HEPARIN AND HEPARAN SULPHATE: FROM SEQUENCE DETERMINATION TO THERAPEUTIC STRATEGIES FOR PARKINSON’S DISEASE

Project Information

HS-SEQ

Grant agreement ID: 899687

DOI

10.3030/899687

Project closed

EC signature date 28 May 2020

Start date 1 October 2020

End date 30 September 2025

Funded under

• EXCELLENT SCIENCE - Future and Emerging Technologies (FET)

Total cost

€ 3 998 385,00

EU contribution

€ 3 848 385,00

Coordinated by

UNIVERSITEIT UTRECHT
Netherlands

Periodic Reporting for period 3 - HS-SEQ (HEPARIN AND HEPARAN SULPHATE: FROM SEQUENCE DETERMINATION TO THERAPEUTIC STRATEGIES FOR PARKINSON’S DISEASE)

Reporting period: 2023-04-01 to 2025-09-30

Summary of the context and overall objectives of the project

A major challenge facing an ageing society is the burden of diseases with unmet medical needs such as neurological conditions including Parkinson’s, Alzheimer’s disease and multiple sclerosis but also cancer, diabetes, wound healing and inflammatory diseases. Addressing these needs requires radical new thinking to create innovative, cost-effective and safe solutions – one emerging possibility is the next generation of heparin and heparan sulfate (HS)-based therapeutics.

HS are highly sulfated polysaccharides found in animal tissues. HS binds a variety of protein ligands and regulates a range of biological activities, including development, angiogenesis, blood coagulation and tumor metastasis. Alterations in HS expression are associated with a multitude of diseases. By understanding their functions, we can unlock a tremendous potential for diverse biomedical applications. However, due to a fundamental technological bottleneck, it has been difficult to harness this potential.

HS-SEQ is a multi-interdisciplinary European consortium that will tackle this bottleneck by developing an integrated technology platform that can simultaneously record multiple molecular properties, such as molecular weight by mass spectrometry, collisional cross sections by ion mobility spectroscopy and vibrational properties by gas-phase infrared ion spectroscopy, and thus effectively sequence heparin/HS to determine the functional codes within. Implementation of the analytical platform requires a large collection of well-defined HS saccharides to generate reference databases that can patch molecular properties to structural features. This will be achieved in this program by implementing automated chemoenzymatic enzymatic synthesis of HS saccharides and fractionation protocols that can provide large collections of well-defined compounds. The collection of well-defined HS saccharides will also provide unique opportunities to develop an antibody toolkit to identify epitopes expressed by cells and tissues. Transformational applications of the new technologies will be pursued including the identification of HS codes that promote generation of dopaminergic neurons from pluripotent stem cells for cell replacement therapy in Parkinson’s disease and to achieve unprecedented in-depth analysis of pharmaceutical and next generation heparins.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

A library of structurally diverse hexa- octa- and deca-saccharides has been prepared by the chemical synthesis of oligosaccharides modified by a benzyl ether at one of the C-6 hydroxyls of GlcN to control enzymatic diversification by sulfotransferases and a C-5 epimerase. Orthogonal methods including size exclusion, strong anion exchange, hydrophilic interaction liquid chromatography, and a reverse phase C18 have been employed to fractionate heparin to give oligosaccharides of varying sizes, which have been characterized by IMS-MS.

A chemoenzymatic methodology has been developed that can provide HS oligosaccharides composed of two or more NS domains separated by NA domains of different length. Competition binding studies showed that the length of an NA domain modulates the binding of the chemokines. Well-defined heparin mimetics of different chain length have been analyzed as inhibitors of cell attachment of SARS-Cov-2.

An automation platform has been developed for the chemo-enzymatic synthesis of HS oligosaccharides based on a catch and release approach in which enzymatic reactions are performed in solution and product purification is accomplished by capture onto a resin followed by washing steps and expeditious release for a subsequent enzymatic transformation.

HS oligosaccharides have been analyzed by IMS-MS and cryogenic IR spectroscopy. It can unambiguously differentiate sulfation patterns in hexuronic acids and patterns of sulfation. To unravel spectral complexity, we employed a machine learning algorithm based on Random Forest. Proof of principle has been provided that the combination of MS-IMS-IR into a single, continuous analytical workflow is effective for HS analysis. A Synapt G2-S (Waters) has been augmented with a cryogenic ion trap for multiplexing of different mass- and/or ion-mobility-selected ions.

Poorly characterised scFv’s (single chain variable fragments) have been produced in E. coli. Single amino acids in scFv’s were substituted using targeted mutagenesis. Analysis of specificity of scFv antibodies has been performed using HS oligosaccharide microarray technology. We have shown the importance of amino acid changes in the complementarity determining region 3 (CDR3) in scFvs for recognition of new patterns in rat kidney tissue. Interactions of HS oligosaccharides with scFvs have been studies by NMR and computational approaches.
A rapid protocol has been developed to generate DA neurons from hPSCs. Eighteen fractionated HS saccharide pools were examined, and lead compounds identified that can promote neuritogenesis in TH+ neurons. To examine the function of sulfatases in this context Parkinson’s disease, SULF1-/-, SULF2-/- and double SULF1-/-;SULF2-/- hPSC cell lines have been generated.
Heparin/LMWH, modified heparins and clinically used heparins have been analyzed by the new analytical methodologies.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Conventional approaches for the analysis of heparin, HS and other GAGs can provide compositions but not molecular structures. HS-Seq will introduce an analytic approach that can identify isomeric structures of heparin, HS and other GAGs. This innovation will be possible by the introduction of new instrumentation that can simultaneously record multiple molecular properties including molecular weight by mass spectrometry (MS), collisional cross sections (CCS) by ion mobility spectroscopy (IMS) and vibrational properties by gas phase infra-red (IR) ion spectroscopy. Another innovation of the HS-Seq program is that that analytical method development will make critical use of synthetic standards that can provide a library of IMS and IR-ion properties.

Conventional methods for the preparation of HS oligosaccharides cannot readily provide collections of HS oligosaccharides for analytic method development, to determine ligand requirements of HS-binding proteins and substrate specificities of HS-biosynthetic enzymes, and to HS based therapeutics. HS-Seq will introduce automated chemo-enzymatic methodologies that can overcome the promiscuity of biosynthetic enzymes and provide large collections of compounds.

The HS saccharides will also be exploited in a unique manner to develop an antibody toolkit to identify HS-epitopes expressed by cells/tissues and harness the potential of HS in regenerative medicine, in particular to underpin cell replacement therapy for PD.

HS-SEQ will be transformative by providing (i) new technologies to characterize pharmaceutical heparin which is a 11 billion dollar industry (30% European share) resulting in its safer use and wider application; (ii) offering new avenues to develop diagnostics and therapeutics for diseases with unmet medical needs and specifically we will develop novel treatment approaches for PD; (iii) training the future European scientific and industrial leadership who can drive glycoscience-based advances in biomedicine; and (iv) increase competitiveness of small and medium sized European companies.

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