Sulfated Polysaccharides with Anticoagulant Potential: A Review Focusing on Structure-Activity Relationship and Action Mechanism.

Thromboembolism is the culprit of cardiovascular diseases, leading to the highest global mortality rate. Anticoagulation emerges as the primary approach for managing thrombotic conditions. Notably, sulfated polysaccharides exhibit favorable anticoagulant efficacy with reduced side effects. This review focuses on the structure-anticoagulant activity relationship of sulfated polysaccharides and the underlying action mechanisms. It is concluded that chlorosulfonicacid-pyridine method serves as the preferred technique to synthesize sulfated polysaccharides. The anticoagulant activity of sulfated polysaccharides is linked to the substitution site of sulfate groups, degree of substitution, molecular weight, main side chain structure, and glycosidic bond conformation. Moreover, sulfated polysaccharides exert anticoagulant activity via various pathways, including the inhibition of blood coagulation factors, activation of antithrombin III and heparin cofactor II, antiplatelet aggregation, and promotion of the fibrinolytic system.

Specificity of a ß-porphyranase produced by the carrageenophyte red alga Chondrus crispus and implications of this unexpected activity on red algal biology.

The carrageenophyte red alga Chondrus crispus produces three family 16 glycoside hydrolases (CcGH16-1, CcGH16-2, and CcGH16-3). Phylogenetically, the red algal GH16 members are closely related to bacterial GH16 homologs from subfamilies 13 and 14, which have characterized marine bacterial ß-carrageenase and ß-porphyranase activities, respectively, yet the functions of these CcGH16 hydrolases have not been determined. Here, we first confirmed the gene locus of the ccgh16-3 gene in the alga to facilitate further investigation. Next, our biochemical characterization of CcGH16-3 revealed an unexpected ß-porphyranase activity, since porphyran is not a known component of the C. crispus extracellular matrix. Kinetic characterization was undertaken on natural porphyran substrate with an experimentally determined molecular weight. We found CcGH16-3 has a pH optimum between 7.5 and 8.0; however, it exhibits reasonably stable activity over a large pH range (pH 7.0-9.0). CcGH16-3 has a KM of 4.0 ± 0.8 µM, a kcat of 79.9 ± 6.9 s-1, and a kcat/KM of 20.1 ± 1.7 µM-1 s-1. We structurally examined fine enzymatic specificity by performing a subsite dissection. CcGH16-3 has a strict requirement for D-galactose and L-galactose-6-sulfate in its -1 and +1 subsites, respectively, whereas the outer subsites are less restrictive. CcGH16-3 is one of a handful of algal enzymes characterized with a specificity for a polysaccharide unknown to be found in their own extracellular matrix. This ß-porphyranase activity in a carrageenophyte red alga may provide defense against red algal pathogens or provide a competitive advantage in niche colonization.

Direct antitumoral effects of sulfated fucans isolated from echinoderms: a possible role of neuropilin-1/ß1 integrin endocytosis and focal adhesion kinase degradation.

Hypercoagulability, a major complication of metastatic cancers, has usually been treated with heparins from natural sources, or with their synthetic derivatives, which are under intense investigation in clinical oncology. However, the use of heparin has been challenging for patients with risk of severe bleeding. While the systemic administration of heparins, in preclinical models, has shown primarily attenuating effects on metastasis, their direct effect on established solid tumors has generated contradictory outcomes. We investigated the direct antitumoral properties of two sulfated fucans isolated from marine echinoderms, FucSulf1 and FucSulf2, which exhibit anticoagulant activity with mild hemorrhagic potential. Unlike heparin, sulfated fucans significantly inhibited tumor cell proliferation (by ~30-50%), and inhibited tumor migration and invasion in vitro. We found that FucSulf1 and FucSulf2 interacted with fibronectin as efficiently as heparin, leading to loss of prostate cancer and melanoma cell spreading. The sulfated fucans increased the endocytosis of ß1 integrin and neuropilin-1 chains, two cell receptors implicated in fibronectin-dependent adhesion. The treatment of cancer cells with both sulfated fucans, but not with heparin, also triggered intracellular focal adhesion kinase (FAK) degradation, with a consequent overall decrease in activated focal adhesion kinase levels. Finally, only sulfated fucans inhibited the growth of B16-F10 melanoma cells implanted in the dermis of syngeneic C57/BL6 mice. FucSulf1 and FucSulf2 arise from this study as candidates for the design of possible alternatives to long-term treatments of cancer patients with heparins, with the advantage of also controlling local growth and invasion of malignant cells.

Spontaneous granulation of moderately halophilic sludge inoculated with saltern sediments from single granule into multi-granule aggregation.

There is very limited research on the application of moderate halophiles for biotreatment of hypersaline wastewater widely generated from some industries. This study demonstrated the development of moderate halophiles inoculated from saltern sediments into aerobic granule sludge (AGS) to treat hypersaline wastewater with a salinity of 100 g/L. The granulation of moderate halophiles can occur without applying the settling velocity selective pressure. The saltern sediment initially aggregated into single small granules and finally developed into 1200 ± 50 µm multiparticle granules. The halophiles affiliated in Halomonas was dominant in the granular bacterial community, with a relative abundance of 94.52%. Halomonas ventosae secreted sulfated polysaccharides. The sulfated polysaccharides content accounted for 63.95 ± 2.10% in the polysaccharides (PS), having an adhesive role in connecting single granules. Multiparticle granules showed the clear stratified structure, with α-D-glucopyranose polysaccharides in the inner bounders and ß-D-glucopyranose polysaccharides in the outer. The moderately granular sludge showed the stable chemical oxygen demand (COD) removal efficiency of >90% and the aerobic total inorganic nitrogen (TIN) removal efficiency (equal to ammonia removal) of 70 ± 5.00%. This paper contributes new insight into the formation of moderately halophilic granular sludge and accelerates the application of moderately halophilic granular sludge to treat hypersaline wastewater.

Marine Sulfated Polysaccharide PMGS Synergizes with Paclitaxel in Inhibiting Cervical Cancer In Vitro.

The incidence and mortality of cervical cancer in female malignancies are second only to breast cancer, which brings a heavy health and economic toll worldwide. Paclitaxel (PTX)-based regimens are the first-class choice; however, severe side effects, poor therapeutic effects, and difficulty in effectively preventing tumor recurrence or metastasis are unavoidable. Therefore, it is necessary to explore effective therapeutic interventions for cervical cancer. Our previous studies have shown that PMGS, a marine sulfated polysaccharide, exhibits promising anti-human papillomavirus (anti-HPV) effects through multiple molecular mechanisms. In this article, a continuous study identified that PMGS, as a novel sensitizer, combined with PTX exerted synergistic anti-tumor effects on cervical cancer associated with HPV in vitro. Both PMGS and PTX inhibited the proliferation of cervical cancer cells, and the combination of PMGS with PTX displayed significant synergistic effects on Hela cells. Mechanistically, PMGS synergizes with PTX by enhancing cytotoxicity, inducing cell apoptosis and inhibiting cell migration in Hela cells. Collectively, the combination of PTX and PMGS potentially provides a novel therapeutic strategy for cervical cancer.

Development of Bi- and Tri-Layer Nanofibrous Membranes Based on the Sulfated Polysaccharide Carrageenan for Periodontal Tissue Regeneration.

Periodontitis is a microbially-induced inflammation of the periodontium that is characterized by the destruction of the periodontal ligament (PDL) and alveolar bone and constitutes the principal cause of teeth loss in adults. Periodontal tissue regeneration can be achieved through guided tissue/bone regeneration (GTR/GBR) membranes that act as a physical barrier preventing epithelial infiltration and providing adequate time and space for PDL cells and osteoblasts to proliferate into the affected area. Electrospun nanofibrous scaffolds, simulating the natural architecture of the extracellular matrix (ECM), have attracted increasing attention in periodontal tissue engineering. Carrageenans are ideal candidates for the development of novel nanofibrous GTR/GBR membranes, since previous studies have highlighted the potential of carrageenans for bone regeneration by promoting the attachment and proliferation of osteoblasts. Herein, we report the development of bi- and tri-layer nanofibrous GTR/GBR membranes based on carrageenans and other biocompatible polymers for the regeneration of periodontal tissue. The fabricated membranes were morphologically characterized, and their thermal and mechanical properties were determined. Their periodontal tissue regeneration potential was investigated through the evaluation of cell attachment, biocompatibility, and osteogenic differentiation of human PDL cells seeded on the prepared membranes.

Sulfated Polysaccharides as a Fighter with Protein Non-Physiological Aggregation: The Role of Polysaccharide Flexibility and Charge Density.

Proteins can lose native functionality due to non-physiological aggregation. In this work, we have shown the power of sulfated polysaccharides as a natural assistant to restore damaged protein structures. Protein aggregates enriched by cross-ß structures are a characteristic of amyloid fibrils related to different health disorders. Our recent studies demonstrated that model fibrils of hen egg white lysozyme (HEWL) can be disaggregated and renatured by some negatively charged polysaccharides. In the current work, using the same model protein system and FTIR spectroscopy, we studied the role of conformation and charge distribution along the polysaccharide chain in the protein secondary structure conversion. The effects of three carrageenans (κ, ι, and λ) possessing from one to three sulfate groups per disaccharide unit were shown to be different. κ-Carrageenan was able to fully eliminate cross-ß structures and complete the renaturation process. ι-Carrageenan only initiated the formation of native-like ß-structures in HEWL, retaining most of the cross-ß structures. In contrast, λ-carrageenan even increased the content of amyloid cross-ß structures. Furthermore, κ-carrageenan in rigid helical conformation loses its capability to restore protein native structures, largely increasing the amount of amyloid cross-ß structures. Our findings create a platform for the design of novel natural chaperons to counteract protein unfolding.

New Insight on In Vitro Biological Activities of Sulfated Polysaccharides from Ulvophyte Green Algae.

Green algae are natural bioresources that have excellent bioactive potential, partly due to sulfated polysaccharides (SPs) which are still rarely explored for their biological activities. There is currently an urgent need for studies exploring the anticancer biological activity of SPs extracted from two Indonesian ulvophyte green algae: the sulfated polysaccharide of Caulerpa racemosa (SPCr) and the sulfated polysaccharide of Caulerpa lentillifera (SPCl). The method of isolating SPs and their assessment of biological activities in this study were based on previous and similar studies. The highest yield sulfate/total sugar ratio was presented by SPCr than that of SPCl. Overall, SPCr exhibits a strong antioxidant activity, as indicated by smaller EC50 values obtained from a series of antioxidant activity assays compared to the EC50 values of Trolox (control). As an anti-obesity and antidiabetic, the overall EC50 value of both SPs was close to the EC50 of the positive control (orlistat and acarbose). Even more interesting was that SPCl displayed wide-ranging anticancer effects on colorectal, hepatoma, breast cancer cell lines, and leukemia. Finally, this study reveals new insights in that SPs from two Indonesian green algae have the potential to be promising nutraceuticals as novel antioxidative actors, and to be able to fight obesity, diabetes, and even cancer.

Biological properties and potential of compounds extracted from red seaweeds.

Macroalgae have been recently used for different applications in the food, cosmetic and pharmaceutical industry since they do not compete for land and freshwater against other resources. Moreover, they have been highlighted as a potential source of bioactive compounds. Red algae (Rhodophyta) are the largest group of seaweeds, including around 6000 different species, thus it can be hypothesized that they are a potential source of bioactive compounds. Sulfated polysaccharides, mainly agar and carrageenans, are the most relevant and exploited compounds of red algae. Other potential molecules are essential fatty acids, phycobiliproteins, vitamins, minerals, and other secondary metabolites. All these compounds have been demonstrated to exert several biological activities, among which antioxidant, anti-inflammatory, antitumor, and antimicrobial properties can be highlighted. Nevertheless, these properties need to be further tested on in vivo experiments and go in-depth in the study of the mechanism of action of the specific molecules and the understanding of the structure-activity relation. At last, the extraction technologies are essential for the correct isolation of the molecules, in a cost-effective way, to facilitate the scale-up of the processes and their further application by the industry. This manuscript is aimed at describing the fundamental composition of red algae and their most studied biological properties to pave the way to the utilization of this underused resource.

Antioxidant Activity of Fucoidan Modified with Gallic Acid Using the Redox Method.

Antioxidant compounds decrease the amount of intracellular reactive oxygen species (ROS) and, consequently, reduce the deleterious effects of ROS in osteoblasts. Here, we modified a 21 kDa fucoidan (FucA) with gallic acid (GA) using the redox method, to potentiate its antioxidant/protective capacity on pre-osteoblast-like cells (MC3T3) against oxidative stress. The 20 kDa FucA-GA contains 37 ± 3.0 mg GA per gram of FucA. FucA-GA was the most efficient antioxidant agent in terms of total antioxidant capacity (2.5 times), reducing power (five times), copper chelation (three times), and superoxide radical scavenging (2 times). Exposure of MC3T3 cells to H2O2 increased ROS levels and activated caspase-3 along with caspase-9. In addition, the cell viability decreased approximately 80%. FucA-GA also provided the most effective protection against oxidative damage caused by H2O2. Treatment with FucA-GA (1.0 mg/mL) increased cell viability (~80%) and decreased intracellular ROS (100%) and caspase activation (~80%). In addition, Fuc-GA (0.1 mg/mL) abolished H2O2-induced oxidative stress in zebra fish embryos. Overall, FucA-GA protected MC3T3 cells from oxidative stress and could represent a possible adjuvant for the treatment of bone fragility by counteracting oxidative phenomena.

Complexes of Cu-Polysaccharide of a Marine Red Microalga Produce Spikes with Antimicrobial Activity.

Metal-polysaccharides have recently raised significant interest due to their multifunctional bioactivities. The antimicrobial activity of a complex of Cu2O with the sulfated polysaccharide (PS) of the marine red microalga Porphyridium sp. was previously attributed to spikes formed on the complex surface (roughness). This hypothesis was further examined here using other Cu-PS complexes (i.e., monovalent-Cu2O, CuCl and divalent-CuO, CuCl2). The nanostructure parameters of the monovalent complexes, namely, longer spikes (1000 nm) and greater density (2000-5000 spikes/µm2) were found to be related to the superior inhibition of microbial growth and viability and biofilm formation. When Escherichia coli TV1061, used as a bioluminescent test organism, was exposed to the monovalent Cu-PS complexes, enhanced bioluminescence accumulation was observed, probably due to membrane perforation by the spikes on the surface of the complexes and consequent cytoplasmic leakage. In addition, differences were found in the surface chemistry of the monovalent and divalent Cu-PS complexes, with the monovalent Cu-PS complexes exhibiting greater stability (ζ-potential, FTIR spectra, and leaching out), which could be related to spike formation. This study thus supports our hypothesis that the spikes protruding from the monovalent Cu-PS surfaces, as characterized by their aspect ratio, are responsible for the antimicrobial and antibiofilm activities of the complexes.

Anti-Inflammatory Effect of Sulfated Polysaccharides Isolated from Codium fragile In Vitro in RAW 264.7 Macrophages and In Vivo in Zebrafish.

In this study, the anti-inflammatory activity of sulfated polysaccharides isolated from the green seaweed Codium fragile (CFCE-PS) was investigated in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and zebrafish. The results demonstrated that CFCE-PS significantly increased the viability of LPS-induced RAW 264.7 cells in a concentration-dependent manner. CFCE-PS remarkably and concentration-dependently reduced the levels of inflammatory molecules including prostaglandin E2, nitric oxide (NO), interleukin-1 beta, tumor necrosis factor-alpha, and interleukin-6 in LPS-stimulated RAW 264.7 cells. In addition, in vivo test results indicated that CFCE-PS effectively reduced reactive oxygen species, cell death, and NO levels in LPS-stimulated zebrafish. Thus, these results indicate that CFCE-PS possesses in vitro and in vivo anti-inflammatory activities and suggest it is a potential ingredient in the functional food and pharmaceutical industries.

Antiviral Activities of Algal-Based Sulfated Polysaccharides.

An antiviral agent is urgently needed based on the high probability of the emergence and re-emergence of future viral disease, highlighted by the recent global COVID-19 pandemic. The emergence may be seen in the discovery of the Alpha, Beta, Gamma, Delta, and recently discovered Omicron variants of SARS-CoV-2. The need for strategies besides testing and isolation, social distancing, and vaccine development is clear. One of the strategies includes searching for an antiviral agent that provides effective results without toxicity, which is well-presented by significant results for carrageenan nasal spray in providing efficacy against human coronavirus-infected patients. As the primary producer of sulfated polysaccharides, marine plants, including macro- and microalgae, offer versatility in culture, production, and post-isolation development in obtaining the needed antiviral agent. Therefore, this review will describe an attempt to highlight the search for practical and safe antiviral agents from algal-based sulfated polysaccharides and to unveil their features for future development.

Pine Pollen Polysaccharides' and Sulfated Polysaccharides' Effects on UC Mice through Modulation of Cell Tight Junctions and RIPK3-Dependent Necroptosis Pathways.

The purpose of this study is to explore the effects of pine pollen polysaccharides and sulfated polysaccharides on mice with ulcerative colitis and whether they could protect mice from inflammation by regulating the tight junctions of colonic epithelial cells and regulating the RIPK3-dependent necroptosis pathways. Pine pollen polysaccharides were prepared by water boiling and ethanol precipitation. After deproteinedization with trichloroacetic acid, the UV spectrum showed that there were no proteins. One polysaccharide component (PPM60-III) was made by gel filtration chromatography, and then sulfated polysaccharide (SPPM60-III) was derived using the chlorosulfonic acid-pyridine method. After treatment with PPM60-III and SPPM60-III, the body weight of mice with ulcerative colitis induced by dextran sodium sulfate increased, the DAI score decreased, the levels of pro-inflammatory factors and inflammation-related enzymes decreased, and the level of anti-inflammatory factors increased. In addition, after treatment, the expressions levels of tight junction proteins increased, the expressions levels of key proteins of programmed necroptosis decreased, while the level of Caspase-8 increased. The results indicated that pine pollen polysaccharides and sulfated polysaccharides have a certain therapeutic effect on UC mice, and the therapeutic effect may be achieved by regulating the tight junction of colonic epithelial cells and regulating the RIPK3-dependent necroptosis pathways.

Ion-exchange purification and structural characterization of five sulfated fucoidans from brown algae.

Fucoidans are a diverse class of sulfated polysaccharides integral to the cell wall of brown algae, and due to their various bioactivities, they are potential drugs. Standardized work with fucoidans is required for structure-function studies, but remains challenging since available fucoidan preparations are often contaminated with other algal compounds. Additionally, fucoidans are structurally diverse depending on species and season, urging the need for standardized purification protocols. Here, we use ion-exchange chromatography to purify different fucoidans and found a high structural diversity between fucoidans. Ion-exchange chromatography efficiently removes the polysaccharides alginate and laminarin and other contaminants such as proteins and phlorotannins across a broad range of fucoidans from major brown algal orders including Ectocarpales, Laminariales and Fucales. By monomer composition, linkage analysis and NMR characterization, we identified galacturonic acid, glucuronic acid and O-acetylation as new structural features of certain fucoidans and provided a novel structure of fucoidan from Durvillaea potatorum with α-1,3-linked fucose backbone and ß-1,6 and ß-1,3 galactose branches. This study emphasizes the use of standardized ion-exchange chromatography to obtain defined fucoidans for subsequent molecular studies.

Carrageenan-containing over-the-counter nasal and oral sprays inhibit SARS-CoV-2 infection of airway epithelial cultures.

Pharmaceutical interventions are urgently needed to prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and transmission. As SARS-CoV-2 infects and spreads via the nasopharyngeal airways, we analyzed the antiviral effect of selected nasal and oral sprays on virus infection in vitro. Two nose sprays showed virucidal activity but were cytotoxic precluding further analysis in cell culture. One nasal and one mouth spray suppressed SARS-CoV-2 infection of TMPRSS2-expressing Vero E6 cells and primary differentiated human airway epithelial cultures. The antiviral activity in both sprays could be attributed to polyanionic ι- and κ-carrageenans. Thus, application of carrageenan-containing nasal and mouth sprays may reduce the risk of acquiring SARS-CoV-2 infection and may limit viral spread, warranting further clinical evaluation.

Effects of sulfated polysaccharides isolated from Codium fragile on inflammatory cytokine gene expression and Edwardsiella tarda infection in rockfish, Sebastes schlegelii.

Sulfated polysaccharides (SPs) derived from Codium fragile (sponge seaweed) can regulate cytokine expression in mammalian macrophages, NK cell lines and olive flounder head kidney primary cells in vitro. In this study, we found that SPs from C. fragile exhibited anti-bacterial activities against fish pathogenic bacteria including Streptococcus parauberis, Lactococcus garvieae, Aeromonas salmonicida and Edwardsiella tarda at a minimum inhibitory concentration of 2 mg/mL, but not against S. iniae or Vibrio anguillarum. Immunostimulatory effects of SPs from C. fragile on rockfish (Sebastes schlegelii) were evaluated by analyzing mRNA expression levels of inflammatory cytokines (interleukin (IL)-1ß, IL-8, IL-6 and tumor necrosis factor (TNF)-α) and anti-inflammatory cytokines (IL-10) both in vitro and in vivo. Results revealed that expression levels of all genes tested were upregulated in rockfish head kidney and spleen cells by SPs from C. fragile in a dose/time-dependent manner in vitro. By contrast, expression levels of these genes were significantly (p < 0.05) downregulated in the head kidney and spleen of rockfish in vivo at 1 and 3 days post intraperitoneal injection of SPs from C. fragile. In the liver, these genes were downregulated on day 1, but upregulated on day 3. Treatment with SPs downregulated the expression of these genes in spleen, but upregulated IL-10 gene expression in the intestine and liver. Meanwhile, when fish were fed with crude SPs for 4 weeks and challenged with E. tarda, infected fish started to die starting from 2 days after immune challenge. The cumulative mortality of the 0.1% group was significantly lower (p < 0.05) than that of the control group without feeding with SPs. Expression levels of IL-1ß and IL-6 genes were significantly (p < 0.05) upregulated in head kidney of the 0.5% group on day 1 while IL-1ß gene expression was downregulated on day 3 in the liver. These results indicate that SPs from C. fragile can regulate the immune gene expression in rockfish and that a diet containing 0.1% crude SPs can reduce the mortality of rockfish caused by E. tarda infection.

Antiviral Activity of Carrageenans and Processing Implications.

Carrageenan and carrageenan oligosaccharides are red seaweed sulfated carbohydrates with well-known antiviral properties, mainly through the blocking of the viral attachment stage. They also exhibit other interesting biological properties and can be used to prepare different drug delivery systems for controlled administration. The most active forms are λ-, ι-, and κ-carrageenans, the degree and sulfation position being determined in their properties. They can be obtained from sustainable worldwide available resources and the influence of manufacturing on composition, structure, and antiviral properties should be considered. This review presents a survey of the antiviral properties of carrageenan in relation to the processing conditions, particularly those assisted by intensification technologies during the extraction stage, and discusses the possibility of further chemical modifications.

Marine Sulfated Polysaccharides as Promising Antiviral Agents: A Comprehensive Report and Modeling Study Focusing on SARS CoV-2.

SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) is a novel coronavirus strain that emerged at the end of 2019, causing millions of deaths so far. Despite enormous efforts being made through various drug discovery campaigns, there is still a desperate need for treatments with high efficacy and selectivity. Recently, marine sulfated polysaccharides (MSPs) have earned significant attention and are widely examined against many viral infections. This article attempted to produce a comprehensive report about MSPs from different marine sources alongside their antiviral effects against various viral species covering the last 25 years of research articles. Additionally, these reported MSPs were subjected to molecular docking and dynamic simulation experiments to ascertain potential interactions with both the receptor-binding domain (RBD) of SARS CoV-2's spike protein (S-protein) and human angiotensin-converting enzyme-2 (ACE2). The possible binding sites on both S-protein's RBD and ACE2 were determined based on how they bind to heparin, which has been reported to exhibit significant antiviral activity against SARS CoV-2 through binding to RBD, preventing the virus from affecting ACE2. Moreover, our modeling results illustrate that heparin can also bind to and block ACE2, acting as a competitor and protective agent against SARS CoV-2 infection. Nine of the investigated MSPs candidates exhibited promising results, taking into consideration the newly emerged SARS CoV-2 variants, of which five were not previously reported to exert antiviral activity against SARS CoV-2, including sulfated galactofucan (1), sulfated polymannuroguluronate (SPMG) (2), sulfated mannan (3), sulfated heterorhamnan (8), and chondroitin sulfate E (CS-E) (9). These results shed light on the importance of sulfated polysaccharides as potential SARS-CoV-2 inhibitors.

Griffithsin and Carrageenan Combination Results in Antiviral Synergy against SARS-CoV-1 and 2 in a Pseudoviral Model.

Over 182 million confirmed cases of COVID-19 and more than 4 million deaths have been reported to date around the world. It is essential to identify broad-spectrum antiviral agents that may prevent or treat infections by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) but also by other coronaviruses that may jump the species barrier in the future. We evaluated the antiviral selectivity of griffithsin and sulfated and non-sulfated polysaccharides against SARS-CoV-1 and SARS-CoV-2 using a cytotoxicity assay and a cell-based pseudoviral model. The half-maximal cytotoxic concentration (CC50) and half-maximal effective concentration (EC50) were determined for each compound, using a dose-response-inhibition analysis on GraphPad Prism v9.0.2 software (San Diego, CA, USA). The therapeutic index (TI = CC50/EC50) was calculated for each compound. The potential synergistic, additive, or antagonistic effect of different compound combinations was determined by CalcuSyn v1 software (Biosoft, Cambridge, UK), which estimated the combination index (CI) values. Iota and lambda carrageenan showed the most potent antiviral activity (EC50 between 3.2 and 7.5 µg/mL). Carrageenan and griffithsin combinations exhibited synergistic activity (EC50 between 0.2 and 3.8 µg/mL; combination index <1), including against recent SARS-CoV-2 mutations. The griffithsin and carrageenan combination is a promising candidate to prevent or treat infections by SARS-CoV-1 and SARS-CoV-2.