*2.6. Structure–Activity Relationship*

The structural complexity of SPs has made the study of their structure–activity relationship quite challenging, and until now it has not been entirely understood; however, certain key bioactivity-related structural features could be concluded from the previously reported studies and the present modeling study.

The first structural determinant that may affect the antiviral potential of this class of compounds is the number of negatively charged groups (e.g., sulfates or carboxylates). Polysaccharides with higher numbers of sulfate or carboxylate groups per monosaccharide were more bioactive. Highly sulfated glucans (either natural or chemically synthesized derivatives) were found to be far more bioactive as antiviral agents than those with lower degrees of sulfation, while non-sulfated glucans were practically inactive [107,108]. Moreover, in our modeling study, MSPs with a single sulfate or carboxylate group per monosaccharide did not achieve stable binding with either S-RBD or ACE2 (ΔG values > −5 kcal/mol); hence, we could conclude that electrostatic and other polar interactions mediated by these

negatively charged groups play a significant role in stabilizing the binding of this class of compounds to S-RBD and ACE2.

**Figure 9.** Binding mode of sulphated galactofucan (compound **1**) inside site 1 (**A**,**B**) and its mutated form (**C**,**D**) together with its RMSDs during 50 ns of MDSs (**E**).

**Figure 10.** Protein–ligand contacts of sulphated galactofucan (compound 1) inside site 1 (**A**) and its mutated form (**B**) during the MDS.

**Figure 11.** Binding mode of sulfated mannan (**3**) inside site 1 (**A**,**B**) and its mutated form (**C**,**D**) together with its RMSDs during 50 ns of MDSs (**E**).

**Figure 13.** Binding mode of chondroitin sulphate E (compound **9**) inside site 1 (**A**,**B**) and its mutated form (**C**,**D**) together with its RMSDs during 50 ns of MDSs (**E**).

**Figure 14.** Protein–ligand contacts of chondroitin sulphate E (compound 9) inside site 1 (**A**) and its mutated form (**B**) during the MDS.

**Figure 15.** Binding mode of sulfated galactofucan (**1**) inside site 2 (**A**,**B**) together with its RMSDs and protein–ligand contacts during 50 ns of MDSs (**C**,**D**).

**Figure 16.** Binding mode of sulfated polymannuroguluronate (SPMG) (compound 2) inside site 2 (**A**,**B**) together with its RMSDs and protein–ligand contacts during 50 ns of MDSs (**C**,**D**).

**Figure 17.** Binding mode of sulfated mannan (compound 3) inside site 2 (**A**,**B**) together with its RMSDs and protein–ligand contacts during 50 ns of MDSs (**C**,**D**).

Secondly, the distribution of these negatively charged moieties played an essential role in their antiviral activity. In previous studies, carrageenans with similar degrees of sulfation (50 mol%) but isolated from different sources showed varying antiviral activities [39,109,110]. Different charge densities were proposed to explain these findings, and accordingly to explain the antiviral activity [71]. Our modeling study found that neither iota- nor kappa-carrageenan achieved stable binding with any proposed binding sites.

This could be attributed to their relatively low degree of sulfation (one sulfate group per onosaccharide); however, both were reported to be bioactive against SARS CoV-2 and other pathogenic viruses. This could be explained by the unique distribution of these limited sulfate groups, which might cause them to be bioactive via different modes of action. In contrast, sulfated galactan (**7**) has a high degree of sulfation (Figure 8); however, it did not achieve stable binding with the proposed binding sites (ΔG value >−5 kcal/mol). Accordingly, the presence of many negatively charged moieties does not guarantee good antiviral activity without the proper orientations and distribution over the polysaccharide backbone.

**Figure 18.** Binding mode of sulfated polymannuroguluronate (SPMG) (compound 2) inside site 3 (**A**,**B**) together with its RMSDs and protein–ligand contacts during 50 ns of MDSs (**C**,**D**).

**Figure 19.** Binding mode of lambda-carrageenan (compound 5) inside site 3 (**A**,**B**) together with its RMSDs and protein– ligand contacts during 50 ns of MDSs (**C**,**D**).

Finally, the general chemical structure of the polysaccharides (e.g., the stereochemistry of the glycosidic linkage and that of monosaccharides and the presence of branching points in the main backbone) has a general impact on the degree of their antiviral activity [111].
