A Heparan Sulfate Mimetic RAFT Copolymer Inhibits SARS-CoV-2 Infection and Ameliorates Viral-Induced Inflammation

Abstract

The high transmissibility and mutation ability of coronaviruses enable them to easily escape existing immune protection and also pose a challenge to existing antiviral drugs. Moreover, drugs only targeting viruses cannot always attenuate the "cytokine storm". Herein, a synthetic heparan sulfate (HS) mimetic, HMSA-06 is reported, that exhibited antiviral activities against both the SARS-CoV-2 prototype and Omicron strains by targeting viral entry and replication. Of particular note, HMSA-06 demonstrated more potent anti-SARS-CoV-2 effects than PG545 and Roneparstat. SARS-CoV-2 is reported to hijack autophagy to facilitate its replication, therefore boosting autophagy can attenuate SARS-CoV-2 infection. It is revealed that HMSA-06, but not a similar HS mimetic that failed to inhibit SARS-CoV-2, can upregulate cellular autophagy flux. In addition, HMSA-06 was found to robustly block the NLRP3-mediated inflammatory reaction in SARS-CoV-2 infected THP-1 derived macrophages as evidenced by a reduction in inflammasome formation and the subsequent decreased secretion of mature caspase-1 and IL-1β. The HMSA-06's inflammation inhibitory function is further confirmed using a LPS/ATP-stimulated THP-1 macrophage model. Altogether, this study has identified a promising HS mimetic to combat SARS-CoV-2-associated diseases by inhibiting viral infection and attenuating viral-induced inflammatory reaction, providing insights into the development of novel anti-coronavirus drugs in the future.

Keywords: NLRP3; SARS‐CoV‐2; coronavirus; heparan sulfate mimetics; poly(SS‐co‐AA).

Figure 1

HMSA‐06 inhibits the SARS‐CoV‐2 prototype infection. A) The illustration of screening the effective compounds with capacities to inhibit the SARS‐CoV‐2 prototype replication by post‐viral infection treatment. Vero E6 cells were grown to 70%–80% confluence and were infected by SARS‐CoV‐2 (MOI = 0.01). After 1 h, cells were washed with culture media and fresh media with different HS mimetics were added. Cells were harvested at 48 h post infection for analyzing the SARS‐CoV‐2 infection. B) The representative blots show the expression of SARS‐CoV‐2 spike (S) protein in the presence of different heparan sulfate mimetics (upper panel) and the intensity of S protein (lower panel). The results were shown as mean ± SD from two independent experiments. C) The structure of HMSA‐06. D) The western blots illustrate the expression of S protein in Vero E6 cells in the presence of distinct concentrations of HMSA‐06‐5 or HMSA‐06‐20. E) The intensity of S protein from two independent experiments from (D) was quantified using the ImageJ. F) The mRNA expression of the SARS‐CoV‐2 envelope E) gene was quantified by q‐PCR from the samples in (D). G) The representative western blots of three independent experiments represent the expression of S protein in the presence of different concentrations of HMSA‐06‐5 or HMSA‐06‐20 in Calu3 cells. The number of plaques caused by SARS‐CoV‐2 in the presence of various concentrations of HMSA‐06 (H) or HMS‐01 (I) was assessed by the plaque assay. The data were normalized by the plaque number in the absence of HMSA‐06 or HMS‐01 from two independent experiments (n = 2). Statistical analysis was performed using one‐way analysis of variance (ANOVA). ^** p ≤ 0.01, ^*** p ≤ 0.001.

Figure 2

HMSA‐06 restricts the SARS‐CoV‐2 Omicron infection. A) Vero E6 cells were infected by SARS‐CoV‐2 Omicron (MOI=0.5), and HMSA‐06‐20 (0.5µm) or heparin (100µg mL⁻¹) were added to the medium at 1h after infection. Cells were fixed at 48 h post‐infection, and the expression of the SARS‐CoV‐2 Omicron S protein in the presence or absence of HMSA‐06 or heparin was examined by Immunofluorescence assay. The representative images from two independent experiments were shown. Scale bar, 30µm. B) Vero E6 cells or C) Caco2 cells were treated with different concentrations of HMSA‐06 as indicated after infection with SARS‐CoV‐2 Omicron (MOI=0.01). The expression of the SARS‐CoV‐2 Omicron S protein and nucleocapsid protein (N) were evaluated by western blot. The representative western blots of two independent experiments were presented. The non‐specific bands detected by the anti‐SARS‐CoV‐2 N protein antibody in the Caco2 cells were indicated by an asterisk. D) The number of plaques formed by the SARS‐CoV‐2 Omicron infection with or without HMSA‐06 incubation at different concentrations. The data were normalized by the plaque number in the absence of HMSA‐06 from two independent experiments.

Figure 3

HMSA‐06 exhibits higher anti‐SARS‐CoV‐2 activities than PG545 and Roneparstat. A) The experiments were set up in a similar way as in Figure 2B. The representative western blots illustrate the expression of the SARS‐CoV‐2 Omicron S protein in the presence or absence of different concentrations of HMSA‐06‐20 and PG545 in Vero E6 cells. B) The mRNA expressions of the SARS‐CoV‐2 E gene from the same samples in (A) were assessed by q‐PCR and data were shown as mean ± SD from two independent experiments. C) The representative western blots show the expression of the SARS‐CoV‐2 Omicron with or without treatments of different concentrations of HMSA‐06‐20, PG545, and Roneparstat in Caco2 cells. D) The mRNA expressions of the SARS‐CoV‐2 E gene from the same samples in (C) were assessed by q‐PCR and data were shown as mean ± SD from two independent experiments.

Figure 4

HMSA‐06 boosts the autophagy. A) The 70%–80% confluent Vero E6 cells were incubated with 09–20K (1 and 2 µm) for 24 and 48 h, the expression of LC3 was assessed by western blot. The representative western blots of three independent experiments were shown. B) The 70%–80% confluent Vero E6 cells were incubated with HMSA‐06‐05 or HMSA‐06‐20 (2 µm) for 48 h. Or CQ (20 µm) was added to the cells at 40 h after HMSA‐06 treatment. The representative western blots of three independent experiments were shown. GAPDH was utilized as a loading control. C) GFP‐LC3 was transfected into Vero E6 cells. After 24 h, the cells were incubated with HMSA‐06‐20 (2 µm) for 24 h and CQ (20 µm) was added to the cells at 16 h after HMSA‐06‐20 treatment. The GFP‐LC3 puncta in the cells in the absence or presence of HMSA‐06 were observed under the microscope. The representative pictures were shown. Scale bar, 20 µm. D) The working mechanism of the autophagic flux probe adapted from the previous study^{[ 26 ]} is shown. A lower GFP/RFP ratio indicates higher autophagic flux. E) The probe pMRX‐IP‐GFP‐LC3‐RFP‐LC3ΔG was transfected into Vero E6 cells and treated with or without HMSA‐06‐20 (2 µm) for 24h. Cells were fixed and the GFP and RFP fluorescence were observed under a microscope. The representative images that merged by GFP and RFP fluorescent channels were shown. F) Quantification of GFP/RFP fluorescence intensity. The Mean ± SD of the ratio of GFP and RFP fluorescence intensity in 50 transfected cells recorded in each condition is shown (n = 50). Scale bar, 20 µm. Statistical analysis was performed using Student's t‐test. ^**** P ≤ 0.0001. G) The representative western blots of two independent experiments illustrating the expression of the SARS‐CoV‐2 S protein, N protein and cellular LC3 with or without treatment of HMSA‐06‐20 were shown. GAPDH was used as a control.

Figure 5

HMSA‐06 attenuates the inflammatory reaction induced by SARS‐CoV‐2 infection. A) The macrophage‐like THP‐1 cells were induced by LPS+ATP to activate the inflammation reaction in the presence or absence of various concentrations of HMSA‐06‐20 or HMSA‐06‐05. The expression of the mature caspase‐1 and IL‐1β in the supernatant as well as the pro‐caspase‐1 and pro IL‐1β in the cell pellets were assessed by western blot. The representative western blots of two independent experiments were shown. B) Macrophage‐like THP‐1 cells were induced by LPS+ATP in the absence or presence of HMSA‐06‐20. The ASC puncta were observed under the microscope and the representative pictures were shown. HMSA‐06 significantly decreased the number of ASC puncta. Scale bar, 30 µm. C) The macrophage‐like THP‐1 cells were treated with LPS+ATP to activate the inflammation reaction with or without adding HMSA‐06‐20 (2 µm) or heparin (100 µg mL⁻¹). The expression of pro‐ or mature caspase‐1 and IL‐1β in cell pellets or supernatants were assessed by western blot. D) Macrophage‐like THP‐1‐ACE2 cells were infected by the SARS‐CoV‐2 prototype (MOI = 0.2) in the absence or presence of HMSA‐06‐20 (1 or 2 µm) with or without prime by LPS. The western blots show the expression of the pro or mature caspase‐1 and IL‐1β in cell pellets or supernatant as well as the expression of the SARS‐CoV‐2 S protein. The representative western blots of two independent experiments were shown. E) The representative images illustrate the ASC puncta in the SARS‐CoV‐2 infected macrophage‐like THP‐1‐ACE2 cells with or without adding the HMSA‐06‐20. Scale bar, 30 µm. F) Quantification of the cells with ASC puncta. The Mean ± SD of the percentage of the cells with ASC puncta from 30 images randomly taken from each condition is shown (n = 30). Statistical analysis was performed using Student's t‐test. ^**** p ≤ 0.0001. G) The representative images show the NLRP3 puncta in the SARS‐2 (SARS‐CoV‐2) infected macrophage‐like THP‐1‐ACE2 cells in the presence or absence of HMSA‐06‐20 (2 µm) or MCC950 (10 µm). Scale bar, 30 µm. H) The Mean ± SD of the percentage of the cells with NLRP3 puncta from 30 images randomly taken from each condition in (E) is shown (n = 30). Statistical analysis was performed using Student's t‐test. ^**** p ≤ 0.0001. ns, not significant. I) The macrophage‐like THP‐1‐ACE2 cells were infected using a high titer of SARS‐CoV‐2 (MOI = 5). The expression of active caspase‐1 and mature IL‐1β in the supernatant and the expression of SARS‐CoV‐2 S protein in cell pellets in the presence or absence of HMSA‐06‐20 (2 µm) were examined by western blot.

Figure 6

The proposed model for the anti‐SARS‐CoV‐2 infection and anti‐virus‐induced inflammation of HMSA‐06. The activation of NLRP3‐mediated inflammatory reactions has been shown in immune cells, tissues, and animal models though the mechanism of how SARS‐CoV‐2 triggers the inflammation is not fully understood.^{[ 41 ]} Based on our findings, we propose a working model describing how HMSA‐06 could act as a good candidate to treat SARS‐CoV‐2‐associated diseases: HMSA‐06 is able to inhibit SARS‐CoV‐2 cell entry (especially for the SARS‐CoV‐2 Omicron strain) and viral replication through boosting autophagic flux, which could function as the first line to prevent the progress of severe COVID‐19. In addition, HMSA‐06 significantly attenuates the activation of NLRP3 inflammasome, which could ameliorate the hyperinflammatory reaction at the late stage of SASR‐CoV‐2 infection. Damage‐associated molecular patterns (DAMP). Heparan sulfate proteoglycan (HSPG).