Skip to main content

Smart Biologics: Developing New Tools in Glycobiology

Periodic Reporting for period 3 - SWEETOOLS (Smart Biologics: Developing New Tools in Glycobiology)

Reporting period: 2019-02-01 to 2020-07-31

Sugars are ubiquitous molecules found throughout all kingdoms of life. Early studies contributed considerably to our appreciation of sugar functions by showing that abnormalities in the glycosylation can develop into pathogenesis and severe disease. Despite the crucial role of sugars in many biological events we still do not have adequate tools to decipher their complexity. To unveil the mysteries in the rapidly emerging field of sugar biology (glycobiology) we aim in this project to develop new tools that will help us to study and understand these important biomolecules. To realize this, we plan to construct unique bioconjugates, which will enable us to target various sugar processing enzymes with unprecedented selectivity. Our goal is to develop a new class of smart probes that will help us to answer fundamental questions in glycobiology. The outcomes of this project will significantly deepen our knowledge of glycoconjugates and in the long term, will allow for the design of efficient vaccines and for the development of better therapeutics.
In the first three project periods, we have successfully elaborated several parts of the project proposal. In the first part of the project devoted to the development of a methodology to find selective glycosidase inhibitors, we performed numerous optimizations of synthetic procedures to obtain the required compounds in sufficient amount and quality. Due to complications encountered in the subsequent screening experiments (and hit identification) using the glycosidases we decided to perform and optimize the procedure using more accessible/cheaper enzymes (carbonic anhydrases which are available within collaboration with the group of Dr. Rezacova from IOCB). These experiments provided us with important information about the efficiency and limitations of the methodology. By comparing different approaches (synthetic vs phage display libraries) we found that the phage display libraries lead to selection of better/more selective inhibitor-peptide conjugates. We plan to further expand the developed methodology to find selective inhibitors also for more challenging enzymes including sugar-processing enzymes. Toward this direction, we initiated experiments with screening of the peptide libraries using whole intact cells instead of purified proteins. In this way, the system will be closer to the natural situation where the target enzymes are located at the cell surface. We have also successfully solved the initial problems with hit peptide sequencing (usually done by Edman sequencing, which is time consuming and low throughput). We now use a ladder-based sequencing based MALDI-MS experiments where larger amounts of hit peptide sequences can be identified.
We also moved forward in the second part of the project devoted to the construction of glycopeptide libraries. After extensive optimizations, we are now able to construct sophisticated heteroglycopeptide libraries based on the proposed click reaction/deprotection steps. We have sucessfully performed screening of such libraries toward model protein (Concanavalin A) and initiated a collaboration where we aim at more interesting, cancer-related proteins (Galectins). First hit glycopeptide sequences have been identified and resynthesized and we will now evluate the influence of the individual parts (sugar moieties and peptide backbone) on the binding and selectivity.
To study glycoconjugates from a different perspective, we succeeded in the synthesis and metabolic incorporation of a new TCO sialic acid derivative. In combination with the developed fluorogenic bioorthogonal reactions, we were able to visualize and inspect glycoconjugates on live cells. Thanks to the favorable incorporation into specific glycostructures of TCO-Sia, we plan to employ this strategy in studies related to the control/inhibition/activation of the attachment and entry of various viruses into host cells. In particular, we plan to use the Influenze A virus as a model system. In addition, the TCO-Sia derivative in combination with other known and complementary metabolic labeling strategies should enable performing studies devoted to identification of e.g. sugar-protein, protein-protein interactions in a cellular context, that will deliver important insight into this type of events at the molecular level.
Our results are in good line with the outlined objectives. We have initiated and developed several new approaches to study the fascinating biology of glycoconjugates. We believe that this project will deliver fundamentally new approach to study sugar processing enzymes, and as such, will provide us with valuable tools which will finally contribute to better treatment and diagnosis of various severe diseases.