/s41467-020-20405-8.

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DC4 - Protein expression and purification of FTs and crystallization of FTs and STs complexed to inhibitors

Laura O’Byrne

Biography

I have a Bachelor’s degree in Chemistry from the University of Turin. I furthered my studies at the University of Milan, where I graduated with a Master’s degree in Pharmaceutical Biotechnology in July 2023, achieving the highest honors. During the completion of my thesis, I had the opportunity to undertake a year-long internship at the CNR in Milan. Here, I was tasked with expressing various proteins, previously identified as potential pharmacological targets. Additionally, I performed molecular biology techniques such as mutagenesis and cloning and conducted biochemical assays to test inhibitory compounds of these proteins.

Research project objectives

The main key points of my project will be:

  • optimize expression of FUT6/FUT7/FUT8 and ST6GalNAcI/ST6GalI for purification using HEK293 cells
  • kinetic characterization and enzyme inhibition studies
  • determination of the Kds for the inhibitors using ITC, Octet
  • obtain crystal structures or cryo-EM structures with inhibitors

Universidad Zaragoza  (UNIZAR)

The host laboratory (BIFI–UNIZAR) has the usual and appropriate research facilities, infrastructure and equipment in order to carry out the expression, purification, biophysical characterization of proteins and the determination of crystal structures. We have all the required equipments to do molecular biology, express and purify proteins and obtain protein crystals. Furthermore, we do have several ITC apparatus, SPR (Biacore), UV and fluorescence spectrophotometers, circular dichroism, fluorometers, luminometers for 96 well plates proteins and our own X-ray diffractometer. Finally, we do have routine access to the synchrotrons ALBA and Diamond.

Selected papers (10): * indicates corresponding authorship

1.-) Lira-Navarrete E, et al., Rovira C*, Hurtado-Guerrero R*. (2014) Substrate-guided front-face reaction revealed by combined structural snapshots and metadynamics for the polypeptide GalNAc-T2. Angew Chem Int Ed Engl 53(31):8206-10. (89 citations/9 yrs)

2.-) Lira-Navarrete E, et al., Hurtado-Guerrero R*. (2015) Dynamic interplay between catalytic and lectin domains of GalNAc-transferases modulates protein O-glycosylation. Nat Commun 6:6937. (92 citations/8 yrs)

3.-) Valero-González J, et al., Hurtado-Guerrero R*. (2016) A proactive role of water molecules in acceptor recognition by protein O-fucosyltransferase 2. Nat Chem Biol 12(4):240-6. (65 citations/7 yrs)

4.-) de Las Rivas M, et al., Hurtado-Guerrero R*. (2017) The interdomain flexible linker of the polypeptide GalNAc transferases dictates their long-range glycosylation preferences. Nat Commun 8(1):1959. (35 citations/6 yrs)

5.-) Park JB, et al., Hurtado-Guerrero R*, Angulo J*, Hardwidge PR, Shin JS*, Cho HS*. (2018) Structural basis for arginine glycosylation of host substrates by bacterial effector proteins. Nat Commun 9(1):4283. (52 citations/5 yrs)

6.-) Fang W, et al., Hurtado-Guerrero R, Arroyo J*, van Aalten DMF*. (2019) Mechanisms of redundancy and specificity of the Aspergillus fumigatus Crh transglycosylases. Nat Commun 10(1):1669. (21 citations/4 yrs)

7.-) García-García A, et al., Hurtado-Guerrero R*. (2020) Structural basis for substrate specificity and catalysis of α1,6-fucosyltransferase. Nat Commun 2020;11(1):973. (37 citations/3 yrs)

8.-) de Las Rivas M, et al., Hurtado-Guerrero R*. (2020) Molecular basis for fibroblast growth factor 23 O-glycosylation by GalNAc-T3. Nat Chem Biol 16(3):351-360. (42 citations/3 yrs)

9.-) González-Ramírez AM, et al., Marcelo F, Corzana F*, Hurtado-Guerrero R*. (2022) Structural basis for the synthesis of the core 1 structure by C1GalT1. Nat Commun 13(1):2398. (6 citations/1 yr)

10.-) Taleb V, et al., Rovira C*, Hurtado-Guerrero R*. (2022) Structural and mechanistic insights into the cleavage of clustered O-glycan patches-containing glycoproteins by mucinases of the human gut. Nat Commun 13(1):4324. (7 citations/1 yr)

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DC3 - Evaluation of glycosyltransferases inhibitors and targeted approaches in cancer cells and tumour spheroids

Jordan Gotti

Biography

After achieving a BSc in Biotechnology, I completed a MSc in Industrial Biotechnology at the University of Milan, graduating with honors. For my master’s thesis, I joined the multidisciplinary lab of Prof. Brinks in Delft (Netherlands) through the Erasmus Traineeship program. Afterward, I worked at San Raffaele Telethon Institute for Gene Therapy in Milan (Italy), focusing my attention on CAR-T cells and the unmet need to find an early molecular biomarker of response that can guide patient selection for the treatment. Now, joining the GlyCanDrug network, I have the exciting opportunity to contribute to the advancement of cancer precision therapeutics.

Research project objectives

The DC3 research project will focus on the evaluation of glycosyltransferases inhibitors and targeted approaches in gastrointestinal cancer models.

1) Analysis of the effects of the selected inhibitors in the glycan biosynthesis mediated by the targeted glycosyltransferases using glycoengineered cancer cell models.
2) Characterization and monitoring of the glycomic and glycoproteomic features induced by the inhibitors in advanced cancer models,
3) Evaluation of the functional biological effect of the glycosyltransferases inhibitors in cancer cells.
4) Analysis of the glycan-based therapeutic efficacy on the cancer models.

Instituto de Investigação e Inovação em Saúde (i3S)

The host laboratories (i3S) are equipped with modern equipment and facilities for analysis of the functional effects in the tumour biology and their potential therapeutic applications. Cell culture, molecular and genetic engineering, protein biochemistry, flow cytometry and FACS, confocal and electron microscopy, HPLCs, Mass (ESI/MS) spectrometers, proteomics, high-throughput screening and animal model facilities. Full access to the literature databases.

Selected papers (10):

  1. Mereiter S., Balmaña M., Campos D., Gomes J., Reis C.A. Glycosylation in the Era of Cancer-Targeted Therapy: Where Are We Heading?. Cancer Cell 36(1):6-16, 2019. DOI: 10.1016/j.ccell.2019.06.006
  2. Duarte H.O., Rodrigues J.G., Gomes C., Hensbergen P.J., Ederveen A.L.H., de Ru A.H., Mereiter S., Polónia A., Fernandes E., Ferreira J.A., van Veelen P.A., Santos L.L., Wuhrer M., Gomes J., Reis C.A. ST6Gal1 targets the ectodomain of ErbB2 in a site-specific manner and regulates gastric cancer cell sensitivity to trastuzumab. Oncogene 40(21):3719-3733, 2021. DOI: 10.1038/s41388-021-01801-w
  3. Rodrigues J.G., Duarte H.O., Gomes C., Balmaña M., Martins Á.M., Hensbergen P.J., de Ru A.H., Lima J., Albergaria A., van Veelen P.A., Wuhrer M., Gomes J., Reis C.A. Terminal a2,6-sialylation of epidermal growth factor receptor modulates antibody therapy response of colorectal cancer cells. Cellular Oncology 44(4):835-850, 2021. DOI: 10.1007/s13402-021-00606-z
  4. Costa A.F., Campos D., Reis C.A., Gomes C. Targeting Glycosylation: A New Road for Cancer Drug Discovery. Trends in Cancer 6(9):757-766, 2020. DOI: 10.1016/j.trecan.2020.04.002
  5. Marcos N.T., Pinho S., Grandela C., Cruz A., Samyn-Petit B., Harduin-Lepers A., Almeida R., Silva F., Morais V., Costa J., Kihlberg J., Clausen H., Reis C.A. Role of the human ST6GalNAc-I and ST6GalNAc-II in the synthesis of the cancer-associated Sialyl-Tn antigen. Cancer Research 64(19):7050-7057, 2004. DOI: 10.1158/0008-5472.CAN-04-1921
  6. Freitas D., Campos D., Gomes J., Pinto F., Macedo J.A., Matos R., Mereiter S., Pinto M.T., Polónia A., Gartner F., Magalhães A., Reis C.A. O-glycans truncation modulates gastric cancer cell signaling and transcription leading to a more aggressive phenotype. EBioMedicine 40:349-362, 2019. DOI: 10.1016/j.ebiom.2019.01.017
  7. Poças J., Marques C., Gomes C., Otake A.H., Pinto F., Ferreira M., Silva T., Faria-Ramos I., Matos R., Ribeiro A.R., Senra E., Cavadas B., Batista S., Maia J., Macedo J.A., Lima L., Afonso L.P., Ferreira J.A., Santos L.L., Polónia A., Osório H., Belting M., Reis C.A. Costa-Silva B., Magalhães A. Syndecan-4 is a maestro of gastric cancer cell invasion and communication that underscores poor survival. Proceedings of the National Academy of Sciences of the United States of America 120(20):, 2023. DOI: 10.1073/pnas.2214853120
  8. Martins Á.M., Lopes T.M., Diniz F., Pires J., Osório H., Pinto F., Freitas D., Reis C.A. Differential Protein and Glycan Packaging into Extracellular Vesicles in Response to 3D Gastric Cancer Cellular Organization. Advanced Science:, 2023. DOI: 10.1002/advs.202300588
  9. Campos D., Freitas D., Gomes J., Magalhães A., Steentoft C., Gomes C., Vester-Christensen M.B., Ferreira J.A., Afonso L.P., Santos L.L., De Sousa J.P., Mandel U., Clausen H., Vakhrushev S.Y., Reis C.A., Probing the O-glycoproteome of gastric cancer cell lines for biomarker discovery. Molecular and Cellular Proteomics 14(6):1616-1629, 2015. DOI: 10.1074/mcp.M114.046862
  10. Pinho S.S., Reis C.A Glycosylation in cancer: Mechanisms and clinical implications. Nature Reviews Cancer 15(9):540-555, 2015. DOI: 10.1038/nrc3982

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DC1- Synthesis of fucose and sialic acid mimetics able to selectively disrupt the function of key glycosyltransferases implicated in cancer

Pedro Miguel Ascenso Vieira

Biography

I began my chemistry studies in 2017 at the Nova School of Science and Technology of the Nova University of Lisbon, gaining my bachelor’s degree in applied chemistry. For my master’s degree I wanted to focus more on organic chemistry and obtained my master´s degree in Bioorganic Chemistry at the same university. To finish my master´s I wanted to focus more on medicinal chemistry, so my master´s thesis was to develop new triazene derivatives against microorganism and biofilm growth.

Research project objectives

This DC position will focus on the identification of small molecules able to selectively interfere with the creation of key glycan motifs implicated in cancer. The DC will synthesize and characterize new glycomimetics. The synthesis be done using an original version of the [4+2] inverse electron demand hetero Diels Alder reaction (ihDA). The DC will also perform structural biology and cancer biology studies to unveil information on the interaction of the synthetic glycomimetics with the target glycosyltransferases.

PhD School in Chemical Science XXXIX Cycle (University of Florence, Department of Chemistry ‘Ugo Schiff’, UNIFI-DICUS)

The host laboratories (DICUS–UNIFI) are equipped with modern equipment for organic synthesis and nanomaterials, including 400MHz NMR spectroscopy facilities, a state of the art cryo-electron microscope (cryo-EM), polarimeter, DLS, microwave reactors, HPLCs, FT-IR, and mass (GC/MS, ESI/MS) spectrometers, UV-Vis spectrophotometers, freeze dryers, and centrifuges. Full access to the literature databases (Reaxys, Web of Science, ScFinder) and on-line access to the primary lit. https://www.chim.unifi.it/vp-363-strumentazione-dipartimentale.html

Selected papers (10):​

  1. Biagiotti G.; G. Toniolo G.; Albino M.; Severi M.; Andreozzi P.; Marelli M.; Kokot H.; Tria G.; Guerri A.; Sangregorio C.; Rojo J.; Berti D.; Marradi M.; Cicchi S.; Urbancˇicˇ I.; van Kooyk Y.; Chiodo F.; Richichi B. Simple engineering of hybrid cellulose nanocrystal–gold nanoparticles results in a functional glyconanomaterial with biomolecular recognition properties. Nanoscale Horizons, 2023, DOI: 10.1039/d3nh00063j​
  2. Tricomi, J.; Cacaci, M.; Biagiotti, G.; Caselli, L.; Niccoli, F.; Torelli, R. Gabbani, A.; Di Vito, M.; Pineider, F.; Severi, M.; Sanguinetti, M.; Menna, E.; Lelli, M.; Berti, D.; Cicchi, S.; Bugli, F.; Richichi, B. Ball Milled Glyco-graphene oxide conjugates markedly disrupted Pseudomonas aeruginosa biofilm, Nanoscale, 2022, DOI: 10.1039/D2NR02027K.​
  3. Biagiotti, G.; Legnani, L.; Aresta, G.; Chiacchio, M.A.; Richichi, B.  Benzo[c][1,2]thiazine-Based Analogs in the Inverse Electron Demand [4+2] Hetero Diels-Alder Reaction with Glycals: Access to Tetracyclic Fused Galactose and Fucose Derivatives. Eur. J. Org. Chem., 2022, e202200769. doi: 10.1002/ejoc.202200769.​
  4. Anderluh, M.; Berti, F.; Bzducha‐Wróbel, A.; Chiodo, F.; Colombo, C.; Compostella, F.; Durlik, K.; Ferhati, X.; Holmdahl, R.; Jovanovic, D.; Kaca, W.; Lay, L.; Marinovic‐Cincovic, M.; Marradi, M.; Ozil, M.; Polito, L.; Reina, J.J.; Reis, C.A.; Sackstein, R.; Silipo, A.; Švajger, U.; Vaněk, O.; Yamamoto, F.; Richichi, B.; S. J. van Vliet, Recent advances on smart glycoconjugate vaccines in infections and cancer. FEBS J., 2021, doi: 10.1111/febs.15909​
  5. Martin, K.C.; Tricomi, J.; Corzana, F.; García-García, A.; Ceballos-Laita, L.; Hicks, T.; Monaco, S.; Angulo, J.; Hurtado-Guerrero, R.; Richichi, B.; Sackstein, R. Fucosyltransferase-specific inhibition via next generation of fucose mimetics. ChemCommun, 2021, 57, 1145-1148.​
  6. Biagiotti G.; Purić E.; Urbančič I.; Krišelj A.; Weiss M.; Mravljak J.; Gellini C.; Lay L.; Chiodo F.; Anderluh M.; Cicchi S.; Richichi B. Combining cross-coupling reaction and Knoevenagel condensation in the synthesis of glyco-BODIPY probes for DC-SIGN super-resolution bioimaging. BioorgChem, 2021, 109, 104730. ​
  7. Andreozzi P.; Simó C.; Moretti P.; Martinez Porcel J.; Ursula Lüdtke T.; de los Angeles Ramirez M.; Tamberi L.; Marradi M.; Amenitsch H.; Llop J.; Grazia Ortore M.; Moya S.E. Novel Core–Shell Polyamine Phosphate Nanoparticles Self-Assembled from PEGylated Poly (allylamine hydrochloride) with low toxicity and increased in vivo circulation time, Small 2021, 17, 2102211. DOI: 10.1002/smll.202102211.​
  8. Vetro M.; Safari, D.; Fallarini, S.; Salsabila K.; Lahmann M.; Penadés, S.; Lay L.; Marradi M.; Compostella F. Preparation and immunogenicity of gold glyco-nanoparticles as antipneumococcal vaccine model Nanomedicine 2017, 12(1), 13–23. DOI: 10.2217/nnm-2016-0306​
  9. Chiodo F.; Marradi M.; Park J.; Ram A.F.J.; Penadés S.; van Die I.; Tefsen B.Galactofuranose-Coated Gold Nanoparticles Elicit a Proinflammatory Response in Human Monocyte-Derived Dendritic Cells and Are Recognized by DC-SIGN. ACS Chem. Biol. 2014, 9, 383−389. DOI: 10.1021/cb4008265​
  10. Terán-Navarro H.; Zeoli A.; Salines-Cuevas D.; Marradi M.; Montoya N.; Gonzalez-Lopez E.; Ocejo-Vinyals J.G.; Dominguez-Esteban M.; Gutierrez-Baños J.L.; Campos-Juanatey F.; Yañez-Diaz S.; Garcia-Castaño A.; Rivera F.; Duran I.; Alvarez-Dominguez C. Gold Glyconanoparticles Combined with 91–99 Peptide of the Bacterial Toxin, Listeriolysin O, Are Efficient Immunotherapies in Experimental Bladder Tumors. Cancers 2022, 14, 2413. DOI: 10.3390/cancers14102413

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