Shotgun functional glycomics of heparan sulphate saccharides: generating diverse libraries to decode biological selectivity

Lead Research Organisation: University of Liverpool
Department Name: Sch of Biological Sciences

Abstract

Complex sugars (glycans) are a highly diverse family of molecules with a broad range of functions in biological processes including cell recognition, adhesion, cell-cell communication and signalling. The study of the glycome - the entire set of glycans expressed by particular cells or tissues - is an emerging field which aims to understand how glycan functions underpin the complexity of human biology, and their involvement in disease processes. The selective interaction of proteins with glycans is one of the keys to their biological functions. A number of new technologies have emerged to support the development of 'glycomics' studies - large-scale studies of glycan structural diversity and the selective interactions of glycans with their matching protein partners which underpins their functions. However, these approaches do not directly reveal functional properties, especially for some complex classes of glycans like the heparan sulphate (HS) family of sulphated glycans which are responsible for regulation of a wide range of biological processes including growth factor signalling, enzyme activity and cell adhesion. We now need to have the tools to ask how specific structures control specific proteins to control biological systems. In this project we propose to develop a new approach to address this question by generating a large library of novel HS glycans with a wide range of structures, and screening them in biological assays, followed by analysis of selected structures. This will permit us to evaluate the structure-activity relationships of HS at higher throughput for the first time. This 'shotgun' library approach will provide a powerful and generic new tool for decoding the function of the HS glycome by allowing specific glycan structures to be matched to specific biological functions. In the future this strategy could provide new information that could be translated into applications in biotechnology and drug development.

Technical Summary

Decoding post-translational modifications of proteins, including the glycome, is a critical facet of the post-genome era, since they modify the functional proteome. Strategies have emerged to support 'glycomics' (large-scale) studies of glycan structure diversity and their selective interactions with cognate proteins. However to date these approaches have lacked the ability to directly generate functional data, particularly in the case of the complex heparan sulphate (HS) glycans. There is growing evidence that functional specificity exists in HS-dependent control of cognate protein activities, but technologies to truly address this question have been a bottleneck. The aim of this project is to develop a 'shotgun' functional glycomics approach for exploring the structure-activity relationships of HS. Generating a large random (unbiased) library of novel and diverse saccharides within HS chemical space, and screening them in test-bed bioassays, will permit detailed evaluation of biological specificity at higher throughput for the first time. We will: 1. Extend the chemical space of natural HS saccharide libraries covered by our existing pilot library (using a wider range of starting materials and saccharide generation methods). 2. Enhance the diversity of the HS library using enzymic approaches (recombinant sulfotransferases in concert with novel natural, semi-synthetic and synthetic substrates). 3. Screen the HS libraries to identify hits in test-bed bioassays (including FGF growth factor and Slit/Robo signalling). 4. Initiate structural analysis of HS library components and screening hits (to confirm structural diversity and identify novel structure-activity relationships, using state-of-the-art MS methods). This project will generate unique resources and a powerful new strategy for decoding the functions of the HS glycome. Such data will provide high value functional information on structure-activity relationships for the HS family.

Planned Impact

Expected Beneficiaries: This project is expected to have wide impact in many areas of biomedicine and biotechnology related to medicine in particular (eg. applications in diagnostics, drug discovery and regenerative medicine). This is due firstly to the relevance of HS biology to many disease processes (eg. cancer, inflammation, neurodegeneration, wound repair) and also fundamental biological processes that are critical for stem cell control and tissue engineering. New methods for determining specific HS targets will open up opportunities for breakthroughs in identification of novel information on HS specifity of biological action which could underpin commercial exploitation. More widely, the glycomics strategies will be of broad interest in view of the wide application potential of glycans in general. Better understanding of HS biology is also relevant to societal impacts. For example, Prof. Turnbull has met with families involved in the UK Hereditary Multiple Exostoses (HME) Support group (www.hmesg.org.uk). HME is caused by genetic deficiencies in HS biosynthesis that result in a multifactorial clinical problems including growth deficiency, bone tumours and premature death. The HME group are interested in promoting better understanding of the disease, current research into its causes and symptoms, and potential new treatments. Communications and Engagement: Commercial: The Turnbull lab has a number of active collaborations with Industry both in the UK and overseas, including IRL Ltd (a partner on this project), and research collaborations with SpheriTech Ltd (Runcorn; BBSRC CASE) and Summit (Dextra) Ltd (Reading). All these projects will benefit from the project and we will actively develop these partnerships as described in the Impact plan statement. Prof. Turnbull is also actively involved in discussions with the NorthWest Development Agency regarding the establishment of a Centre of Excellence in Glycosciences, aimed at networking of the high level of academic expertise in this field in the northwest of England with commercial partners. Societal: The UK Hereditary Multiple Exostoses (HME) Support group has a Liverpool group contact, Tina Read, who is developing web-based resources and information for families with children affected by this disease. The Turnbull group plan to assist them with information on the molecular basis of the disease and how future research might help with new disease treatments, and hosting of a meeting of the national organisation in Liverpool in 2010. General dissemination: We will actively seek to disseminate information about our research efforts to both industry and the general public, through websites (Liverpool Centre for Glycobiology, and University Business Gateway); press releases and opportunities for public speaking. Collaboration: The principal commercial partnership within the proposed project is with IRL Ltd (Wellington, NZ). We have an existing research agreement initiated in 2009 with IRL Ltd on identification of synthetic targets with potential commercial applications in Alzheimers disease and cancer. IRL will have an active interest in outputs regarding new targets and enzymic modification of their synthetic saccharides. Exploitation and Application: There is considerable potential for commercial exploitation of outputs from this project, for example in drug development, stem cell exploitation and tissue engineering. The tools and strategy development aspect of the project may also yield new intellectual property of commercial potential. We will actively and regularly monitor our research output and potential publications with ULive Ltd (the University of Liverpool IP and tech transfer company), protect by patenting, and exploit via out-licencing or development of a spin-out company. For full details see the Impact Plan appended

Publications

10 25 50

publication icon
Puvirajesinghe TM (2012) Array-based functional screening of heparin glycans. in Chemistry & biology

 
Description Developed novel approaches for making and screening HS saccharide libraries
Exploitation Route Further research and application
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Title Engineered heparins 
Description Libraries of selectively chemically modified heparins for applications in heparin-based drug discovery. 
Type Of Material Technology assay or reagent 
Year Produced 2006 
Provided To Others? Yes  
Impact Exploited for a number of publications and to develop drug leads for AD, malaria, ant-cancer therapeutics and spinal repair therapies. 
 
Title Heparan sulphate saccharide libraries 
Description Development of technologies for and production of complex libraries of HS saccharides 
Type Of Material Biological samples 
Year Produced 2006 
Provided To Others? Yes  
Impact Novel discoveries of structure-activity relationships for complex HS saccharides of relevance to biological and disease processes 
 
Description Advanced mass spectrometry for glycomics 
Organisation Imperial College London
Department Department of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of compounds for analysis by MS
Collaborator Contribution Collaboration on advanced glycan analysis by mass spectrometry
Impact Network grants and publications
 
Description Glycomics strategies for heparan sulphates 
Organisation University of Liverpool
Department School of Biological Sciences Liverpool
Country United Kingdom 
Sector Academic/University 
PI Contribution Expertise on structure and activity relationships and glycomics strategies
Collaborator Contribution Collaboration on molecular interactions and interactomics approaches.
Impact Publications, grants and ongoing research collaborations.
 
Company Name IntelliHep Ltd 
Description Spin-out company from University of Liverpool aiming to exploit engineered heparins in drug discovery and biotechnology applications. Company has a number of collaborative projects with external partners, and is also developing an internal pipeline of projects. Company is pre-investment and established a lab in 2010 and seeking further grant and investment income.; http://www.intellihep.com/ 
Year Established 2006 
Impact Early stage development of hit compounds as beta-secretase inhibitors for treating the underlying cause of Alzheimers disease. Option to licence IP from Liverpool University on an anticancer metastatic drug. Employment of research staff and regular funded projects sub-contracted into University labs. Collaborating with China partners on commercial applications of heparin by-products.
Website http://www.intellihep.com/
 
Description Hosting visit to Liverpool University by local ARUK funding supporters 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Regional
Primary Audience Supporters
Results and Impact Contact was invaluable in informing supporters directly about research activities supported in Liverpool by their fund raising efforts

Funding supporters were able to gain insights into new potential treatments for AD, and how their funding is used for research.
Year(s) Of Engagement Activity 2013
 
Description Media releases on research findings 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact University press releases on the novel Alzheimer drug hits we discovered, along with developments in synthetic chemistry of our sugars, and applications in nerve repair and cancer therapeutics

International press coverage, radio interviews (Radio City Liverpool), and TV news (BBC NW Tonight).
Year(s) Of Engagement Activity 2006,2007,2008,2012,2013
 
Description Outreach to Business Community 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Health professionals
Results and Impact Article in magazine Research Intelligence by UoL Business Gateway,(see website link "Sweet Smell of Success" http://www.liv.ac.uk/researchintelligence/issue30/alzheimers.html)

Dissemination of information to business community
Year(s) Of Engagement Activity 2007
URL http://www.liv.ac.uk/researchintelligence/issue30/alzheimers.html
 
Description Public Lecture to Garston Rotary Club 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Lecture to Garston Rotary Club

Contributing the Public Understanding of Science
Year(s) Of Engagement Activity 2008
 
Description Public lecture to Liverpool Soroptimists Club 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Talk sparked many questions and discussion of AD and potential new drugs and diagnostics

Contribution to Public Understanding of Science
Year(s) Of Engagement Activity 2014
 
Description RCUK/BBSRC event: Lifelong Health: Bioscience of Ageing (at Westminster) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Poster highlighting discovery of new class of drugs for AD at the RCUK/BBSRC event: 2007 Lifelong Health: Bioscience of Ageing (at Westminster)

Dissemination of information on research success to MPs and policymakers
Year(s) Of Engagement Activity 2007
 
Description Supporting ARUK fund raising publicity with major London donor 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach National
Primary Audience Supporters
Results and Impact Supporters engaged directly with researcher undertaking research activity.

As above
Year(s) Of Engagement Activity 2013
 
Description Visit to Coleg Cambria, Wrexham. Meet the Scientist & Careers Talk. February 2019. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Visit to Coleg Cambria, Wrexham. Meet the Scientist & Careers Talk. February 2019.
Year(s) Of Engagement Activity 2019