Periodic Reporting for period 3 - CARBOCENTRE (Activity-Based Profiling of Glycoprocessing Enzymes for Human Health and a Sustainable Society)
Reporting period: 2024-06-01 to 2025-11-30
What is the problem being addressed?
Carbohydrates, in the form of oligosaccharides and glycoconjugates, represent the most abundant and diverse class of biomolecules on Earth. The enzymes responsible for building and breaking down these complex sugars — known as carbohydrate-active enzymes (CAZymes) — are found across all forms of life. Despite their ubiquity and biological importance, our ability to study and manipulate these enzymes is limited by the lack of specific molecular tools that can target them in living systems and in industrial settings. This prevents impact in medical and biotechnological sectors.
Why is it important for society?
Understanding and controlling CAZymes has far-reaching implications for both human health and sustainable industry. In medicine, these enzymes are key players in diseases such as cancer and lysosomal storage disorders. In biotechnology, they are central to applications such as biomass conversion to biofuels and the development of enzymes for use in food and cleaning products. Developing new tools to visualize, modulate, and investigate these enzymes can thus lead to improved diagnostics, therapies, and greener industrial processes.
What are the overall objectives?
The Carbocentre project aims to revolutionize our understanding and control of glycoprocessing enzymes through a multidisciplinary approach. By integrating structural biology, enzymology, computational chemistry, organic synthesis, and chemical biology, the project has three core scientific objectives:
1. To design molecular tools that selectively capture the active sites of three key CAZyme families — retaining glycosidases, inverting glycosidases, and glycosyltransferases.
2. To combine biochemical and 3D structural analyses with computational models to map the reaction mechanisms of these enzymes.
3. To use this mechanistic understanding to develop probes and inhibitors equipped with fluorescent and bio-orthogonal tags for enzyme imaging, manipulation, and discovery.
These tools will be applied in two major domains:
• In human health, to enable visualization, diagnosis, and therapeutic targeting of enzymes involved in viral infection, cancer and genetic diseases.
• In biotechnology, to explore, discover and harness natural enzyme diversity for sustainable applications, including biofuel production and household product formulation.
Through open sharing of reagents and knowledge, Carbocentre also promotes collaboration and impact across the international glycobiology community, accelerating both scientific discovery and practical innovation.
Carbohydrates, in the form of oligosaccharides and glycoconjugates, represent the most abundant and diverse class of biomolecules on Earth. The enzymes responsible for building and breaking down these complex sugars — known as carbohydrate-active enzymes (CAZymes) — are found across all forms of life. Despite their ubiquity and biological importance, our ability to study and manipulate these enzymes is limited by the lack of specific molecular tools that can target them in living systems and in industrial settings. This prevents impact in medical and biotechnological sectors.
Why is it important for society?
Understanding and controlling CAZymes has far-reaching implications for both human health and sustainable industry. In medicine, these enzymes are key players in diseases such as cancer and lysosomal storage disorders. In biotechnology, they are central to applications such as biomass conversion to biofuels and the development of enzymes for use in food and cleaning products. Developing new tools to visualize, modulate, and investigate these enzymes can thus lead to improved diagnostics, therapies, and greener industrial processes.
What are the overall objectives?
The Carbocentre project aims to revolutionize our understanding and control of glycoprocessing enzymes through a multidisciplinary approach. By integrating structural biology, enzymology, computational chemistry, organic synthesis, and chemical biology, the project has three core scientific objectives:
1. To design molecular tools that selectively capture the active sites of three key CAZyme families — retaining glycosidases, inverting glycosidases, and glycosyltransferases.
2. To combine biochemical and 3D structural analyses with computational models to map the reaction mechanisms of these enzymes.
3. To use this mechanistic understanding to develop probes and inhibitors equipped with fluorescent and bio-orthogonal tags for enzyme imaging, manipulation, and discovery.
These tools will be applied in two major domains:
• In human health, to enable visualization, diagnosis, and therapeutic targeting of enzymes involved in viral infection, cancer and genetic diseases.
• In biotechnology, to explore, discover and harness natural enzyme diversity for sustainable applications, including biofuel production and household product formulation.
Through open sharing of reagents and knowledge, Carbocentre also promotes collaboration and impact across the international glycobiology community, accelerating both scientific discovery and practical innovation.
The global objective of CARBOCENTRE is the design and application of activity-based probes for characterization, detection and inhibition of glycoprocessing enzymes, focusing on three major strands of increasing complexity: (1) retaining glycoside hydrolases (ret-GHs, 1st research strand), inverting glycoside hydrolases (inv-GHs, 2nd research strand, challenging as no ABP ideas exist) and glycosyltransferases (GTs, 3rd research strand, extremely challenging as even inhibitors for this class are lacking).
These compounds are then applied across two domains of application: Biotechnology and Medicine. In biomedicine, we expect the reagents and methods developed to find application in diagnosis (such as for lysosomal storage diseases, and other diseases where lack of enzyme activity is key), as readouts for drug discovery (such as in competitive and high throughput approaches), as enzyme stabilisers (pharmacological chaperones) and as drugs themselves, such as in anti-cancer and anti-viral applications. In biotechnology we expect application in enzyme discovery in complex systems (soil, microbiota, secretomes etc), industrial enzyme characterisation, as readouts for directed evolution; essentially to provide functional data to the wealth of sequence data.
The strands and the application domains are based around a common ethos of understanding and exploiting enzyme mechanism.
Since the start of the program, most progress has been made on strand 1, retaining glycoside hydrolases. By harnessing 3-D structure and computation, ABPs have designed and synthesized for a considerable area of enzymes. In biomedicine, we have diagnostics and chaperones for very many families of enzymes involved in genetic disease, we have readouts for high throughput fluorescence polarization assays allowing for high throughput drug screening. We have developed enzyme inhibitors, based upon probe designs, for application in anti-cancer applications through heparanase inhibition (a spin-out company has been established, aided by ERC proof of concept funds), we have developed inhibitors of human enzymes involved in virus maturation − initially as anti-Sars-Cov2 compounds − and achieved first-in class neuraminidase inhibitors (IP protection in progress). In the biotechnology arena, we have focused our work on discovery (enzymes in bacterial and fungal secretomes, enzymes in animal microbiota), on the enzyme responsible for the degradation of highly recalcitrant plant polysaccharides (cellulose, xylan, xyloglucan, starch) and on the key industrial challenges of enzyme stability and engineering. In research strand 2 (inverting glycoside hydrolases), we need to overcome the challenges of the absence of a catalytic nucleophile. We are working on human glucosidase I (again an anti-HIV/Covid target) and screening newly designed libraries for compounds – including computer modeling − which are looking promising. In strand 3 (glycosyltransferases), we have been focusing on expressing enzymes for study, which is being very challenging and fundamental studies of reaction coordinate, though computation and 3D structures – including AI-generated structures. We also designed libraries of inhibitors, competitive in the first instance and will work on introducing covalency going forward. We have successfully designed competitive inhibitors of glycogen phosphorylase (a “simplified” glycosyltransferase) and α4-galactosyltransferase and hope to build upon this in the future.
These compounds are then applied across two domains of application: Biotechnology and Medicine. In biomedicine, we expect the reagents and methods developed to find application in diagnosis (such as for lysosomal storage diseases, and other diseases where lack of enzyme activity is key), as readouts for drug discovery (such as in competitive and high throughput approaches), as enzyme stabilisers (pharmacological chaperones) and as drugs themselves, such as in anti-cancer and anti-viral applications. In biotechnology we expect application in enzyme discovery in complex systems (soil, microbiota, secretomes etc), industrial enzyme characterisation, as readouts for directed evolution; essentially to provide functional data to the wealth of sequence data.
The strands and the application domains are based around a common ethos of understanding and exploiting enzyme mechanism.
Since the start of the program, most progress has been made on strand 1, retaining glycoside hydrolases. By harnessing 3-D structure and computation, ABPs have designed and synthesized for a considerable area of enzymes. In biomedicine, we have diagnostics and chaperones for very many families of enzymes involved in genetic disease, we have readouts for high throughput fluorescence polarization assays allowing for high throughput drug screening. We have developed enzyme inhibitors, based upon probe designs, for application in anti-cancer applications through heparanase inhibition (a spin-out company has been established, aided by ERC proof of concept funds), we have developed inhibitors of human enzymes involved in virus maturation − initially as anti-Sars-Cov2 compounds − and achieved first-in class neuraminidase inhibitors (IP protection in progress). In the biotechnology arena, we have focused our work on discovery (enzymes in bacterial and fungal secretomes, enzymes in animal microbiota), on the enzyme responsible for the degradation of highly recalcitrant plant polysaccharides (cellulose, xylan, xyloglucan, starch) and on the key industrial challenges of enzyme stability and engineering. In research strand 2 (inverting glycoside hydrolases), we need to overcome the challenges of the absence of a catalytic nucleophile. We are working on human glucosidase I (again an anti-HIV/Covid target) and screening newly designed libraries for compounds – including computer modeling − which are looking promising. In strand 3 (glycosyltransferases), we have been focusing on expressing enzymes for study, which is being very challenging and fundamental studies of reaction coordinate, though computation and 3D structures – including AI-generated structures. We also designed libraries of inhibitors, competitive in the first instance and will work on introducing covalency going forward. We have successfully designed competitive inhibitors of glycogen phosphorylase (a “simplified” glycosyltransferase) and α4-galactosyltransferase and hope to build upon this in the future.
At the start of the project, the state of the art was activity-based probes for retaining, predominantly beta- predominantly exo-glycosidases involved in human disease. We have gone beyond that with extensive probes for alpha glycosidases, and for multiple subsite endoglycosidases. We have developed ABPs that work on more complex systems, with non-canonical mechanism with cysteine and tyrosine nucleophiles and have rationalized their molecular mechanisms. We have extended medical work into successful anti-cancer and anti-viral applications, into clinically relevant biofilm degradation, the novelty allowing the establishment of strong IP positions. We have taken these technologies into the biotechnology sector focusing on the key societal challenges of green technologies, energy security and microbiotal health. Though collaboration, we have established diverse new projects in enzyme design and optimization by artificial intelligence, and in a “personalized medicine” human gut health – for example reactivation of glucuronidated drugs by gut organisms and cataloging of the individual enzymes of diverse microbiomes at an individual organism level. Sharing our probes with the wider research community was a central commitment of the original grant application, and we are pleased to report that this goal has been achieved with striking success. The widespread uptake of our compounds by researchers working beyond the conceptual scope of the Carbocentre program highlights both the broad utility and the unbiased nature of our reagents, which reliably report on glycoprocessing enzyme activity irrespective of biological context or intended application.
In the biotechnology strand we expected to see developments into (a) the degradation of recalcitrant – heavy substituted, xylanases – a key industrial challenge (b) looking at (beta) mannan degradation, another societal challenge (c) moving into individual enzymes of the microbiota in live animal systems, (d) bridging biotechnology and medicine though more studies into biofilm degrading enzymes and (e) moving into the relatively unstudied area of marine polysaccharides. In biomedicine, we hope to crack (a) challenges in N-acyl sugar degrading systems (b) 3D and computing will be heavily applied to the inverting glucosidase I arena, (c) we will new establish methods to allow development of compounds that allow discrimination between human enzymes with similar specificity (such as GBA1 and GBA2) and we will build on our anti-virus work for next generation anti-virals and diagnostics. A key focus will be glycosyltransferases where we hope to establish specific inhibitors though synthetics libraries, bump and hole variants, and application of computation and structural analysis.
We are confident that AI and directed evolution approaches using the probes as readout, will lead to major breakthroughs in enzyme engineering.
In the biotechnology strand we expected to see developments into (a) the degradation of recalcitrant – heavy substituted, xylanases – a key industrial challenge (b) looking at (beta) mannan degradation, another societal challenge (c) moving into individual enzymes of the microbiota in live animal systems, (d) bridging biotechnology and medicine though more studies into biofilm degrading enzymes and (e) moving into the relatively unstudied area of marine polysaccharides. In biomedicine, we hope to crack (a) challenges in N-acyl sugar degrading systems (b) 3D and computing will be heavily applied to the inverting glucosidase I arena, (c) we will new establish methods to allow development of compounds that allow discrimination between human enzymes with similar specificity (such as GBA1 and GBA2) and we will build on our anti-virus work for next generation anti-virals and diagnostics. A key focus will be glycosyltransferases where we hope to establish specific inhibitors though synthetics libraries, bump and hole variants, and application of computation and structural analysis.
We are confident that AI and directed evolution approaches using the probes as readout, will lead to major breakthroughs in enzyme engineering.