**1. Introduction**

Mother Nature remains an outstanding hub for valuable natural compounds that have been used for multiple purposes since ancient times by our ancestors, including pharmaceutical, biomedical, nutritional, and cosmetics applications. Among the dozens of natural product groups, polysaccharides are macromolecular polymeric carbohydrate molecules comprised of large chains of monosaccharide units. They are widely found in animals, plants, and microorganisms, and their functions are mainly either structureor storage-related. Many studies have revealed that natural polysaccharides and their

**Citation:** Salih, A.E.M.; Thissera, B.; Yaseen, M.; Hassane, A.S.I.; El-Seedi, H.R.; Sayed, A.M.; Rateb, M.E. Marine Sulfated Polysaccharides as Promising Antiviral Agents: A Comprehensive Report and Modeling Study Focusing on SARS CoV-2. *Mar. Drugs* **2021**, *19*, 406. https://doi.org/ 10.3390/md19080406

Academic Editors: Khaled A. Shaaban and Vassilios Roussis

Received: 31 May 2021 Accepted: 20 July 2021 Published: 22 July 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

chemically modified derivatives have significant inhibitory activities against viral diseases such as human immunodeficiency virus (HIV) and herpes simplex virus (HSV) [1,2].

Sulfated polysaccharides (SPs) are a class of negatively charged polysaccharides comprising natural or modified sulfate moieties in their structural carbohydrate backbone. They possess significant biological activities such as antioxidant, anti-allergic, antiviral, anticancer, and anticoagulant abilities; hence, the study of SPs has significant importance for drug discovery campaigns [1,3]. SPs are mainly found in the cell walls of marine algae or seaweeds; they are less common in some mammals, such as fish skins, and rare in mangrove plants. The seaweed cell wall comprises about 40% of sulfated polysaccharides, which is relatively higher than the average content in other sources. The most interesting marine algal SPs are sourced from ulvans from green macroalgae, carrageenans and agar from red macroalgae, and fucoidans and laminarians from brown macroalgae. These SPs have shown antiviral activity against herpes simplex virus (HSV), human immunodeficiency virus type-1 (HIV-1), chikungunya virus, cytomegalovirus (CMV), influenza virus, and hepatitis virus, in addition to other enveloped and non-enveloped viruses. Moreover, the antiviral activity of SPs against the current COVID-19 pandemic has also been reported [1,4,5].

Marine sulfated polysaccharides (MSPs) have recently received increasing attention due to their antiviral activity. In particular, carrageenans have exhibited promising inhibitory effects on many viral strains, effectively preventing the internalization of virus particles by interfering with the interactions between the virus and host cell receptors. Carrageenan nasal spray (Boots Dual Defence® in the UK market) has been proven effective in patients with common cold infected by human coronaviruses beta and alpha. In contrast, the impact was attributed to the increased viral clearance and the reduced relapses of symptoms in children and adults [6]. Another nasal spray formulation (xylometazoline HCl) containing iota-carrageenan (**4**) can effectively relieve nasal congestion of the upper respiratory tract and protect the respiratory mucosa against viral infection [7]. Coldamaris® lozenges, comprising iota-carrageenan (**4**) as the active pharmaceutical ingredient, cause denaturation of glycoproteins on the coronavirus surface, inhibiting the virucidal effects of coronavirus [8].

It remains challenging to develop novel drugs within a limited timeframe, as drug discovery is time- and resource-consuming; however, the process of drug discovery is immensely enhanced by the modern era of computational technology, which has accelerated drug discovery and drug repurposing. Computer-assisted or in silico design employs computational methods in drug discovery and is being applied to streamline and speed up hit-to-lead optimization and hit identification. Several computational techniques have been introduced to predict and select therapeutic targets, study the interactions between drug and receptor, characterize and determine ligand binding sites on the targets, and determine hit compounds using ligand- and structure-based virtual screening [9,10].

Molecular docking and dynamic simulations are the most intriguing among the computational tools employed for structure-based drug discovery, an approach that evaluates the binding affinities between two candidates: small molecules and macromolecular targets (protein). Many drugs currently in the market were developed based on in silico strategies, such as zanamivir (used to treat influenza), nelfinavir, and saquinavir (used in the treatment of HIV); therefore, computational methods have been receiving popularity in the pharmaceutical industry as being crucial in the drug discovery process as reliable and effective techniques [11–13].

In the present investigation, we aimed to shed light on the antiviral potential of MSPs, illustrating their modes of interaction against different targets with the aid of several in silico tools, highlighting the most potential candidates for further investigation against SARS CoV-2.

#### **2. Results and Discussion**

Herein, we have summarized the findings of approximately 80 research studies published in the last 25 years on MSPs that exhibited potential antiviral effects against a total

of 22 different viral strains (Table 1, Figure S1). These antiviral MSPs were sourced from various marine sources, including brown algae, red algae, green algae, blue-green algae, microalgae, sea cucumber, and squid cartilage. According to the findings, 40% of the antiviral MSPs were isolated from the red algae, followed by 24% from brown algae, 14% from green algae, 10% from blue-green algae, 9% from microalgae, 2% from squid cartilage, and 1% from sea cucumber (Figure 1). The red-algae-derived MSPs exhibited greater efficacy against several viruses; however, the distribution was highly skewed towards herpes simplex viruses (HSV), which received most of the research effort (Figure S1).

**Figure 1.** The systematic study of marine sources of MSPs that have shown antiviral activities in the last 25 years.

#### *2.1. MSPs from Red Algae*

Red macroalgae were commercially considered more valuable than brown and green macroalgae and are widely used in manufacturing hydrocolloids, such as carrageenan and agar, which in turn are involved many applications, such as food, pharmaceutical, and biotechnological industries. The principal polysaccharide components of the red algae are sulfated galactans that are produced extracellularly; however, they are made up of a linear backbone of alternating 3-linked β-D-galactopyranose and 4-linked α-D-galactopyranose, with a few exceptions, such as DL-hybrid sulfated galactan. The various structural types of sulfated galactan (**7**) have revealed potent antiviral activity against several types of enveloped viruses, such as HSV-1, HSV-2, DENV-2, HIV-1, and HIV-2 (Table 1).


*Mar. Drugs* **2021**, *19*, 406

**Table 1.** The antiviral activity levels and modes of action of different MSPs derived from various marine sources.






Carrageenans, which are isolated from the cell walls, represent about 30–70% of the red algal dry weight, and their structure consists of linear chains of alternating galactopyranose units with linkages between 1 and 3 monomeric positions and other galactopyranose units with linkages between 1 and 4 monomeric positions or 3,6-galactopyranose units. The most important and extensively studied carrageenans are kappa (κ), iota, and lambda (λ), which vary in the number and position of sulfate ester groups (S), in accordance with the presence of 3,6-anhydrous-D-galactopyranose units [4]. Carrageenans are broadspectrum antivirals and have recently shown significant inhibitory effects by preventing the physical binding and entry of viral particles. Carrageenans were found to be effective against 12 different viruses (HSV, SARS-CoV-2, InfV, hRV, hCoV-OC43, HIV, DENV, hCV, HPV, RVFV, JEV, and TMV). Additionally, carrageenans are the most investigated class in human clinical trials against various virus diseases, such as sexually transmitted HIV, HPV, and HSV, in addition to rhinoviruses [101]. The most successful antiviral preparations of carrageenans were the nasal spray dosage forms that have recently been developed against rhinoviruses and SARS-CoV-2 [6,7,18,43]. Alongside carrageenan and sulfated galactans, other MSPs extracted from red algae and showed considerable activities against HSV, such as sulfated xylomannans, sulfated mannan, sulfated xylogalac tans, and sulfated xylan (Table 1, Figure 2).

**Figure 2.** Representative MSPs from red algae.

#### *2.2. MSPs from Brown Algae*

Brown algae come in second place after red algae in terms of antiviral activities and research attention (Figure 1). The main reported MSPs from this genus were fucoidans, a group of heterogeneous sulfated polysaccharides that represent about 25–30% of the algae dry weight and are made up of a backbone of α-(1→3)-L-fucopyranose residues or alternating α-(1→3) and α-(1→4)-linked L-fucopyranosyls with a sulfate group mainly substituted on C-2 or C-4 fucopyranose residues [4]. Fucoidans were found to be effective against 7 different viruses (HSV, SARS-CoV-2, InfAV, HIV-1, DENV-2, NDV, and HCMV) (Table 1, Figure 3 and Figure S1).

Generally, fucoidans block viral infection by preventing viral entry through competing for the positive charge attachment site of the envelope glycoproteins. The extent of antiviral activity is related to the number of sulfate groups present in the fucoidan structure [102]. As illustrated in Table 1, fucoidans have been tested in vivo using mice or mouse models for their activity against InfAV and HSV-2 and showed promising results. A recent in vitro study of two different fucoidans revealed that these compounds could be potent inhibitors of SARS-CoV-2 [17,18].

#### *2.3. MSPs from Green Algae*

Green-algae-derived MSPs come in third place as the most-investigated algae. Most of the examined compounds for antiviral activities belong to Ulvanes (Monostroma, Ulva, Enteromorpha), *Codium*, and *Caulerpa*. Different green-algae-derived MSPs were studied in vitro and in vivo against 11 viruses, including avian InfAV, HSV, HCMV, DENV-2, EV71, MeV, NDV, MuV, hCV, HIV, and JEV (Table 1, Figure 4). Song and his colleagues examined the antiviral activity of *Ulva pertusa* against avian InfAV and found that it exhibited a mild antiviral effect (40% viral inhibition). When combined with a vaccine against the same virus, it generated a synergistic effect, which significantly enhanced the production of antibodies by more than two-fold (~100%) [86]. Although the antiviral activities of green algae have received less attention than the red and brown, they possess unique antiviral properties against diverse viruses, such as NDV [78] and JEV [85].

**Figure 4.** Representative MSPs from green algae (green structures), blue-green algae, and marine-animal-derived SPs (violet structures).

#### *2.4. MSPs from Miscellaneous Marine Sources*

Blue-green algae, microalgae, sea cucumbers, and squid cartilage (Table 1, Figure 4) are studied much less than the former algae for their MSPs as potential antiviral agents. Among them, the novel sulfated polysaccharide calcium spirulan (Ca-SP) was isolated from the blue-green alga *Spirulina platensis* and found to be an inhibitor for several viruses, including HSV-1, HCMV, InfAV, MeV, HIV-1, and Muv [88]. Recently, a clinical trial confirmed the potential of Ca-SP (Spirularin® HS) against herpes viruses through inhibiting the attachment and penetration of HSV-1 into mammalian epithelial cells and blocking the entry of Kaposi sarcoma-associated herpesvirus HSV-8 [90]. The highly sulfated polysaccharide p-KG03 isolated from the microalgae *Gyrodinium impudium* showed potent inhibitory activity against InfAV by blocking the early stage of replication and entry [95]. Sea cucumber sulfated polysaccharide (SCSP) is one of the most recent MSPs extracted from the sea cucumber *Stichopus japonicus*. Song and colleagues confirmed its activity against SARS-CoV-2 in an in vitro study through binding to the S-glycoprotein, preventing SARS-CoV-2 host cell entry [18].

#### *2.5. In Silico Investigation of MSPs against SARS CoV-2*

Molecular modeling of polysaccharides is not an easy task due to their high diversity, complexity, and flexibility; however, the recent advances with in silico tools can relieve the

complexity of the process to a greater extent. Recently, heparin (**10**), an example of sulfated polysaccharide, was reported to exhibit a promising antiviral activity against SARS CoV-2 (EC50 = 36 μg/mL) through S-protein binding; hence, it suppresses the viral attachment to ACE2 (SARS CoV-2 S-protein receptor) and subsequently its entry inside the host cell (Figure 5) [17,103]. Similarly, most of the reported MSPs were found to mediate their antiviral activity via the exact mechanism, particularly those reported as anti-SARS CoV-2.

**Figure 5.** Schematic representation of the structure of SARS CoV-2 S-protein and how it can bind to the human ACE2. (PDB code: 6VXX) [104].

Accordingly, in this study, we shed light on MSPs as potential SARS CoV-2 antiviral agents by speculating on their plausible mode of action at the molecular level using a series of molecular docking and dynamic simulation experiments. Firstly, we determined the possible binding sites on both the S-protein receptor-binding domain (S-RBD) and ACE2 for MSPs. To do so, we utilized ClusPro [105], a software specialized in the prediction of heparin (**10**) (i.e., as an example of sulfated polysaccharide) binding sites in any given protein via molecular docking. The predicted heparin-S-RBD and heparin-ACE2 complexes were then subjected to 50 ns molecular dynamic simulation (MDS) experiments to select the most stable binding modes with each protein (S-RBD and ACE2). As shown in Figure 6, heparin was predicted to achieve stable binding with S-RBD at two sites (sites 1 and 2). Site 1 is located in a region that can interact with ACE2 directly and has a moderate positive charge. In contrast, site 2 is shallower than site 1 and is located in a wide positively charged region. Heparin-S-RBD complexes in these two sites were significantly stable over the course of MDS with low deviations from the original poses (RMSD ~ 2.5 Å) and minimal fluctuations (site 1 and site 2, Figure 6). This apparent stability resulted from the networks of H-bonds and ionic interactions formed between heparin and each binding site (Tables 2 and 3).

**Figure 6.** SARS CoV-2s S-RBD–ACE2 complex showing heparin binding sites (sites 1, 2 and 3).

**Table 2.** Docking scores and binding free energies of the top-scoring MSPS against S-RBD of the original and mutated SARS CoV-2 S-protein (site 1).


Note: \* Δ*G* was calculated using FEP method (see Materials and Methods for further information). \*\* N501Y-mutated strain of SARS CoV-2. \*\*\* This compound dissociated early at the beginning of MDS experiments (at ~25 ns). # The reported scores are the averages of three independent docking experiments (standard errors were between 0.1 and 0.3).

> On the other hand, heparin was predicted to interact with a small pocket (site 3) on ACE2 located near the S-RBD binding region (Figure 6). Similar to sites 1 and 2, heparin achieved stable binding with site 3 during the MDS (RMSD ~ 1.9 Å) through extensive H-bonds and ionic interactions (site 3, Table 4 and Figure 6).


**Table 3.** Docking scores and binding free energies of top-scoring MSPS against RBD of SARS CoV-2 S-protein (site 2).

Note: \* Δ*G* was calculated using the FEP method (see Materials and Methods for further information). \*\* This compound dissociated early at the beginning of MDS (at ~25 ns).

> **Table 4.** Docking scores and binding free energies of the top-scoring MSPS against ACE2 (site 3).


Note: \* Δ*G* was calculated using FEP method (see Materials and Methods for further information).

> The complex stability of RDB–ACE2 was also studied upon heparin binding to each site. As depicted in Figure 4, the distance between S-RBD and ACE2 (calculated as the distance between GLN-493 and GLU-35, respectively) remained constant (~5.1 Å) over 30 ns of MDS, while binding of heparin to S-RBD or ACE2 via sites 1, 2, or 3 led to significant instability of the complex and gradual dissociation (i.e., increased distance between GLN-493 and GLU-35). Heparin binding to site 1 on S-RBD or site 3 on ACE2 showed the greatest effects as S-RBD dissociated almost completely from ACE2 after 20 ns of simulation (Figure 7). Accordingly, it can be concluded that stable binding of SPs to sites 1, 2, or 3 destabilizes the S-RBD–ACE2 complex.

**Figure 7.** The calculated distance between S-RBD and ACE2 (i.e., between GLN-493 and GLU-35, respectively) during 30 ns of MDS in the absence and presence of heparin in site 1, 2, or 3.

It was recently reported that heparin can bind to S-RBD, preventing SARS CoV-2 from reaching ACE2 and entering the host cell; however, according to our modeling results, heparin can also bind to and block ACE2, acting as a protective agen<sup>t</sup> against SARS CoV-2 infection. To study the mode of action of all reported MSPs against SARS CoV-2 to sugges<sup>t</sup> previously unreported candidates, we subjected all collected compounds (Table 1) to dock against the proposed binding sites (sites 1, 2, and 3). Top-scoring hits (Figure 8) were selected according to the following criteria: (i) Docking score < −5 kcal/mol. Scores >−5 (i.e., −4.9 to −1.2 kcal/mol) showed unstable binding with the corresponding protein (RMSD > 20 Å at the first 20 ns); (ii) ΔG value < −5 kcal/mol, (iii) the docking pose remains stable over 50 ns of MDS. Some compounds achieved docking scores <−5 kcal mol (e.g., −6.4 kcal mol), although they were significantly unstable during the MDS experiments (RMSD > 15 Å at the first 20 ns) and presented significantly higher ΔG values (~−1.3 kcal/mol). As such, we made the binding stability at least 50 ns alongside ΔG value < −5 kcal/mol another selection criterion to discriminate between binders from non-binders, ensuring that all selected hits can achieve stable binding with the corresponding protein.

**Figure 8.** Sulphated polysaccharides with docking scores <−5 kcal/mol. Red compounds were significantly unstable during the course of MDS experiments. Black compound (compound 9) achieved stable binding with site 1 only. Blue compounds achieved stable bindings with sites 1 and 2. Green compounds achieved stable bindings with sites 1, 2, and 3.

Additionally, we took into consideration the newly emerged SARS CoV-2 variants during our docking experiments. Upon reviewing the recent mutations of the viral Sprotein, we found that the UK variant (i.e., B.1.1.7) has a mutation in its S-RBD (i.e., N501Y) that was associated with its higher affinity to the ACE2 receptor [106]. This mutation was the replacement of the amino acid ASP-501 with TYR. We applied this mutation to the S-RBD structure to study its effect on the binding with MSPs upon docking and MDS.

As shown in Table 2, docking against site 1 (with and without the N501Y mutation) resulted in nine SPs with scores <−5 kcal/mol (Figure 8). Four of these hits were previously reported to exert antiviral activity against SARS CoV-2. Further MDS and ΔG experiments revealed that the best binding modes for iota-carrageenan (**4**), kappa-carrageenan (**6**), sulfated galactan (**7**), and sulfated heterorhamnan (**8**) (Figure 8) with site 1 were significantly unstable (their average RMSD values were higher than 15 Å and their ΔG values were higher than −5 kcal/mol) and they could easily dissociate from site 1 (with and without the N501Y mutation). Sulfated galactofucan (**1**), sulfated mannan (**3**), and chondroitin sulphate E (CS-E) (**9**) were among the best-scoring hits that were significantly stable during the MDS experiments, either with the mutated or non-mutated RBD. Additionally, they had not been previously reported for their antiviral efficacy against SARS CoV-2; hence, they were considered good candidates for future evaluation.

As shown in Figures 9–14, the prevalent interactions of these MSPs and RBD site 1 in its mutated and non-mutated forms were H-bonds and water bridges, where the sulfate esters were the main contributors in these interactions with site 1 key amino acid residues (Table 2). Moreover, their fluctuations and deviations from the starting docking poses were almost identical to both the mutated and non-mutated form of site 1 during the MDS, except for chondroitin sulphate E, which achieved slightly more stability and less deviation from the starting binding pose upon binding to the non-mutated form of site 1 (Figure 13).

Regarding site 2, only five sulfated polysaccharides presented docking scores <−5 kcal/mol, of which iota-carrageenan (**4**) was unstable on MDS (got an average RMSD >15 Å) and presented a ΔG value higher than −5 kcal/mol (i.e., 1.7 kcal/mol). Sulfated galactofucan (**1**), sulfated polymannuroguluronate (SPMG) (**2**), and sulfated mannan (**3**) were among the best MSPs that achieved stable binding with S-RBD site 2 and were not previously reported against inhibitory effects of SARS CoV-2 (Table 3). In this site (i.e., site 2), ionic interactions between the negatively charged sulfate moieties and the positively charged key amino acid residues of this site were crucial, along with the other polar interactions (e.g., H-bonding and water bridging), in maintaining stable binding with such shallow binding site (Figures 15–17).

On the other hand, docking against the ACE2 binding site (site 3) revealed that both (SPMG) and lambda-carrageenan were the best-performing MSPs. Additionally, they were also stable over the course of MDS, achieving low deviations from their starting binding poses (RMSD ~ 2.9 Å) and ΔG values <−5 kcal/mol (~−5.2 kcal/mol). Lambdacarrageenan was previously identified to bind to SARS CoV-2 S-protein [44]; however, there have been no reports on the ACE2 binding potential of MSPs so far. As such, focusing on this human protein target will be of grea<sup>t</sup> interest in future investigations of SPs as anti-COVID-19 therapeutics. Similarly to site 1 and 2, the dominant interactions between these MSPs and the key amino acid residues inside site 3 were also of the polar type (e.g., H-bonds, water bridges, and ionic interactions) (Figures 18 and 19).
