DC8 - Structural and dynamics characterization of the complexes of bioactive molecules with GTs and scFv antibodies by NMR

Adelyn María Betances Mora

Biography

I began my studies in 2018 at the Pontifical Catholic University Mother and Teacher (PUCMM), Santiago Campus, Dominican Republic, where I graduated with a Bachelor’s degree in Chemistry in 2022. I then received a scholarship from the Ministry of Higher Education, Science, and Technology (Mescyt) of the Dominican Republic to pursue a Master’s degree in Advanced Studies in Chemistry at the University of Seville. I specialized in the structural determination of chemical substances and molecular chemistry, covering aspects from synthesis to applications. In my Master’s thesis, I used advanced NMR techniques (specifically saturation transfer difference STD-NMR spectroscopy) to investigate the interactions between glycomimetics and proteins.

Research project objectives

In this DC position, my research will focus on the application of advanced NMR techniques to validate and characterize molecular interactions involving enzymes related to cancer. I will start with NMR validation of identified hits and provide detailed pharmacophore information of these validated hits using multifrequency NMR spectroscopic approaches like DEEP-STD NMR fingerprinting approaches. I will further elucidate structural details of enzyme-inhibitor and scFv-combotope complexes employing also novel multisolvent-STD NMR techniques. Additionally, I will explore “on-cell” double-difference STD NMR techniques on scFv-loaded engineered NK cells. My training will include structural biology, advanced NMR techniques, state-of-the-art high-throughput screening (HTS), and NK cell engineering.

Chemical Science at the University of Seville (University of Seville) Consejo Superior de Investigaciones Científicas (CSIC)/ Instituto de Investigaciones Químicas (IIQ).

The host department and laboratories fully meet the requirements for the successful execution of the research project. Access to the facilities at the Chemical Research Institute (IIQ) will be available.
IIQ: The laboratories are well equipped with a full range of spectroscopic (5xNMR instruments, UV-Vis, FT-IR, 2xMS, Maldi-TOF), X-ray diffraction and analytical techniques.

Selected papers:

1.Rocha, G.; Ramírez-Cárdenas, J.; Padilla-Pérez, M. C.; Walpole, S.; Nepravishta, R.; García-Moreno, M. I.; Sánchez-Fernández, E. M.; Ortiz Mellet, C.; Angulo, J.; Muñoz-García, J. C. Speeding-up the Determination of Protein–Ligand Affinities by STD NMR: The Reduced Data Set STD NMR Approach (Rd-STD NMR). Anal. Chem. 2024. https://doi.org/10.1021/acs.analchem.3c03980.

2.Silva-Díaz, A.; Ramírez-Cárdenas, J.; Muñoz-García, J. C.; de la Fuente, M. C.; Thépaut, M.; Fieschi, F.; Ramos-Soriano, J.; Angulo, J.; Rojo, J. Fluorinated Man9 as a High Mannose Mimetic to Unravel Its Recognition by DC-SIGN Using NMR. J. Am. Chem. Soc. 2023, 145 (48), 26009–26015. https://doi.org/10.1021/jacs.3c06204.

3.Nepravishta, R.; Ramírez-Cárdenas, J.; Rocha, G.; Walpole, S.; Hicks, T.; Muñoz-García, J. C.; Angulo, J. Fast Validation of Static and Dynamic 3D Models of Weak Protein Ligand Complexes from STD NMR Spectroscopy. ChemRxiv 2023. https://doi.org/10.26434/chemrxiv-2022-b7s0x-v2.

4.Monaco, S.; Angulo, J.; Wallace, M. Imaging Saturation Transfer Difference (STD) NMR: Affinity and Specificity of Protein–Ligand Interactions from a Single NMR Sample. J. Am. Chem. Soc. 2023, 145 (30), 16391–16397. https://doi.org/10.1021/jacs.3c02218.

5.Gabrielli, V.; Kuraite, A.; da Silva, M. A.; Edler, K. J.; Angulo, J.; Nepravishta, R.; Muñoz–García, J. C.; Khimyak, Y. Z. Spin Diffusion Transfer Difference (SDTD) NMR: An Advanced Method for the Characterisation of Water Structuration within Particle Networks. J. Colloid Interface Sci. 2021, 594, 217–227. https://doi.org/https://doi.org/10.1016/j.jcis.2021.02.094.

6.Gabrielli, V.; Muñoz-García, J. C.; Pergolizzi, G.; de Andrade, P.; Khimyak, Y. Z.; Field, R. A.; Angulo, J. Molecular Recognition of Natural and Non-Natural Substrates by Cellodextrin Phosphorylase from Ruminiclostridium Thermocellum Investigated by NMR Spectroscopy. Chem. – A Eur. J. 2021, 27 (63), 15688–15698. https://doi.org/https://doi.org/10.1002/chem.202102039.

7.Park, J. B.; Kim, Y. H.; Yoo, Y.; Kim, J.; Jun, S.-H.; Cho, J. W.; El Qaidi, S.; Walpole, S.; Monaco, S.; García-García, A. A.; et al. Structural Basis for Arginine Glycosylation of Host Substrates by Bacterial Effector Proteins. Nat. Commun. 2018, 9 (1), 4283. https://doi.org/10.1038/s41467-018-06680-6.

8.Sequeira, S.; Kavanaugh, D.; MacKenzie, D. A.; Šuligoj, T.; Walpole, S.; Leclaire, C.; Gunning, A. P.; Latousakis, D.; Willats, W. G. T.; Angulo, J.; et al. Structural Basis for the Role of Serine-Rich Repeat Proteins from Lactobacillus Reuteri in Gut Microbe–Host Interactions. Proc. Natl. Acad. Sci. 2018, 115 (12), E2706–E2715. https://doi.org/10.1073/pnas.1715016115.

9.Monaco, S.; Tailford, L. E.; Juge, N.; Angulo, J. Differential Epitope Mapping by STD NMR Spectroscopy To Reveal the Nature of  Protein-Ligand Contacts. Angew. Chem. Int. Ed. Engl. 2017, 56 (48), 15289–15293. https://doi.org/10.1002/anie.201707682.

10.Muñoz-García, J. C.; Chabrol, E.; Vivès, R. R.; Thomas, A.; de Paz, J. L.; Rojo, J.; Imberty, A.; Fieschi, F.; Nieto, P. M.; Angulo, J. Langerin–Heparin Interaction: Two Binding Sites for Small and Large Ligands As Revealed by a Combination of NMR Spectroscopy and Cross-Linking Mapping Experiments. J. Am. Chem. Soc. 2015, 137 (12), 4100–4110. https://doi.org/10.1021/ja511529x.

11.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.

12.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.

13.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.

14.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.

15.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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DC9 - Expression, purification and characterization of sialyltransferases, and validation of glycosyltransferases inhibitors using microplate assays

Sushmaa Dangudubiyyam

Biography

I come from India and am passionate to pursue a career as a research scientist. In the future, I hope to make contributions in the field of molecular medicine – in developing targeted therapies against human pathologies. I have a Masters in Biochemistry and Molecular Biology, with over 3 years’ research experience, wherein I had the opportunity to work with human diseases (Non-alcoholic fatty liver disease) and Carbohydrate Active Enzymes (CAZymes). I had been looking forward to focus my research pursuits towards using molecular approaches aimed at treating human pathologies, for which the GlyCanDrug is a perfect opportunity. When I am not working in the lab, I love to curl up with a book, or hit the gym. I am always curious to learn something new, to meet new people and engage in interesting conversations.

Research project objectives

The DC9 project aims at the biochemical and structural characterization of recombinantly expressed and purified glycosyl transferases, and to screen potential inhibitor molecules against these enzymes to aid in the development of precision cancer therapeutic agents.

Key objectives include the following:

  1. Expression and purification of recombinant glycosyl transferases (STs: ST6Gal I and ST6GalNAcI).
  2. Biochemical characterization of recombinant enzymes and development of microplate assays for STs and FTs (which includes preparation of glycoprotein acceptor substrates and synthesis of sugar donors for the identification of potential specific STs and FTs inhibitors)
  3. Organic synthesis and structural biology by enzyme expression and purification, inhibitors characterization in cell-based assays.

Université de Lille/ Unité de Glycobiologie Structurale et Fonctionnelle (UGSF)

The Joint Research Unit (JRU) 8576 – Unité de Glycobiologie Structurale et Fonctionnelle (UGSF) is a joint unit of research between CNRS and Univ. Lille. It is a research institute entirely dedicated to Glycoscience that covers the study of carbohydrates and promotes a highly multidisciplinary research and scientists are from a variety of different fields including biochemistry, chemistry, computational sciences, developmental biology, genetics, microbiology aiming to a better understanding of the structure to functions relationships of complex carbohydrates.

Selected papers (10):

  1. HARDUIN-LEPERS A., KRZEWINSKI-RECCHI M.A., COLOMB F., FOULQUIER F., GROUX-DEGROOTE S. and DELANNOY, P. Sialyltransferases function in cancer. Front. Biosci. (2012) 4, 499-515. doi: 10.2741/e396
  2. PETIT D., TEPPA R.E., MIR A.-M., VICOGNE D., THISSE C., THISSE B., FILLOUX C. and HARDUIN-LEPERS A. Integrative view of β–galactoside α2,3-sialyltransferases (ST3Gal) evolutionary relationships and functional divergence in Chordates: Massive st3gal gene loss in Homo sapiens. Mol Biol Evol. (2015) Apr;32(4):906-27. doi:10.1093/molbev/msu395
  3. HARDUIN-LEPERS A., MOLLICONE R, DELANNOY P and ORIOL R. The animal sialyltransferases and sialyltransferase-related genes: a phylogenetic approach. Glycobiology (2005) 15 (8), 805-817. DOI: 10.1093/glycob/cwi063
  4. GILORMINI P.-A., LION C., NOEL M., KRZEWINSKI-RECCHI M.-A., HARDUIN-LEPERS A., GUERARDEL Y, BIOT C. Improved workflow for the efficient preparation of ready to use CMP-activated sialic acid  Glycobiology (2016) Nov;26(11):1151-1156. doi: 10.1093/glycob/cww084
  5. YAMAKAWA N., VANBESELAERE J., CHANG L-Y., YU S.-Y., DUCROCQ L., HARDUIN-LEPERS A., KURATA J., AOKI-KINOSHITA K., SATO C., KHOO K-H., KITAJIMA K., GUERARDEL Y. Systems glycomics of adult zebrafish identifies organ specific sialylation and glycosylation patterns. Nature communications (2018) November 07, 9, article number 4647. doi:10.1038/s41467-018-06950-3.
  6. NOEL M., GILORMINI P.-A., COGEZ V., YAMAKAWA N., VICOGNE D., LION C., BIOT C., GUERARDEL Y, HARDUIN-LEPERS A. Probing the CMP-sialic acid donor specificity of two human –D-galactoside sialyltransferases (ST3Gal I and ST6Gal I) selectively acting on O- and N-glycosylproteins. ChemBioChem (2017) Jul 4;18(13):1251-1259. doi: 10.1002/cbic.201700024
  7. NOEL M., GILORMINI P.-A., COGEZ V., LION C., BIOT C., HARDUIN-LEPERS A., GUERARDEL Y. MicroPlate Sialyltransferase Assay (MPSA): a rapid and sensitive assay based on an unnatural sialic acid donor and bioorthogonal chemistry. Bioconjugate journal (2018) Oct 17;29(10):3377-3384. doi: 10.1021/acs.bioconjchem.8b00529.
  8. GROUX-DEGROOTE S, VICOGNE D, COGEZ V, SCHULZ C, HARDUIN-LEPERS A. B4GALNT2 controls Sda and sLex antigens biosynthesis in healthy and cancer human colon ChemBioChem (2021) 22(24):3381-3390 doi: 101002/cbic202100363.
  9. HARDUIN-LEPERS A. The vertebrate sialylation machinery: structure-function and molecular evolution of GT-29 sialyltransferases. Glycoconjugate J. (2023) 40, 473–492. doi.org/10.1007/s10719-023-10123-w
  10. DECLOQUEMENT M, VENUTO MT, COGEZ V, STEINMETZ A., SCHULZ C, LION C, NOEL M, RIGOLOT V, TEPPA RE, BIOT C, REBL A, GALUSKA SP and HARDUIN-LEPERS A. Salmonid polysialyltransferases to generate a variety of sialic acid polymers. Sci. Rep. (2023) 13, 15610. doi.org/10.1038/s41598-023-42095-0.

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