Dwivedi R , Samanta P , Sharma P , Zhang F , Mishra SK , Kucheryavy P , Kim SB , Aderibigbe AO , Linhardt RJ , Tandon R , Doerksen RJ , Pomin VH .

P20 GM130460 NIGMS NIH HHS , R03 NS110996 NINDS NIH HHS , S10 OD028523 NIH HHS

	Figure 1. Purification, electrophoretic mobility, and 1D1H NMR spectra of the polyanionic glycosidic fractions obtained from Pentacta pygmaea.A, crude polysaccharides (20 mg) obtained from the body wall of the sea cucumber P. pygmaea through nonspecific proteolytic (papain) digestion were fractionated in a DEAE cellulose column (2.5 × 20 cm) equilibrated with 100 mM sodium acetate buffer (pH 6.0). Multiple fractions (1 ml each) were obtained using a linear gradient of NaCl, from 0 to 3 M (---). Fractions were individually analyzed by Metachromasy using 1,9-dimethylmethylene blue (Abs 525 nm, ○) for the presence of sulfated glycan, by Dubois reaction (Abs 490 nm, •) for hexose, by Ehrlich assay (Abs 420 nm, Δ) for sialic acid, and by Carbazole reaction (Abs 550 nm, ▲) for uronic acid. The fractions corresponding to the respective peaks, labeled as nonsulfated glycan and the P. pygmaea-derived fucosylated chondroitin sulfate (PpFucCS), were pooled, dialyzed, and lyophilized for further characterization. B, molecular weight distribution of the purified PpFucCS was analyzed by polyacrylamide gel electrophoresis along with a series of molecular markers: low-molecular-weight heparin (LMWH) (∼8 kDa), unfractionated heparin (UFH) (∼15 kDa), chondroitin sulfate-A (CS-A) (∼40 kDa), chondroitin sulfate-C (∼60 kDa), and the sea cucumber Isostichopus badionotus-derived fucosylated chondroitin sulfate (IbFucCS) and sulfated fucan (IbSF). Samples (10 μg/each) were loaded on a 12% polyacrylamide gel and stained by 0.1% (w/v) toluidine blue (in 1% acetic acid) after electrophoretic migration. C, one-dimensional (1D) 1H NMR spectrum (δH expansion 6.0–0.0 ppm) of the purified PpFucCS were recorded in D2O, at 50 °C, on a 600 MHz Bruker NMR instrument. The NMR signals corresponding to characteristic peaks were properly labeled, as seen for the four composing α-fucose (Fuc) units (2,4S, 2,4S, 4S, and 0S) and the three composing β-N-acetylgalactosamine (GalNAc, N) units (4S, 6S, and 0S), all based on the diagnostic 1H signals present in the anomeric region, glucuronic acid (U) and GalNAc and Fuc methyl protons. S denotes sulfation in which the preceding number indicates the site or lack of occurrence.
	Figure 2. 2D1H-1H and1H-13C NMR spectra of the composing units of PpFucCS.A, the overlayed COSY (green), TOCSY (magenta), and 150 ms mixing time NOESY (gray) spectral strip from the anomeric region (δH/δH expansions 5.8–5.0/6.0–3.25 ppm) shows the presence of four spin systems (vertical dashed lines) corresponding to four α-fucose (Fuc) units labeled as A–D, respectively, Fuc2,4S, Fuc2,4S, Fuc 4S, and Fuc. U denotes signals corresponding to glucuronic acid. B, δH/δH expansions 4.8 to 4.5/6.0 to 3.25 ppm in TOCSY spectrum covering the anomeric region of N-acetylgalactosamine (GalNAc) units, showing the presence of three spin systems (vertical dashed lines) corresponding to the non-, 6-, and 4-sulfated GalNAc units labeled, respectively, as N0S, N6S, and N4S. Pairs of numbers between parentheses indicate position of the 1H nuclei assigned in the cross-peaks, marked by circles. C and D, 1H-13C HSQC spectrum (δH/δC expansions 6.0–3.25/112.0–45.0 ppm, and δH/δC expansions 2.5–1.0/30.0–10.0 ppm), showing all 1H-13C cross-peaks of PpFucCS. All 2D NMR spectra were acquired at 50 °C on a 600 MHz Bruker NMR instrument. Labels on 1H-13C cross-peaks of (C) and (D) follow the same patterns of (A) and (B).
	Figure 3. Structural representation of sulfated glycans.A, PpFucCS is composed of a chondroitin sulfate backbone of alternating N-acetylgalactosamine (GalNAc) and glucuronic acid (GlcA) in repeating disaccharide units of [→3)-β-GalNAc-(1→4)-β-GlcA-(1→], where the GalNAc units are mostly 4-sulfated (80%), and, to a much lesser extent, 4,6-disulfated (10%) or nonsulfated (10%). The GlcA units are substituted at the C3 position by three types of α-fucose (Fuc) branches: 40% Fuc2,4S-(1→ (A unit in NMR), 30% Fuc2,4S-(1→4)-Fuc-(1→ (B-D units in NMR), and 30% Fuc4S-(1→ (C unit in NMR), where S = SO3−. B, unfractionated heparin (UFH) is mostly composed of alternating α-glucosamine (GlcN) and α-iduronic acid (IdoA) in repeating disaccharide units of [→4)-GlcN-(1→4)-IdoA-(1→]. Sulfation occurs mostly at the C6 and N-positions of GlcN and C2 position of IdoA. Although more rare, other substitutions like sulfation at the C3 position and acetylation at the N-position of the GlcN unit as well as nonsulfation in both monosaccharides can also occur. C, the sulfated α-fucan isolated from Isostichopus badionotus (IbSF) is composed of the following tetrasaccharide unit [→3)-Fuc2,4S-(1→3)-Fuc2S-(1→3)-Fuc2S-(1→3)-Fuc-(1→].
	Figure 4. Anti-SARS-CoV-2 activity of different sulfated glycans. Plots showing (A) quantitation of GFP-transduced cells in the presence of different mammalian GAGs: unfractionated heparin, UFH (black); chondroitin sulfates CS-A, CS-B (dermatan sulfate), and CS-C (purple); and marine sulfated glycans: algal BoSG and sea urchin LvSF (gray), and holothurian PpFucCS (blue), IbFucCS (red), and IbSF (green) at an examined concentration of 50 mg/l. The average for the vehicle (control) is indicated with a dotted line. The numbers between parentheses on top of each bar indicate the average value. B, curves show percentage of SARS-CoV-2 inhibition of the three holothurian sulfated glycans and unfractionated heparin in a concentration-dependent manner. Numbers in parentheses indicate the increasing times observed in the IC50 values of the curves compared with the curve of the lowest IC50 value. C, plots showing percentage cell viability of HEK293T cells when treated with holothurian sulfated glycans at the highest concentration of 50 mg/l. ns stands for nonsignificant.
	Figure 5. SPR bar plots. The bar plots (based on triplicate experiments for standard deviation) indicate normalized S-protein (A and C–F) or N501Y mutant (B) binding to surface heparin inhibited with different sulfated glycans and concentrations. Control bars are in gray, UFH in black, PpFucCS in blue, IbFucCS in red, and IbSF in green. The numbers on top of each bar (except for control bars used for normalization) indicate the average value obtained in the experiments.
	Figure 6. Molecular modeling of the oligosaccharide building blocks from the holothurian sulfated glycans in their unbound and bound states with SARS-CoV-2 S-protein RBD.A and B, PpFucCS1, (C and D) PpFucCS2, (E and F) PpFucCS3, (G and H) IbSF. A, C, E, and G, 2D plots of the distribution of glycosidic torsion angles of the marine glycans in their unbound state, from 1 μs MD simulations. B, D, F, and H, 2D plots of the distribution of glycosidic torsion angles of the marine glycans in their bound state with S-protein RBD, from 200 ns MD simulations. 3D conformations of the composing oligosaccharide structures of the holothurian sulfated glycans are shown in sticks. Bound glycan glycosidic torsional plots are shown in blue (WT) and orange (N501Y).
	Figure 7. Predicted binding poses of sulfated glycans bound to wildtype (left panel) and N501Y mutant (right panel) of SARS-CoV2 S-protein RBD. Docked glycan in WT (pink) and in N501 mutant (green) and selected neighboring interacting residues (gray) are shown in sticks for (A) PpFucCS1, (B) PpFucCS2, (C) PpFucCS3, (D) IbSF, and (E) heparin. Dashed lines indicate polar interactions between the RBD and glycan. F, average docking scores from five independent docking runs as obtained from AutoDock Vina. Error bars represent ±standard deviation.
	Figure 8. Anticoagulant concentration-dependent response of holothurian sulfated glycans.A, aPTT, (B) AT-mediated factor IIa inhibition, (C) AT-mediated factor Xa inhibition, and (D) HCII-mediated factor IIa inhibition. The sulfated glycans tested (in a concentration range up to 100 μg/ml) were unfractionated heparin (UFH; in black), chondroitin sulfate B-type (CS-B, also known as DS; in purple), and the three holothurian sulfated glycans, PpFucCS (blue), IbFucCS (red), and IbSF (green). Values in parentheses in (A) indicate the percentage of IU/mg calculated from the aPTT curves related to the standard for heparin of 180 IU/mg. Numbers in parentheses in B–D indicate the increasing times observed in the IC50 values of the curves compared with the curve of the lowest IC50 value. The concentrations of coagulation (co)factors used in the experiments were 10 nM of AT or HCII and 2 nM of factors IIa or Xa.

Ahmed, Heparin induced thrombocytopenia: diagnosis and management update. 2007, Pubmed

Ahmed, Heparin induced thrombocytopenia: diagnosis and management update. 2007, Pubmed
Beaudet, Impact of autoclave sterilization on the activity and structure of formulated heparin. 2011, Pubmed
Bezerra, Conformational properties of l-fucose and the tetrasaccharide building block of the sulfated l-fucan from Lytechinus variegatus. 2020, Pubmed , Echinobase
Bose, Role of heparan sulfate in human parainfluenza virus type 3 infection. 2002, Pubmed
Cagno, Heparan Sulfate Proteoglycans and Viral Attachment: True Receptors or Adaptation Bias? 2019, Pubmed
Chen, Sulfation pattern of the fucose branch is important for the anticoagulant and antithrombotic activities of fucosylated chondroitin sulfates. 2013, Pubmed , Echinobase
Chen, Sequence determination and anticoagulant and antithrombotic activities of a novel sulfated fucan isolated from the sea cucumber Isostichopus badionotus. 2012, Pubmed , Echinobase
Clausen, SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2. 2020, Pubmed
Compton, Initiation of human cytomegalovirus infection requires initial interaction with cell surface heparan sulfate. 1993, Pubmed
Dechecchi, Heparan sulfate glycosaminoglycans are involved in adenovirus type 5 and 2-host cell interactions. 2000, Pubmed
Dwivedi, Structural and kinetic analyses of holothurian sulfated glycans suggest potential treatment for SARS-CoV-2 infection. 2021, Pubmed
Farias, Structure and anticoagulant activity of sulfated galactans. Isolation of a unique sulfated galactan from the red algae Botryocladia occidentalis and comparison of its anticoagulant action with that of sulfated galactans from invertebrates. 2000, Pubmed
Farndale, A direct spectrophotometric microassay for sulfated glycosaminoglycans in cartilage cultures. 1982, Pubmed
Fiorentini, First detection of SARS-CoV-2 spike protein N501 mutation in Italy in August, 2020. 2021, Pubmed
Fonseca, Effects of polysaccharides enriched in 2,4-disulfated fucose units on coagulation, thrombosis and bleeding. Practical and conceptual implications. 2009, Pubmed , Echinobase
Gong, Structural elucidation and antidiabetic activity of fucosylated chondroitin sulfate from sea cucumber Stichopus japonicas. 2021, Pubmed , Echinobase
Gupta, Heparin: A simplistic repurposing to prevent SARS-CoV-2 transmission in light of its in-vitro nanomolar efficacy. 2021, Pubmed
Hao, Binding of the SARS-CoV-2 spike protein to glycans. 2021, Pubmed
Hirsh, Guide to anticoagulant therapy: Heparin : a statement for healthcare professionals from the American Heart Association. 2001, Pubmed
Hu, Characteristics of SARS-CoV-2 and COVID-19. 2021, Pubmed
Humphrey, VMD: visual molecular dynamics. 1996, Pubmed
Jin, The structure-activity relationship of the interactions of SARS-CoV-2 spike glycoproteins with glucuronomannan and sulfated galactofucan from Saccharina japonica. 2020, Pubmed
Kim, Characterization of heparin and severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) spike glycoprotein binding interactions. 2020, Pubmed
Kirschner, GLYCAM06: a generalizable biomolecular force field. Carbohydrates. 2008, Pubmed
Kwon, Sulfated polysaccharides effectively inhibit SARS-CoV-2 in vitro. 2020, Pubmed
Kyriakidis, SARS-CoV-2 vaccines strategies: a comprehensive review of phase 3 candidates. 2021, Pubmed
Lan, Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. 2020, Pubmed
Li, Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. 2003, Pubmed
Li, Fucosylated chondroitin sulfate oligosaccharides exert anticoagulant activity by targeting at intrinsic tenase complex with low FXII activation: Importance of sulfation pattern and molecular size. 2017, Pubmed , Echinobase
Liu, Heparan Sulfate Proteoglycans as Attachment Factor for SARS-CoV-2. 2021, Pubmed
Lovekamp, Stability and function of glycosaminoglycans in porcine bioprosthetic heart valves. 2006, Pubmed
Luan, Enhanced binding of the N501Y-mutated SARS-CoV-2 spike protein to the human ACE2 receptor: insights from molecular dynamics simulations. 2021, Pubmed
Maier, ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB. 2015, Pubmed
Melo, An algal sulfated galactan has an unusual dual effect on venous thrombosis due to activation of factor XII and inhibition of the coagulation proteases. 2008, Pubmed
Melo, Antithrombin-mediated anticoagulant activity of sulfated polysaccharides: different mechanisms for heparin and sulfated galactans. 2004, Pubmed
Morris, AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. 2009, Pubmed
Mourão, Structure and anticoagulant activity of a fucosylated chondroitin sulfate from echinoderm. Sulfated fucose branches on the polysaccharide account for its high anticoagulant action. 1996, Pubmed , Echinobase
Mourão, Perspective on the use of sulfated polysaccharides from marine organisms as a source of new antithrombotic drugs. 2015, Pubmed
Mycroft-West, Heparin Inhibits Cellular Invasion by SARS-CoV-2: Structural Dependence of the Interaction of the Spike S1 Receptor-Binding Domain with Heparin. 2020, Pubmed
Ngernyuang, A Heparin Binding Motif Rich in Arginine and Lysine is the Functional Domain of YKL-40. 2018, Pubmed
Ni, Role of angiotensin-converting enzyme 2 (ACE2) in COVID-19. 2020, Pubmed
Niu, Two different fucosylated chondroitin sulfates: Structural elucidation, stimulating hematopoiesis and immune-enhancing effects. 2020, Pubmed , Echinobase
Ockelford, Comparison of the in vivo hemorrhagic and antithrombotic effects of a low antithrombin-III affinity heparin fraction. 1982, Pubmed
Pacheco, Different antithrombotic mechanisms among glycosaminoglycans revealed with a new fucosylated chondroitin sulfate from an echinoderm. 2000, Pubmed , Echinobase
Panagos, Fucosylated chondroitin sulfates from the body wall of the sea cucumber Holothuria forskali: conformation, selectin binding, and biological activity. 2014, Pubmed , Echinobase
Pomin, Holothurian fucosylated chondroitin sulfate. 2014, Pubmed , Echinobase
Pomin, Galactosaminoglycans: Medical Applications and Drawbacks. 2019, Pubmed , Echinobase
Pomin, Structure, biology, evolution, and medical importance of sulfated fucans and galactans. 2008, Pubmed , Echinobase
Pomin, NMR structural determination of unique invertebrate glycosaminoglycans endowed with medical properties. 2015, Pubmed
Pomin, Fucanomics and galactanomics: current status in drug discovery, mechanisms of action and role of the well-defined structures. 2012, Pubmed
Pomin, Glycosaminoglycans and Proteoglycans. 2018, Pubmed
Pomin, Selective cleavage and anticoagulant activity of a sulfated fucan: stereospecific removal of a 2-sulfate ester from the polysaccharide by mild acid hydrolysis, preparation of oligosaccharides, and heparin cofactor II-dependent anticoagulant activity. 2005, Pubmed , Echinobase
Pomin, Specific sulfation and glycosylation-a structural combination for the anticoagulation of marine carbohydrates. 2014, Pubmed , Echinobase
Pomin, Anticoagulant motifs of marine sulfated glycans. 2014, Pubmed
Pomin, Antimicrobial Sulfated Glycans: Structure and Function. 2017, Pubmed
Pomin, Exploiting enzyme specificities in digestions of chondroitin sulfates A and C: production of well-defined hexasaccharides. 2012, Pubmed
Pomin, Marine Non-Glycosaminoglycan Sulfated Glycans as Potential Pharmaceuticals. 2015, Pubmed
Pomin, Fucanomics and galactanomics: marine distribution, medicinal impact, conceptions, and challenges. 2012, Pubmed
Pramanik, Aptamer Conjugated Gold Nanostar-Based Distance-Dependent Nanoparticle Surface Energy Transfer Spectroscopy for Ultrasensitive Detection and Inactivation of Corona Virus. 2021, Pubmed
Pramanik, The rapid diagnosis and effective inhibition of coronavirus using spike antibody attached gold nanoparticles. 2021, Pubmed
Rezagholizadeh, Remdesivir for treatment of COVID-19; an updated systematic review and meta-analysis. 2021, Pubmed
Schuurs, Evidence of a putative glycosaminoglycan binding site on the glycosylated SARS-CoV-2 spike protein N-terminal domain. 2021, Pubmed
Shang, Structural basis of receptor recognition by SARS-CoV-2. 2020, Pubmed
Shukla, Herpesviruses and heparan sulfate: an intimate relationship in aid of viral entry. 2001, Pubmed
Soares, A unique fucosylated chondroitin sulfate type II with strikingly homogeneous and neatly distributed α-fucose branches. 2018, Pubmed , Echinobase
Song, Inhibitory activities of marine sulfated polysaccharides against SARS-CoV-2. 2020, Pubmed , Echinobase
Summerford, Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions. 1998, Pubmed
Tandon, Effective Inhibition of SARS-CoV-2 Entry by Heparin and Enoxaparin Derivatives. 2021, Pubmed
Tandon, Effective screening of SARS-CoV-2 neutralizing antibodies in patient serum using lentivirus particles pseudotyped with SARS-CoV-2 spike glycoprotein. 2020, Pubmed
Trott, AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. 2010, Pubmed
Tyagi, Internalization of HIV-1 tat requires cell surface heparan sulfate proteoglycans. 2001, Pubmed
Ustyuzhanina, Two fucosylated chondroitin sulfates from the sea cucumber Eupentacta fraudatrix. 2017, Pubmed , Echinobase
Ustyuzhanina, Fucosylated Chondroitin Sulfates from the Sea Cucumbers Paracaudina chilensis and Holothuria hilla: Structures and Anticoagulant Activity. 2020, Pubmed , Echinobase
Vabret, Immunology of COVID-19: Current State of the Science. 2020, Pubmed
Vasconcelos, Anticoagulant and Antithrombotic Properties of Three Structurally Correlated Sea Urchin Sulfated Glycans and Their Low-Molecular-Weight Derivatives. 2018, Pubmed , Echinobase
Vasconcelos, The Sea as a Rich Source of Structurally Unique Glycosaminoglycans and Mimetics. 2017, Pubmed , Echinobase
Vasconcelos, Marine Carbohydrate-Based Compounds with Medicinal Properties. 2018, Pubmed
Vivès, Heparan sulfate targets the HIV-1 envelope glycoprotein gp120 coreceptor binding site. 2005, Pubmed
Wang, Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2. 2020, Pubmed
Warkentin, Heparin-induced thrombocytopenia in patients treated with low-molecular-weight heparin or unfractionated heparin. 1995, Pubmed
Welte, Current evidence for COVID-19 therapies: a systematic literature review. 2021, Pubmed
Wrapp, Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. 2020, Pubmed
Xu, Modulating the degree of fucosylation of fucosylated chondroitin sulfate enhances heparin cofactor II-dependent thrombin inhibition. 2018, Pubmed
Yan, Heparan sulfates from bat and human lung and their binding to the spike protein of SARS-CoV-2 virus. 2021, Pubmed
Yim, Inhibition of SARS-CoV-2 Virus Entry by the Crude Polysaccharides of Seaweeds and Abalone Viscera In Vitro. 2021, Pubmed
Yu, Elucidating the Interactions Between Heparin/Heparan Sulfate and SARS-CoV-2-Related Proteins-An Important Strategy for Developing Novel Therapeutics for the COVID-19 Pandemic. 2020, Pubmed
Zamorano Cuervo, ACE2: Evidence of role as entry receptor for SARS-CoV-2 and implications in comorbidities. 2020, Pubmed
Zoepfl, Antiviral activities of four marine sulfated glycans against adenovirus and human cytomegalovirus. 2021, Pubmed , Echinobase
Zong, Anticancer polysaccharides from natural resources: a review of recent research. 2012, Pubmed