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1. Recombinant Heparin: An Old Drug for the Modern World

   Thacker, Bryan ; Thorne, Kristen ; Cartwright, Colin ; Park, Jeeyoung ; Glass, Kimberly ; Chea, Annie ; Jeske, Walter ; Walenga, Jeanine ; Sharfstein, Susan ; Esko, Jeffrey ; et al ( October 2021 , Research and practice in thrombosis and haemostasis)

   null (Ed.)

   Background: Although most biologics are produced using recombinant technologies, heparin persists as a product purified from animal tissues. A cell based system for production of heparin would eliminate risk of supply shortage and contamination. Additionally, genetic engineering could yield heparin with improved qualities such as reduced risk of heparin-induced thrombocytopenia. Aims: This work is focused on engineering mammalian cell lines and bioprocess methods to produce recombinant heparin. Methods: The heparan sulfate biosynthetic pathway of mastocytoma cells was genetically engineered to alter the expression of heparan sulfate sulfotransferases. The resulting cell lines were screened for production of anti-FXa activity. Heparan sulfate production from a candidate cell line was tested in chemically defined medium. The recombinant product was characterized structurally and in clotting, anti-protease and heparin induced thrombocytopenia assays. Results: Engineered cells produced heparan sulfate in chemically defined medium with anti-Xa and anti-IIa activity exceeding the requirement for unfractionated heparin despite having lower sulfate content. Chain length was longer than unfractionated heparin. Additionally, binding to platelet factor 4 was reduced compared to unfractionated heparin, suggesting less risk of heparin-induced thrombocytopenia. Conclusion: These results demonstrate the feasibility of producing a substitute for unfractionated heparin from recombinant cell culture. Additionally, recombinant technology may allow production of heparin substitutes with improved properties such as reduced side effects. 

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2. Sulfated Glycans Inhibit the Interaction of MERS-CoV Receptor Binding Domain with Heparin

   https://doi.org/10.3390/v16020237  

   Yang, Jiyuan ; Song, Yuefan ; Jin, Weihua ; Xia, Ke ; Burnett, Grace C. ; Qiao, Wanjin ; Bates, John T. ; Pomin, Vitor H. ; Wang, Chunyu ; Qiao, Mingqiang ; et al ( February 2024 , Viruses)

   Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic virus with high contagion and mortality rates. Heparan sulfate proteoglycans (HSPGs) are ubiquitously expressed on the surface of mammalian cells. Owing to its high negatively charged property, heparan sulfate (HS) on the surface of host cells is used by many viruses as cofactor to facilitate viral attachment and initiate cellular entry. Therefore, inhibition of the interaction between viruses and HS could be a promising target to inhibit viral infection. In the current study, the interaction between the receptor-binding domain (RBD) of MERS-CoV and heparin was exploited to assess the inhibitory activity of various sulfated glycans such as glycosaminoglycans, marine-sourced glycans (sulfated fucans, fucosylated chondroitin sulfates, fucoidans, and rhamnan sulfate), pentosan polysulfate, and mucopolysaccharide using Surface Plasmon Resonance. We believe this study provides valuable insights for the development of sulfated glycan-based inhibitors as potential antiviral agents.

    

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3. Heavy Heparin: A Stable Isotope‐Enriched, Chemoenzymatically‐Synthesized, Poly‐Component Drug

   https://doi.org/10.1002/ange.201900768  

   Cress, Brady F. ; Bhaskar, Ujjwal ; Vaidyanathan, Deepika ; Williams, Asher ; Cai, Chao ; Liu, Xinyue ; Fu, Li ; M‐Chari, Vandhana ; Zhang, Fuming ; Mousa, Shaker A. ; et al ( April 2019 , Angewandte Chemie)

   Heparin is a highly sulfated, complex polysaccharide and widely used anticoagulant pharmaceutical. In this work, we chemoenzymatically synthesized perdeuteroheparin from biosynthetically enriched heparosan precursor obtained from microbial culture in deuterated medium. Chemical de‐N‐acetylation, chemicalN‐sulfation, enzymatic epimerization, and enzymatic sulfation with recombinant heparin biosynthetic enzymes afforded perdeuteroheparin comparable to pharmaceutical heparin. A series of applications for heavy heparin and its heavy biosynthetic intermediates are demonstrated, including generation of stable isotope labeled disaccharide standards, development of a non‐radioactive NMR assay for glucuronosyl‐C5‐epimerase, and background‐free quantification of in vivo half‐life following administration to rabbits. We anticipate that this approach can be extended to produce other isotope‐enriched glycosaminoglycans.

    

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4. Heavy Heparin: A Stable Isotope‐Enriched, Chemoenzymatically‐Synthesized, Poly‐Component Drug

   https://doi.org/10.1002/anie.201900768  

   Cress, Brady F. ; Bhaskar, Ujjwal ; Vaidyanathan, Deepika ; Williams, Asher ; Cai, Chao ; Liu, Xinyue ; Fu, Li ; M‐Chari, Vandhana ; Zhang, Fuming ; Mousa, Shaker A. ; et al ( April 2019 , Angewandte Chemie International Edition)

   Heparin is a highly sulfated, complex polysaccharide and widely used anticoagulant pharmaceutical. In this work, we chemoenzymatically synthesized perdeuteroheparin from biosynthetically enriched heparosan precursor obtained from microbial culture in deuterated medium. Chemical de‐N‐acetylation, chemicalN‐sulfation, enzymatic epimerization, and enzymatic sulfation with recombinant heparin biosynthetic enzymes afforded perdeuteroheparin comparable to pharmaceutical heparin. A series of applications for heavy heparin and its heavy biosynthetic intermediates are demonstrated, including generation of stable isotope labeled disaccharide standards, development of a non‐radioactive NMR assay for glucuronosyl‐C5‐epimerase, and background‐free quantification of in vivo half‐life following administration to rabbits. We anticipate that this approach can be extended to produce other isotope‐enriched glycosaminoglycans.

    

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5. SPR Sensor-Based Analysis of the Inhibition of Marine Sulfated Glycans on Interactions between Monkeypox Virus Proteins and Glycosaminoglycans

   https://doi.org/10.3390/md21050264  

   He, Peng ; Shi, Deling ; Li, Yunran ; Xia, Ke ; Kim, Seon Beom ; Dwivedi, Rohini ; Farrag, Marwa ; Pomin, Vitor H. ; Linhardt, Robert J. ; Dordick, Jonathan S. ; et al ( May 2023 , Marine Drugs)

   Sulfated glycans from marine organisms are excellent sources of naturally occurring glycosaminoglycan (GAG) mimetics that demonstrate therapeutic activities, such as antiviral/microbial infection, anticoagulant, anticancer, and anti-inflammation activities. Many viruses use the heparan sulfate (HS) GAG on the surface of host cells as co-receptors for attachment and initiating cell entry. Therefore, virion–HS interactions have been targeted to develop broad-spectrum antiviral therapeutics. Here we report the potential anti-monkeypox virus (MPXV) activities of eight defined marine sulfated glycans, three fucosylated chondroitin sulfates, and three sulfated fucans extracted from the sea cucumber species Isostichopus badionotus, Holothuria floridana, and Pentacta pygmaea, and the sea urchin Lytechinus variegatus, as well as two chemically desulfated derivatives. The inhibitions of these marine sulfated glycans on MPXV A29 and A35 protein–heparin interactions were evaluated using surface plasmon resonance (SPR). These results demonstrated that the viral surface proteins of MPXV A29 and A35 bound to heparin, which is a highly sulfated HS, and sulfated glycans from sea cucumbers showed strong inhibition of MPXV A29 and A35 interactions. The study of molecular interactions between viral proteins and host cell GAGs is important in developing therapeutics for the prevention and treatment of MPXV.

    

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