Perhydrolase-polydopamine composite coatings possessed potent antiviral activity, and dramatically reduced the infectivity of a SARS-CoV-2 pseudovirus within minutes [Wang et al. Sci. Rep. 11, 12410, 2021]. The single-step approach enables rapid and facile fabrication of enzyme-based disinfectant composite coatings with high activity and stability, which enables reuse following surface washing.
Rapid deactivation of virus particles on contaminated surfaces is critical to achieve reduced viral load and transmission. To this end, we examined two viruses, including a VSV-g pseudotyped lentivirus and a SARS-CoV-2 spike pseudotyped lentivirus, both carrying an Egfp reporter gene. Perhydrolase-polydopamine composite coatings possessed potent antiviral activity, and dramatically reduced the infectivity of a SARS-CoV-2 pseudovirus within minutes (Figure 1). Specifically, AcT-PDA composite coatings at 10 µg∙mL−1 AcT, 5 mM PGD, and 2.5 mM H2O2 were capable of eliminating >80% of functional viral titer for a lentivirus (using HEK293T as susceptible target cells) and >70% of functional viral titer for a SARS-CoV-2 pseudovirus (using HEK293T-Ace2 as susceptible target cells) upon exposure to the composites and the substrates in just 5 min, which is consistent with rapid decontamination. Thus, the AcT-PDA composite was effective in neutralizing both VSV-g and SARS-CoV-2 pseudovirus particles in a very short time, which mimics simple surface contact.
Figure 1. Antiviral activity of AcT (10 μg∙mL−1)-PDA composite against different viruses. (a) VSV-g pseudotyped lentivirus and (b) SARS-CoV-2 pseudovirus in 300 s at 25oC. The x-axis shows the condition tested and the y-axis shows the % infected. The black bars represent t = 90 s and t = 45 s for the lentivirus and pseudovirus, respectively, while the gray bars represent t = 300 s. The representative images show the virus-infected cells after treatment with AcT-PDA at initial time point (t = 90 s for lentivirus and t = 45 s for pseudovirus) and at 300 s. Nuclei are stained blue with Hoechst 33342. A student’s t-test was used to calculate statistical significance. Error bars represent mean ± SEM across three replicates. * p < 0.05, ** p < 0.01, ns-not significant. [Wang et al. Sci. Rep. 11, 12410, 2021]
COVID-19, caused by the SARS-CoV-2 virus, has now spread worldwide with catastrophic human and economic impacts. In an effort to mitigate disease symptoms and impede viral spread, efforts in vaccine development and drug discovery are being conducted at a rapid pace. Recently, we showed that the well-known anticoagulant heparin has exceptional binding affinity to the spike protein (S-protein) of SARS-CoV-2 [Kim et al. Antiviral Res. 181, 104873 (2020)]. The S-protein of SARS-CoV-2 bound more tightly to immobilized heparin (KD=~10-11 M) than the S-proteins of either SARS-CoV (KD=~10-7 M) or MERS-CoV (KD=~10-9 M). However, it is not known whether the tight binding of heparin to the SARS-CoV-2 S-protein translates into potent antiviral activity. Therefore, we evaluated the in vitro antiviral properties of heparin and other closely related polysaccharides to assess the relevance of heparin-related GAGs and other sulfated polysaccharides as part of the pharmacopeia of potential therapeutics that target SARS-CoV-2. Vero-CCL81, which expresses both ACE2 and TMPRSS, were used for viral replication at high titer for use in antiviral assays.
Our results reveal that specific sulfated polysaccharides bind tightly to the S-protein of SARS-CoV-2 in vitro, which suggests they can act as decoys to interfere with S-protein binding to the heparan sulfate co-receptor in host tissues, inhibiting viral infection [Kwon et al. Cell Discov. 6, 50 (2020)]. Specifically, heparin, heparan sulfates, other glycosaminoglycans (GAGs), and fucoidan and other highly sulfated polysaccharides were screened using surface plasmon resonance (SPR) to measure binding affinity to the SARS-CoV-2 S-protein (Figures 2 and 3).
To model this, we constructed a docking model between heparin and the S-protein receptor binding site (RBD) using the crystal structure of the chimeric RBD-ACE2 complex (PDB ID: 6VW1). The RBD’s amino acid residues involved in binding the ACE2 (angiotensin-converting enzyme 2) receptor also participated in heparin binding, suggesting a mechanism of viral entry inhibition by heparin. Antiviral activities correlated with the SPR results. The most potent compound tested, RPI-27, is a high molecular weight, branched polysaccharide related to the known compound fucoidan, and had an EC50 of 8.3 ± 4.6 μg/mL, which corresponds to approx. 83 nM. This is substantially more potent than remdesivir having a reported in vitro EC50 value of 770 nM in Vero-E6 cells and 11.4 µM in Vero-CCL81 cells, currently approved for emergency use for severe COVID-19 infections. The smaller RPI-28 has the same basic structure as RPI-27 but a lower molecular weight and, thus, a lower activity (1.2 μM). Heparin and the TriS-heparin (an intermediate in the bioengineered heparin synthesis pathway) also have potent antiviral activity withEC50 values of approx. 2.1 and 5.0 μM, while the lower molecular weight NACH had an approx. EC50 of 55 μM. Similar antiviral activity of heparin has also been demonstrated recently. Heparin and TriS-heparin are similar, with the latter devoid of the relatively small fraction of 3-O-sulfate groups present on heparin. Thus, their similar activity is expected. However, the low molecular weight NACH had far lower antiviral activity. Less sulfated GAGs, such as heparan sulfate and various chondroitin sulfates, because of their very low S-protein binding were not tested in the antiviral assay.
Figure 2. a) Surface plasmon resonance (SPR) experiments were used to screen polysaccharides that outcompete immobilized heparin binding to SARS-CoV-2 S-protein. Data are presented as mean ± s.d., n = 3 biologically independent samples. A two-sided t-test was performed to test significance against the control (P1 < 0.0001, P2 = 0.0003, P3 = 0.0016, P4 = 0.0041). b) Structural units comprising polysaccharides used for in vitro antiviral studies.
Figure 3. c) Focus reduction assay images of virus infection on treatment of indicated polysaccharides. At 48 h after infection, Vero cells were fixed and probed with SARS-CoV-2 spike primary antibody (1:10000, Sino Bio Inc.) and HRP-conjugated goat rabbit (1:10000, Abcam) secondary antibody. d) Vero cells were infected with SARS-CoV2 at a MOI of 2.5 × 10-3 at different doses of each polysaccharide for 48 h. The viral yield was quantified using a focus reduction assay. Cytotoxicity in Vero cells was measured using a WST-1 assay. The left and right y-axis of the graphs represent mean % inhibition of virus yield and cytotoxicity of the polysaccharides, respectively. Cytotoxicity experiments were performed in duplicate with n = 3 biologically independent samples. Focus reduction assay experiments were performed in mean ± s.d. (quadruplicate measurements) with n = 3 biologically independent samples. e) The RBD-ACE2 binding interface is stabilized by an extensive hydrogen bonding network involving sidechains of several residues on both RBD and ACE2. Polar sidechains of N487, Y489, Q493, Q498 and Y505 on the spike protein RBD along with other residues would be able to bind to heparin and inhibit RBD-ACE2 interaction. Heparin (here an octasaccharide) forms a hydrogen bond network with N448, N450, Q493 and N501 that aids in its occupancy of this binding regions and sterically restrict access to Q498, Y489 and Y505 necessary for ACE2 receptor binding. [Kwon et al. Cell Discov. 6, 50 (2020)]
Current Collaborators:
Robert Linhardt and Fuming Zhang – Rensselaer Polytechnic Institute
Jung Joo Hong – Korea Research Institute of Bioscience and Biotechnology
