*3.3. OA-10 Partly Blocks IAV HA Adsorption to Chicken RBCs at High Concentrations*

IAV is able to adsorb to chicken RBC, resulting in hemagglutination through the interaction of the receptor-binding domain in viral HA1 subunits with the sialic acid receptors on RBC membrane. To see whether HA is the potential target of OA-10, we questioned whether OA-10 could inhibit hemagglutination by interfering with H5 HA adsorption to RBC. OA-10 did not inhibit HA adsorption to chicken RBCs at concentrations of 10 and 20 μM. At the concentrations of OA-10 ≥ 40 μM, it was observed that a portion of RBCs settled to the bottom of the wells but the settled RBCs were not able to flow, as shown in Figure 5A. Meanwhile, OA-10 treatment did not affect RBC properties in the absence of H5 virus, and the positive control antisera against H5 hemagglutinin (Anti-H5) could effectively inhibit H5 HA adsorption to chicken RBCs with a titer at 1:16, and the settled RBCs could move when the plate was tilted (Figure 5A). These results indicated that OA-10 might have a weak interaction with the receptor binding domain of H5 HA1, which needs further demonstration.

**Figure 5.** HA is a potential target of OA-10. (**A**) Comparisons of the behaviors of OA-10 vs. antisera against H5 HA in inhibition of H5N1 IAV-induced aggregation of chicken RBCs. OA-10 partially blocked H5N1 virus adsorption to chicken RBCs at concentrations ≥40 μM. (**B**) Characterization of the affinity between OA-10 and HA protein using surface plasmon resonance (SPR) analysis. (**C**) Inhibition of OA-10 on HA-mediated chicken RBCs hemolysis using arbidol as the positive control.

#### *3.4. OA-10 Exhibits a Strong Interaction with HA Protein*

To investigate the interaction of OA-10 with HA, we studied the affinities of OA-10 with HA by surface plasmon resonance (SPR). A recombinant HA protein from virus strain A/Vietnam/1203/2004 (H5N1) was used as representative HA of H5N1 IAV strains, because the amino acid sequence identity of the full-length HA proteins between A/Vietnam/1203/2004 strain and A/Duck/Guangdong/99(H5N1) strain used in above antiviral assay was 97.0% (Supplementary Material 5) [35]. As shown in Figure 5B, OA-10 interacted with HA in a dose-dependent manner, and the equilibrium dissociation constant (KD) of the interaction was 2.98 <sup>×</sup> <sup>10</sup>−<sup>12</sup> M, indicating a strong affinity between OA-10 with HA. OA-10 did not interact with negative control protein FKBP12 (FK506 binding protein 1A, 12 kDa), indicating that interactions of HA with OA-10 were specific [36]. This result indicates that HA is most likely the target of OA-10 by which it inhibits virus entry and subsequent replication.
