Application of activity-based glycosidase probes for mechanism, enzyme discovery and clinical diagnosis

An expanding human civilization is under enormous pressure to find environmentally sustainable approaches to providing food, energy, diagnostics and medicines. By 2030, approximately 50% more food will need to be produced from the same area of land, but with less water, more expensive energy, and with the constraints and impact of climate change. The demand to replace environmentally damaging fossil fuels by renewable energy and sustainable feed stocks is equally urgent. An expanding and ageing global population faces accelerating health problems such as infectious and inherited disease, antimicrobial resistance and dementia, placing an enormous burden on health and social services, unless new therapeutic approaches are developed.

The development of new approaches to dissect the diverse and wonderful roles for carbohydrates in living cells is now a major challenge for modern cell biology, biochemistry and medicine. The colossal potential of carbohydrates is reflected in the multiplicity of functions for glycans; in addition to acting as energy sources, carbohydrates play central roles in cell and organism structure, communication (carbohydrates are "the language of the cell"). Indeed, we stand on the cusp of an exciting new "glyco-age" which expands beyond the understanding of cellular roles for glycans, through to the development of new carbohydrate-based vaccines and drugs and diagnostics. What scientists and society need, are tools that allow us to probe and utilize carbohydrate chemistry.

This grant submission aims to develop and apply a new class of chemical reporter we have developed. These "activity-based probes" will allow us to make breakthroughs in different areas of carbohydrate science; linked by a common method. We will

1. Develop and apply chemical tools for enzyme discovery - allowing us to identify the enzymes industry needs for sustainable energy production
2. Expand these tools into areas of human health and disease: building upon fundamental academic insight to deliver diagnostics for inherited disease and cancer

Research in these two areas will be highly synergistic with medical tools aiding biotechnology and vice versa. Carbohydrates have for a long time been a challenging area of "Cinderella science". New tools, new approaches and new ways of working will help society benefit from the power and promise of carbohydrates.

The development of new approaches to dissect the diverse roles for carbohydrates in living cells is now a major challenge for modern cell biology, biochemistry and for applications in biotechnology and medicine. It is clear that despite the plethora of genomic data, relevant functional work is still lacking in these challenging fields. The grant will therefore design and apply aziridine activity-based probes (ABPs) of glycosidases for enzyme discovery, identification and quantification across a range of cutting-edge biological challenges. Central to the ethos is the harnessing of knowledge and experience from different areas of glycosidase research to impact on both biotechnology and cellular biology. Our objectives are to provide a fundamental academic dissection of the mechanism, conformational analysis and reactivity of aziridine and epoxide probes of diverse glycosidases and then apply this knowledge both for the discovery of novel plant cell wall degrading enzymes for biotechnology and to quantify mannosidase and heparanase levels in health and disease; notably genetic disorders and metastasis. We aim to deliver relevant new enzymes, clinical and cellular diagnostics and a method for the assessment of drug regimens in complex cellular samples.

Who will benefit from this research?

1. Industry working in diverse areas of polysaccharide degradation
The degradation of plant-derived biomass is one of the key objectives of BBSRC policy, UK government policy and of many UK and European Biotechnology companies. Therefore the spectrum of "stakeholders" in this work is very large indeed. UK biotechnology, albeit belatedly compared to Scandinavian enterprises, is embarked on major expansions in the "enzymatic treatment of plant polysaccharide" sector. Such enzymes also find everyday application that touches on the general public in their paper and packaging products, in household detergents and washing products. These "day-to-day" benefits of the wider public are in addition to the potential massive benefits of reducing fossil fuel usage, allowing greener industries, and fuel security as part of a balanced UK energy portfolio.

2. Clinicians, clinical diagnostics, patient groups for lysosomal storage disease and the pharmaceutical industry for means of assessing inhibitor regimens.
Lysosomal storage diseases such as Gaucher, Fabry, Pompe are amongst the most common genetically inherited disorders currently. Heparanase drugs have been accelerated into phrase III; so this is a valid target for which our laboratory provided the first 3D structure of the drug target.

How will they benefit from this research?
1. Previous interaction with these industries (following pathways to impact for, previous BBSRC grants) has highlighted key areas of industrial weakness:

New beta-glucosidases and means of assessing them in diverse conditions and in mixtures of enzymes (that render coloured substrate work misleading)

New enzymes for xylan degradation
These are the two areas that WP1 will address and so the industrial beneficiaries will be considerable.
It is clear that information derived from this project will provide UK and European industry with key enzymes, and a basis for their engineering and exploitation, in the plant polysaccharide sector. What the work described in the application provides is a new enzyme portfolio and the release into the public sector of sequences, knowledge of activities and a conceptual methodological framework for enzyme optimisation will be based. In particular this knowledge is in an area "hemi-cellulose degradation" that the key industrial leaders and US National Renewable Energy Laboratory has highlighted as a major bottleneck in their applications.
Results from this grant will be in the public domain within months of publication submission. Industry and policy leaders will therefore have almost immediate access to raw data.

2. Rapid lysosomal storage disease diagnostics are essential for rapid detection and treatment. The ability to visualize, selectively, active proteins makes ABP profiling more advantageous than immune-blotting techniques, which also detect mis-folded proteins and inactive isoforms. Crucially, compared to activity assays, ABPs can also distinguish between multiple proteins or isoforms that are active on the same substrate, allowing for dissection of the contribution of each to overall activity. All of these facets mean that clinicians, clinical diagnostics, patient groups for lysosomal storage disease and the pharmaceutical industry will all benefit greatly when such techniques are demonstrated
Similarly, heparanase is not active in traditional colorimetric or fluorometric assays, and require cumbersome and expensive multistep assays to determine their activities. Therefore there is a desperate need for novel chemical tools that can accurately assess the activities of these enzymes. ABPs offer many advantages over both immunoblotting type techniques (e.g. ELISA) and activity assays, which may make them useful for diagnostic purposes and for assessing the efficiency of inhibitor treatments. Thus we will provide a methodological framework from which the UK pharmaceutical industry could benefit.