Structure, anti-SARS-CoV-2, and anticoagulant effects of two sulfated galactans from the red alga Botryocladia occidentalis.

The structure of the sulfated galactan from the red alga Botryocladia occidentalis (BoSG) was originally proposed as a simple repeating disaccharide of alternating 4-linked α-galactopyranose (Galp) and 3-linked ß-Galp units with variable sulfation pattern. Abundance was estimated only for the α-Galp units: one-third of 2,3-disulfation and one-third of 2-monosulfation. Here, we isolated again the same BoSG fractions from the anion-exchange chromatography, obtaining the same NMR profile of the first report. More careful NMR analysis led us to revise the structure. A more complex sulfation pattern was noted along with the occurrence of 4-linked α-3,6-anhydro-Galp (AnGalp) units. Interestingly, the more sulfated BoSG fraction showed slightly reduced in vitro anti-SARS-CoV-2 activities against both wild-type and delta variants, and significantly reduced anticoagulant activity. The BoSG fractions showed no cytotoxic effects. The reduction in both bioactivities is attributed to the presence of the AnGalp unit. Docking scores from computational simulations using BoSG disaccharide constructs on wild-type and delta S-proteins, and binding analysis through competitive SPR assays using blood (co)-factors (antithrombin, heparin cofactor II and thrombin) and four S-proteins (wild-type, delta, gamma, and omicron) strongly support the conclusion about the deleterious impact of the AnGalp unit.

Fractionation of sulfated galactan from the red alga Botryocladia occidentalis separates its anticoagulant and anti-SARS-CoV-2 properties.

Sulfation pattern and molecular weight (MW) play a key role in the biological actions of sulfated glycans. Besides anticoagulant effects, certain sulfated glycans can also exhibit anti-SARS-CoV-2 properties. To develop a more selective antiviral carbohydrate, an efficient strategy to separate these two actions is required. In this work, low MW fractions derived from the red alga Botryocladia occidentalis sulfated galactan (BoSG) were generated, structurally characterized, and tested for activity against SARS-CoV-2 and blood coagulation. The lowest MW fraction was found to be primarily composed of octasaccharides of monosulfated monosaccharides. Unlike heparin or native BoSG, we found that hydrolyzed BoSG products had weak anticoagulant activities as seen by aPTT and inhibitory assays using purified cofactors. In contrast, lower MW BoSG-derivatives retained anti-SARS-CoV-2 activity using SARS-CoV-2 spike (S)-protein pseudotyped lentivirus vector in HEK-293T-hACE2 cells monitored by GFP. Surface plasmon resonance confirmed that longer chains are necessary for BoSG to interact with coagulation cofactors but is not required for interactions with certain S-protein variants. We observed distinct affinities of BoSG derivatives for the S-proteins of different SARS-CoV-2 strains, including WT, N501Y (Alpha), K417T/E484K/N501Y (Gamma), and L542R (Delta) mutants, and stronger affinity for the N501Y-containing variants. Docking of the four possible monosulfated BoSG disaccharides in interactions with the N501Y mutant S-protein predicted potential binding poses of the BoSG constructs and favorable binding in close proximity to the 501Y residue. Our results demonstrate that depolymerization and fractionation of BoSG are an effective strategy to segregate its anticoagulant property from its anti-SARS-CoV-2 action.

Fractionation of sulfated galactan from the red alga Botryocladia occidentalis separates its anticoagulant and anti-SARS-CoV-2 properties

Sulfation pattern and molecular weight (MW) play a key role in the biological actions of sulfated glycans. Besides anticoagulant effects, certain sulfated glycans can also exhibit anti-SARS-CoV-2 properties. To develop a more selective antiviral carbohydrate, an efficient strategy to separate these two actions is required. In this work, low MW fractions derived from the red alga Botryocladia occidentalis sulfated galactan (BoSG) were generated, structurally characterized, and tested for activity against SARS-CoV-2 and blood coagulation. The lowest MW fraction was found to be primarily composed of octasaccharides of monosulfated monosaccharides. Unlike heparin or native BoSG, we found that hydrolyzed BoSG products had weak anticoagulant activities as seen by aPTT and inhibitory assays using purified co-factors. In contrast, lower MW BoSG-derivatives retained anti-SARS-CoV-2 activity using SARS-CoV-2 spike (S)-protein pseudotyped lentivirus vector in HEK293T cells monitored by green fluorescent protein. Surface plasmon resonance confirmed that longer chains are necessary for BoSG to interact with coagulation co-factors but is not required for interactions with certain S-protein variants. We observed distinct affinities of BoSG derivatives for the S-proteins of different SARS-CoV-2 strains, including wild type, N501Y (Alpha), K417T/E484K/N501Y (Gamma), and L542R (Delta) mutants, and stronger affinity for the N501Y-containing variants. Docking of the four possible monosulfated BoSG disaccharides in interactions with the N501Y mutant S-protein predicted potential binding poses of the BoSG constructs and favorable binding in close proximity to the 501Y residue. Our results demonstrate that depolymerization and fractionation of BoSG are an effective strategy to segregate its anticoagulant property from its anti-SARS-CoV-2 action.

Structural and kinetic analyses of holothurian sulfated glycans suggest potential treatment for SARS-CoV-2 infection.

Certain sulfated glycans, including those from marine sources, can show potential effects against SARS-CoV-2. Here, a new fucosylated chondroitin sulfate (FucCS) from the sea cucumber Pentacta pygmaea (PpFucCS) (MW ∼10-60 kDa) was isolated and structurally characterized by NMR. PpFucCS is composed of {→3)-ß-GalNAcX-(1→4)-ß-GlcA-[(3→1)Y]-(1→}, where X = 4S (80%), 6S (10%) or nonsulfated (10%), Y = α-Fuc2,4S (40%), α-Fuc2,4S-(1→4)-α-Fuc (30%), or α-Fuc4S (30%), and S = SO3-. The anti-SARS-CoV-2 activity of PpFucCS and those of the FucCS and sulfated fucan isolated from Isostichopus badionotus (IbFucCS and IbSF) were compared with that of heparin. IC50 values demonstrated the activity of the three holothurian sulfated glycans to be ∼12 times more efficient than heparin, with no cytotoxic effects. The dissociation constant (KD) values obtained by surface plasmon resonance of the wildtype SARS-CoV-2 spike (S)-protein receptor-binding domain (RBD) and N501Y mutant RBD in interactions with the heparin-immobilized sensor chip were 94 and 1.8 × 103 nM, respectively. Competitive surface plasmon resonance inhibition analysis of PpFucCS, IbFucCS, and IbSF against heparin binding to wildtype S-protein showed IC50 values (in the nanomolar range) 6, 25, and 6 times more efficient than heparin, respectively. Data from computational simulations suggest an influence of the sulfation patterns of the Fuc units on hydrogen bonding with GlcA and that conformational change of some of the oligosaccharide structures occurs upon S-protein RBD binding. Compared with heparin, negligible anticoagulant action was observed for IbSF. Our results suggest that IbSF may represent a promising molecule for future investigations against SARS-CoV-2.