Dual Effects of 3- epi-betulin from Daphniphyllum glaucescens in Suppressing SARS-CoV-2-Induced Inflammation and Inhibiting Virus Entry

Abstract

The continuous emergence of SARS-CoV-2 variants has led to a protracted global COVID-19 pandemic with significant impacts on public health and global economy. While there are currently available SARS-CoV-2 vaccines and therapeutics, most of the FDA-approved antiviral agents directly target viral proteins. However, inflammation is the initial immune pathogenesis induced by SARS-CoV-2 infection, there is still a need to find additional agents that can control the virus in the early stages of infection to alleviate disease progression for the next pandemic. Here, we find that both the spike protein and its receptor CD147 are crucial for inducing inflammation by SARS-CoV-2 in THP-1 monocytic cells. Moreover, we find that 3-epi-betulin, isolated from Daphniphyllum glaucescens, reduces the level of proinflammatory cytokines induced by SARS-CoV-2, consequently resulting in a decreased viral RNA accumulation and plaque formation. In addition, 3-epi-betulin displays a broad-spectrum inhibition of entry of SARS-CoV-2 pseudoviruses, including Alpha (B.1.1.7), Eplison (B.1.429), Gamma (P1), Delta (B.1.617.2) and Omicron (BA.1). Moreover, 3-epi-betulin potently inhibits SARS-CoV-2 infection with an EC₅₀ of <20 μM in Calu-3 lung epithelial cells. Bioinformatic analysis reveals the chemical interaction between the 3-epi-betulin and the spike protein, along with the critical amino acid residues in the spike protein that contribute to the inhibitory activity of 3-epi-betulin against virus entry. Taken together, our results suggest that 3-epi-betulin exhibits dual effect: it reduces SARS-CoV-2-induced inflammation and inhibits virus entry, positioning it as a potential antiviral agent against SARS-CoV-2.

Keywords: 3-epi-betulin; SARS-CoV-2; inflammation; virus entry inhibitor.

Figure 1

Stimulation of THP-1 cells with SARS-CoV-2 pseudovirus results in an inflammatory response. (A) Differentiated THP-1 cells were stimulated by pseudoviruses bearing either SARS-CoV-2 spike, envelope, or membrane protein (SEMpv) for 4 h. Cell lysates were collected and analyzed by Western blotting by indicated antibody. The ratio of phosphorylated p65 versus nonphosphorylated p65 is indicated. β-Actin was used as a normalized control. (B) THP-1 cells were stimulated for 16 h using pseudoviruses, and the expression levels of pro-inflammatory genes (IL1B, IL6, and TNF) and viral RNA were detected by RT-qPCR. GAPDH was used as a normalized control. (C) In parallel, the concentration of IL-1β cytokine was detected by ELISA in the supernatant. Data are presented as the mean ± S.D. (n = 3; *, p < 0.05; ***, p < 0.001).

Figure 2

The structural proteins of SARS-CoV-2 pseudovirus induce an inflammatory response. (A) Differentiated THP-1 cells were stimulated with either UV-inactivated or active SARS-CoV-2 pseudovirus for 4 h, and the cell lysates were collected for Western blotting by indicated antibodies. (B) Differentiated THP-1 cells were stimulated with either UV-inactivated or active SARS-CoV-2 pseudovirus for 16 h, and the cellular RNA was collected to detect the expression of pro-inflammatory genes (IL1B, IL6, and TNF) and viral RNA by RT-qPCR. GAPDH was used as a normalized control. (C) The concentration of IL-1β cytokine in the supernatant was analyzed by ELISA. Data are presented as the mean ± S.D. (n = 3; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

Figure 3

CD147 facilitates SARS-CoV-2-induced inflammation response in differentiated THP-1 cells. (A) Expression of ACE2 and CD147 in Calu-3 and differentiated THP-1 were detected by immunofluorescence staining with indicated antibody. DAPI stain as a nuclear marker. (B) THP-1 cells were knocked down by lenti-based shCD147 for 48 h and the CD147 expression level was evaluated using antibody detection and analyzed by flow cytometry. (C) CD147-knockdown THP-1 cells were stimulated by SARS-CoV-2 pseudotyped virus. The protein lysates were collected after 4 h virus incubation and the Western blotting was used to detect the phosphorylation status of p65 by indicated antibody (left panel). The RNA was collected after 16 h stimulation, and the level of proinflammation gene IL1b was detected by RT-qPCR (middle panel) and the level of IL1B cytokine in supernatant was detected by ELISA (right panel). Data are presented as the mean ± S.D. (n = 3; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant).

Figure 4

Compound 5 inhibits SARS-CoV-2-induced inflammation and viral replication. (A) Differentiated THP-1 cells were treated with potential anti-inflammation compounds for 48 h. The cell viability was determined by MTS assay (left panel). Differentiated THP-1 cells were infected with SARS-CoV-2 pseudovirus and treated with potential anti-inflammation compounds for 16 h. Gene expression of IL-1B was detected by RT-qPCR (right panel). (B) 293T cells were treated with indicated dose of compound 5. After 48 h, the cell viability was determined by MTS assay (left panel). 293T cells were transfected with pBAC-SARS-CoV-2 bearing luciferase gene and pCAG2-NP-HA. After 5 h, the cells were reseeded and compound 5 was added into the culture medium for 24 h. Viral replication was detected by luciferase activity assay, while cell viability was determined using an MTS assay. The results are presented as the ratio of luciferase activity to cell viability (right panel). (C) Calu-3 cells were infected with SARS-CoV-2 (hCoV-19/Taiwan/NTU49/2020) at MOI of 0.005 for 1 h. After infection, the cells were treated with either DMSO or 10 μM compound 5 for 24 h. The cell lysate was collected to detect the expression levels of pro-inflammatory genes (IL-6 and TNF) and different sense of viral RNA via RT-qPCR. (D) The culture supernatant was then harvested to determine the virus titer using RT-qPCR and plaque assay. Data are presented as the mean ± S.D. (n = 3; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant).

Figure 5

Compound 5 inhibits SARS-CoV-2-induced inflammation not related to virus entry. (A) Schematic representation of the timeline to evaluate the anti-inflammatory activity of compound 5. After 16 h incubation, cellular RNA was collected, and the expression levels of pro-inflammatory genes were determined by RT-qPCR. (B) Differentiated THP-1 cells were treated with compound 5 for 1 h before infection with SARS-CoV-2 pseudovirus. (C) Compound 5 was mixed with SARS-CoV-2 pseudovirus for 1 h prior to infecting the differentiated THP-1 cells. (D) Differentiated THP-1 cells were infected with SARS-CoV-2 pseudovirus and subsequently treated with compound for 16 h. GAPDH was used as a normalization control. The data are presented as the expression ratio of the inflammatory gene to GAPDH. Data are presented as the mean ± S.D. (n = 3; *, p < 0.05; **, p < 0.01; ns, not significant).

Figure 6

Compound 5 has the potential to inhibit virus entry. (A) The docked poses of compound 5 and betulinic acid anchored with the wild-type SARS-CoV-2 spike proteins. The receptor-binding domain of wild-type spike proteins (yellow) was complexed with the human ACE2 protein structure (PDB ID: 6M0J, chain A, gray). Two compounds, compound 5 (light purple) and betulinic acid (orange), docked with wild-type spike proteins by iGEMDOCK, have −76.9 and −72.8 kcal/mol binding energy, respectively. (B) BHK-ACE2 cells were treated with indicated compounds for 48 h. Cell viability was then determined by MTS assay. (C) SARS-CoV-2 pseudoviruses were pretreated with DMSO or each of indicated compound at 37 °C for 1 h. Subsequently, BHK-ACE2 cells were incubated with the compound–virus mixture for additional 48 h followed by luciferase activity assay. (n = 3; ***, p < 0.001, ns, not significant).

Figure 7

Compound 5 exhibits inhibitory effects on virus entry. (A) Pseudovirus bearing spike from different variants of SARS-CoV-2 was pre-incubated with serially diluted compound at 37 °C for 1 h. The mixture was then put into BHK-ACE2 cells for 48 h followed by luciferase activity assay to determine the EC50 of the compound to each variant of pseudovirus. (B) Each of SARS-CoV-2 variants was pre-incubated with a serial dilution of the compounds at 37 °C for 1 h. Subsequently, VeroE6 cells were incubated with the compound–virus mixture for an additional hour, followed by incubation without compound treatment for 120 h. The plaque formation ratio was calculated by counting the number of plaques and normalizing them to the numbers obtained from the DMSO control. (C) SARS-CoV-2 (NTU49) with MOI at 0.001 was pretreated with DMSO, 10 μM, or 20 μM compound at 37 °C for 1 h. Calu-3 cells were then incubated with the compound–virus mixture for an additional 1 h, followed by incubation without compound treatment for 24 h. The culture supernatant was collected, and the virus titer was determined using qRT-PCR (left) and plaque assay (right). Data are presented as the mean ± S.D. (n = 3; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant).

Figure 8

Interaction profile and 2-D interaction plot of compound 5 binding to wild-type SARS-CoV-2 spike protein. (A) The interaction profiles of compound 5, 6, 7, and 8 by iGEMDOCK. Each interactive residue listed on the x-axis has four codes separated by hyphens. The first code of the interactive residue stands for the force between compounds and residues, H for hydrogen bond force, and V for van der Waals forces. The second code represents the interaction in the main chain (M) or side chain (S). The third code represents the spike protein’s residue type, and the fourth code is the residue serial number. The interactions are represented in green when the energy ≤ –2.5 for H and the energy < –4 for V. The absent interactions are colored in black. Three red triangles indicate these interactive residues interact with compound 5 but not with other compounds. (B) 2-D interaction plot of the interactions between docked compound 5 and the wild-type spike protein visualized by PoseView.

Figure 9

Sequence alignment and structural comparison of compound 5 binding to SARS-CoV-2 spike protein. (A) The multiple sequence alignment of the receptor-binding domain in the SARS-CoV-2 spike proteins was presented. The title of each protein sequence is in the format “ Abbreviations_PDB ID_Chain” listed in table in Section 4.13. The amino acid residues that compound bind to spike protein are marked blue. (B) The receptor-binding domains of wild-type (yellow) and Omicron (pink) spike proteins are superimposed. The human ACE2 protein structure (PDB ID: 6M0J, chain A), which was complexed with the wild-type spike protein in the crystal structure, is colored in gray. Compound 5, with light green or light purple sticks, is indicated as anchored with wild-type or Omicron strains of SARS-CoV-2 spike proteins, respectively.