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Final Report Summary - PROBING SULFATION (Probing the role of sulfation)

Proteins, lipids and glycans are, after their synthesis, often processed enzymatically. Examples of this are the phosphorylation, acylation and glycosylation of proteins. Such modifications often have a regulatory function. They can affect the localization and activity of the protein or alter protein complex formation. It has been hypothesized that sulfation, modification of biomolecules with a negatively charged sulfate group, plays a similar regulatory role. It has been suggested that this specific modification affects receptor recognition and cell-cell signaling. The importance of the specific pattern of the sulfate groups and how altering this pattern by removal of sulfate groups influences the outcome of signaling is not fully understood and it is only being unraveled slowly. It is difficult to study these processes with traditional methods and the proposed research therefore aimed at developing chemical tools that can be used (1) to determine the activity of the enzymes involved in sulfation, (2) to introduce sulfate groups site-selectively onto biomolecules in a controlled manner and (3) to identify proteins that bind to these molecules. For the first objective, we started by identifying molecules that could serve as leads for tools that label the enzymes. Screening a small set of potential lead compounds based on fragments of existing inhibitors did not result in suitable leads. We therefore started to design targeted reagents that modify a conserved residue in the active site. Initial experiments with these reagents looked promising and in control experiments we found by serendipity that the reactive group might function as a mechanism-based inhibitor. However, further studies towards the mechanism of action of these compounds suggest that decomposition products likely inhibit the target enzymes rather than the compound. As such, these leads are not suitable for the chemical probes of objective 1 and we abandoned them as leads for chemical probes. However, they did find use in selective functionalization of proteins (objective 2, vide infra). Our failures to identify a suitable lead necessitated an adjustment of the approach. We reasoned that we could obtain the desired probes for objective 1 using a more classical approach, namely functionalizing known inhibitors with a reactive group. However, this is often a time consuming process since the success of the probe largely depends on the inhibitor-reactive group combination and therefore a library of compounds has to be synthesized. Anticipating that similar problems would be faced for objective 3, we decided to first focus our attention on addressing this bottleneck in chemical probe development. We reasoned that a mix-and-match probe synthesis approach would facilitate screening of combinations. By linking probe fragments in situ using orthogonal chemistries and screening the reaction mixture, the synthetic efforts would be minimal. The proof-of-concept studies demonstrate that this strategy indeed can be applied on a variety of model proteins. Not only places this methodology us in the excellent position to revisit the synthesis of probes for objectives 1 and 3, but it also a major step forward for the field in general. The simplicity of this approach – probes are simply prepared by mixing a reactive group with an appropriate ligand/inhibitor – makes it likely that it will be implemented by a large group of scientist. We will provide our first library of reactive groups to researchers that express interest. For the second-generation reagents, we may establish a probe-development kit and commercialize this kit. Also the synthesized probes will find use in academia to address fundamental questions and pharmaceutical companies – to develop assays
For objective 2, we started to develop methodologies that can be used to functionalize proteins and glycans of interest. During the funding period, we reported a method for the site-selective azidation of proteins. The reaction conditions have recently been optimized to reduce non-specific labeling and to enable on cell labeling these results will be published in the near future. The introduced azido group can be modified with functional groups of interest using click chemistry and it may therefore be exploited for the preparation of well-defined posttranlationally modified proteins. Furthermore this strategy will find use in the preparation of bioconjugates and target identification studies. For the modification of glycans – a virtually uncharted field – we established a method that can be used to modify glucans selectively. By studying the factors that affect the selectivity, we created a model that predicts the selectivity of the reaction for any substrate of interest. This a major step forward, since the model makes it feasible to predict the products of more complex substrates. With this model, the methodology may become an attractive tool to prepare modified enzyme substrates and well-defined labeled glycans for fundamental studies, to modify selectively existing glycosylated drugs, and to prepare linker molecules that can be used for bioconjugate drugs.
Overall, the funding period did not result in the desired tools yet. Unforeseen challenges with respect to probe development necessitate us to address these hurdles. This resulted in a variety of innovative technologies that simplify probe synthesis, enable selective azidation of proteins and a predictable method for the selective functionalization of glycans. These developed methodologies form an excellent stepping-stone towards developing reagents to study sulfation and this will place us finally in the position to study the role of sulfation in a biologically relevant context. Furthermore, we foresee that these methodologies will be adopted by the chemical biology and life science field in general.

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Life Sciences
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