*3.5. OA-10 Inhibits H5N1 IAV-Mediated Hemolysis at Low pH*

We next investigated whether OA-10 was able to inhibit HA-induced chicken RBC hemolysis at a low pH [33]. To trigger hemolysis, the virus-cell suspension was acidified (pH 5.2) to initiate HA subunit HA2 conformational changes that lead to the lysis of chicken RBCs and their hemoglobin release. Arbidol, a licensed antiviral agent against IAV infection in Russia and China, was demonstrated to inhibit IAV replication by binding to HA2 in acidic condition and used as a positive hemolysis inhibitor control in this study [37]. As shown in Figure 5C, as expected, arbidol showed hemolysis inhibition with an IC50 of 51 μM. Interestingly, OA-10 more potently inhibited HA-induced hemolysis in a dose-dependent manner, with an IC50 of 26 μM. Taken together, these results show that OA-10 interacts with HA2.

#### *3.6. Molecular Model of HA-OA-10 Binding*

In order to further understand the molecular basis of OA-10 interacting with HA, we performed docking experiments using CDOCKER protocol to predict the binding modes of OA-10 into the CR6261 antibody binding site which is a highly conserved hydrophobic groove at the HA1-HA2 interface in the HA stem region [19]. The docked conformation of OA-10 was determined based on the minimum

CDOCKER interaction energy. As shown in Figure 6, the modeled structure of HA complexed with OA-10 indicated that C2-OH and C3-OH of the L-rhamnose moiety linked to C2-OH of D-glucose were positioned within appropriate distance (2.46 Å, 1.99 Å and 2.18 Å) to make three strong hydrogen bonds with Gly20 and Val18 in HA2, respectively. Furthermore, C3-OH of D-glucose and the C2-OH of the L-rhamnose moiety linked to C4-OH of D-glucose formed hydrogen bonds with Asp19 and Gln42 with the distances 2.69 Å and 2.78 Å, respectively. In addition, oleanane scaffold also form numerous hydrophobic contacts with the HA1 residue (His38) and HA2 residues (Trp21, Lys38 and Ile45). According to docked conformation of OA-10, 3-O-β-chacotriosyl moiety obviously plays an important role in binding residues in HA2 with higher docking score.

**Figure 6.** Structural model of OA-10 binding site in H5 HA. (**A**) CR6261 antibody binding site surface. The modeling was based on the published crystal structure of the A/Vietnam/1203/2004 (H5N1) (PDB: 6CFG). HA1 and HA2 regions are highlighted in green and blue, respectively. Compound OA-10 is shown as yellow sticks. (**B**) Modeled structure of HA complexed with OA-10. Green lines represent hydrogen bond interaction, with the distances indicated; and pink lines represent hydrophobic interaction between OA-10 and HA.
