Periodic Reporting for period 4 - RibiTool (Ribitol-phosphate: chemical tools to probe the biology of a unique mammalian carbohydrate)
Periodo di rendicontazione: 2024-08-01 al 2025-07-31
Sugars are commonly known for the nutritional role they play in our bodies. Aside from providing much-needed energy that fuels our cells, however, sugars are important for many other biological processes, including for structural stability and communication between cells and their surroundings. This project focuses on a specific type of sugar that helps muscle cells to make vital connections with the surrounding tissue. It is well established that genetic variations affecting the cell’s ability to produce these sugars leads to a group of inherited muscular dystrophies termed alpha-dystroglycanopathies. Yet many questions remain about exactly how cells control the production of these sugars and how various genetic changes lead to the wide spectrum of symptoms and disease outcomes that has been observed in patients suffering from alpha-dystroglycanopathies.
This ERC-funded research programme aims to address a gap in available technologies to study these sugars, thereby contributing to a more comprehensive understanding of the processes that are at the basis of the associated disorders. It achieves this by developing a new toolbox to study the sugar and the cell’s machinery responsible for producing it. Specific objectives include:
1) generation of small molecules that can label, or flag up, the sugar of interest in the context of cells
2) in vitro production of individual components of the cellular machinery that is responsible for the production of the sugar
3) generation of small molecules that can interfere with the machinery from point (2) above
4) application of the generated technology to study the sugar in its natural environment and assess the effects of interfering with its production machinery
The project has provided new knowledge about how we can detect the sugar of interest in cells and how we can interfere with its production. It has delivered a set of new synthetic molecules that enable visualisation of the sugars directly in cells by fluorescence imaging. Additionally, it has established new approaches that can be applied to study how manipulation or loss of individual components of the cellular machinery responsible for the production of the sugar impacts on sugar levels and downstream cellular functions. Upon further optimisation, the established technologies will enable us to unravel the molecular consequences of disease-causing genetic changes on the sugar of interest, providing new insights into the disease process and possible avenues for new therapeutic strategies.
This ERC-funded research programme aims to address a gap in available technologies to study these sugars, thereby contributing to a more comprehensive understanding of the processes that are at the basis of the associated disorders. It achieves this by developing a new toolbox to study the sugar and the cell’s machinery responsible for producing it. Specific objectives include:
1) generation of small molecules that can label, or flag up, the sugar of interest in the context of cells
2) in vitro production of individual components of the cellular machinery that is responsible for the production of the sugar
3) generation of small molecules that can interfere with the machinery from point (2) above
4) application of the generated technology to study the sugar in its natural environment and assess the effects of interfering with its production machinery
The project has provided new knowledge about how we can detect the sugar of interest in cells and how we can interfere with its production. It has delivered a set of new synthetic molecules that enable visualisation of the sugars directly in cells by fluorescence imaging. Additionally, it has established new approaches that can be applied to study how manipulation or loss of individual components of the cellular machinery responsible for the production of the sugar impacts on sugar levels and downstream cellular functions. Upon further optimisation, the established technologies will enable us to unravel the molecular consequences of disease-causing genetic changes on the sugar of interest, providing new insights into the disease process and possible avenues for new therapeutic strategies.
A set of unnatural sugar molecules has been generated that have been equipped with a chemical identification tag allowing us to study the target sugar in a biological context. We published a review on the concept of using unnatural sugars to study biological processes in Biochemical Society Transactions (2021). Additionally, as part of the work towards these molecules, we developed a new chemical synthesis methodology which has been published in Chemical Science (2023).
Secondly, the proposal aimed to study a set of human proteins that are involved in the production of the sugar of interest by biochemical analysis and 3-D structure analysis. Because the production and particulary the isolation, in stable form, of these proteins is very challenging, most of our work has focused on a set of homologous bacterial proteins (TarI, TarK, TarL) that perform similar functions to the human targets. We have established a working pipeline for the successful isolation of each of these proteins and developed assays that enable us to test their functional integrity with the use of synthetic substrates produced in house. One of these assays has also been applied to inhibition studies to test if the molecules produced below can perform their desired blocking effect on the protein TarI. As part of this work, we have additionally developed a new enzymatic method for the generation of one of the protein substrates.
Furthermore, two different sets of synthetic molecules have been delivered that are designed to block and label the human proteins mentioned above. These will find use as research tools to dissect the consequences of interfering with sugar production in cells without the need for genetic manipulation, as well as for the detection of protein levels under normal vs disease-mimicking conditions. We published a review on the topic of activity-based protein profiling in Molecules (2020). Initial proof of concept analysis using the TarI assay described above has revealed that some of these molecules do indeed act as blockers of their anticipated target protein.
Finally, a cellular assay has been established for the testing of the unnatural sugars that have been generated above. The results show that three of these molecules label a number of proteins inside cells. The identity of these unknown proteins will be the topic of future research. Additionally, we have demonstrated that one of the molecules can label the intended sugar when attached to its target protein. A manuscript on this work has been published in RSC Chemical Biology (2025). This new platform will enable investigations into the levels and localisation of the sugar of interest upon manipulation of the sugar production pathway or in response to disease-causing mutations.
Aside from the publications mentioned above, the results of the project have also been disseminated through presentations at a number of scientific conferences in the UK (e.g. the RSC Carbohydrate Group Winter meeting, RSC Organic Division North East regional meeting, Biochemical Society meeting) and the US (Gordon Conference on Carbohydrates, ACS Spring National Meeting).
Secondly, the proposal aimed to study a set of human proteins that are involved in the production of the sugar of interest by biochemical analysis and 3-D structure analysis. Because the production and particulary the isolation, in stable form, of these proteins is very challenging, most of our work has focused on a set of homologous bacterial proteins (TarI, TarK, TarL) that perform similar functions to the human targets. We have established a working pipeline for the successful isolation of each of these proteins and developed assays that enable us to test their functional integrity with the use of synthetic substrates produced in house. One of these assays has also been applied to inhibition studies to test if the molecules produced below can perform their desired blocking effect on the protein TarI. As part of this work, we have additionally developed a new enzymatic method for the generation of one of the protein substrates.
Furthermore, two different sets of synthetic molecules have been delivered that are designed to block and label the human proteins mentioned above. These will find use as research tools to dissect the consequences of interfering with sugar production in cells without the need for genetic manipulation, as well as for the detection of protein levels under normal vs disease-mimicking conditions. We published a review on the topic of activity-based protein profiling in Molecules (2020). Initial proof of concept analysis using the TarI assay described above has revealed that some of these molecules do indeed act as blockers of their anticipated target protein.
Finally, a cellular assay has been established for the testing of the unnatural sugars that have been generated above. The results show that three of these molecules label a number of proteins inside cells. The identity of these unknown proteins will be the topic of future research. Additionally, we have demonstrated that one of the molecules can label the intended sugar when attached to its target protein. A manuscript on this work has been published in RSC Chemical Biology (2025). This new platform will enable investigations into the levels and localisation of the sugar of interest upon manipulation of the sugar production pathway or in response to disease-causing mutations.
Aside from the publications mentioned above, the results of the project have also been disseminated through presentations at a number of scientific conferences in the UK (e.g. the RSC Carbohydrate Group Winter meeting, RSC Organic Division North East regional meeting, Biochemical Society meeting) and the US (Gordon Conference on Carbohydrates, ACS Spring National Meeting).
The unnatural sugars, inhibitors and probes generated in the project are new molecules. We have additionally established a new synthetic methodology that will have wide impact in the field of biological and medicinal chemistry. In particular, one step of the synthetic strategy towards our synthetic sugar molecules presents a significant advance to the state of the art and has as such been published as a methodological research paper.
Having identified successful research tools and established that some of these can label the target sugar in live cells, we have begun to proceed with follow-up experiments in which the tools are used for in-depth characterisation of the sugar of interest, the cell’s machinery responsible for producing it, and the consequences of genetic variations that cause muscular dystrophy. This work provides a conceptually completely new approach, based on innovative tools and underpinned by novel chemistry, to complement and strengthen the existing toolkit for the study of the study of this sugar and its role in disease.
Another impact of our work is in the area of microbial glycobiology. Some gram-positive bacteria, including the pathogen Staphylococcus aureus, contain a cell surface polymer made up of the same sugar as that subject of this study and which plays established roles in infectivity and antimicrobial resistance. Therefore, the compounds synthesised in this work will also be applicable to the study of these bacterial sugars and their cellular production. Likewise, the work on characterisation and inhibition of the proteins TarI, TarK and TarL will have direct relevance to research aimed at targeting these proteins for new antibacterial strategies.
Having identified successful research tools and established that some of these can label the target sugar in live cells, we have begun to proceed with follow-up experiments in which the tools are used for in-depth characterisation of the sugar of interest, the cell’s machinery responsible for producing it, and the consequences of genetic variations that cause muscular dystrophy. This work provides a conceptually completely new approach, based on innovative tools and underpinned by novel chemistry, to complement and strengthen the existing toolkit for the study of the study of this sugar and its role in disease.
Another impact of our work is in the area of microbial glycobiology. Some gram-positive bacteria, including the pathogen Staphylococcus aureus, contain a cell surface polymer made up of the same sugar as that subject of this study and which plays established roles in infectivity and antimicrobial resistance. Therefore, the compounds synthesised in this work will also be applicable to the study of these bacterial sugars and their cellular production. Likewise, the work on characterisation and inhibition of the proteins TarI, TarK and TarL will have direct relevance to research aimed at targeting these proteins for new antibacterial strategies.