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. 2023 Dec 17;15(12):2791.
doi: 10.3390/pharmaceutics15122791.

SARS-CoV-2 Fusion Peptide Conjugated to a Tetravalent Dendrimer Selectively Inhibits Viral Infection

Carla Zannella  1 , Annalisa Chianese  1 , Alessandra Monti  2 , Rosa Giugliano  1 , Maria Vittoria Morone  1 , Francesco Secci  3 , Giuseppina Sanna  4 , Aldo Manzin  4 , Anna De Filippis  1 , Nunzianna Doti  2 , Massimiliano Galdiero  1   5
Affiliations

SARS-CoV-2 Fusion Peptide Conjugated to a Tetravalent Dendrimer Selectively Inhibits Viral Infection

Carla Zannella et al. Pharmaceutics. .

Abstract

Fusion is a key event for enveloped viruses, through which viral and cell membranes come into close contact. This event is mediated by viral fusion proteins, which are divided into three structural and functional classes. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein belongs to class I fusion proteins, characterized by a trimer of helical hairpins and an internal fusion peptide (FP), which is exposed once fusion occurs. Many efforts have been directed at finding antivirals capable of interfering with the fusion mechanism, mainly by designing peptides on the two heptad-repeat regions present in class I viral fusion proteins. Here, we aimed to evaluate the anti-SARS-CoV-2 activity of the FP sequence conjugated to a tetravalent dendrimer through a classical organic nucleophilic substitution reaction (SN2) using a synthetic bromoacetylated peptide mimicking the FP and a branched scaffold of poly-L-Lysine functionalized with cysteine residues. We found that the FP peptide conjugated to the dendrimer, unlike the monomeric FP sequence, has virucidal activity by impairing the attachment of SARS-CoV-2 to cells. Furthermore, we found that the peptide dendrimer does not have the same effects on other coronaviruses, demonstrating that it is selective against SARS-CoV-2.

Keywords: SARS-CoV-2; dendrimer; fusion; fusion peptide; inhibitors; peptide; spike; viral fusion proteins.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(A) Schematic representation of the site-specific nucleophilic substitution reaction (SN2) between the bromoacetylated monomeric peptide R1 (Br-CH2CO-R1) and the thiol (-SH) group of cysteine residues of the branched amino acid core. (B) Chemical structure of the peptide dendrimer R1. All the amino acids have an L configuration.
Figure 2
Figure 2
Toxicity evaluation on Vero-76 cells. Cell monolayers were treated with compounds at different concentrations (5, 10, 20, 50, and 100 μM). After 24 h, cell viability was determined via MTT assay. CTRL+ refers to untreated cells, and CTRL− indicates DMSO-treated cells. **** p < 0.0001; *** p = 0.003; ns: not significant.
Figure 3
Figure 3
Anti-SARS-CoV-2 activity. (A) Co-treatment assay; (B) virucidal assay; (C) cell pre-treatment assay; and (D) post-treatment assay. Cells were first infected, and then, treated with the compound. Cells were infected and treated with ivermectin [20] at 12 μM were used as an internal control and reported as CTRL+, while CTRL− refers to infected cells. The data shown in each column are the means ± standard deviations (SD; error bars) from three independent experiments performed in duplicate. **** p < 0.0001; ns: not significant.
Figure 4
Figure 4
Kinetics assay. (A) Representative plaques of SARS-CoV-2 on Vero-76 cells stained with crystal violet. Dendrimer R1 was incubated with SARS-CoV-2 for 30, 60, 90, and 120 min at different concentrations (5, 10, 20, and 50 µM). Therefore, untreated and treated virus suspensions were added to Vero-76 cells. After further incubation, cells were fixed and stained for visualization of viral plaques. Virus-infected cells are indicated as CTRL− and used as internal control. (B) Quantitative analysis of plaque reduction in virucidal assay at different virus + compound incubation times and at different compound concentrations. CTRL+ refers to cells infected and treated with ivermectin at 12 μM [20]. All values represent the means ± standard deviations (SD; error bars) of three independent experiments performed in duplicate. **** p < 0.0001; ns: not significant.
Figure 5
Figure 5
SARS-CoV-2 entry and attachment tests. In the entry assay, Vero-76 cells were previously infected at 4 °C, and then, the compound incubation was shifted at 37 °C. In the attachment assay, cells were simultaneously incubated with each compound and virus at 4 °C, and then, the infection proceeded at 37 °C. CTRL+ refers to virus-infected cells treated with heparin at 10 μM [13], and CTRL− indicates virus-infected cells. The data shown in each column are the means ± standard deviations (SD; error bars) from three independent experiments performed in duplicate. **** p < 0.0001; ns: not significant.
Figure 6
Figure 6
Anti-HCoV-229E and anti-HCoV-OC43 activity. (A) Co-treatment and (B) virucidal assays against HCoV-229E; (C) co-treatment and (D) virucidal assays against HCoV-OC43. CTRL+ refers to cells treated with ivermectin at 12 μM [20], and CTRL− indicates virus-infected cells. The data shown in each column are the means ± standard deviations (SD; error bars) from three independent experiments performed in duplicate. **** p < 0.0001; ns: not significant.
Figure 7
Figure 7
Anti-SARS-CoV-2 activity on Calu-3 cells. Each compound at different concentrations was incubated with the virus (200 TCID50/mL) during the adsorption step, and then, the resulting mixtures were diluted on Calu-3 cells. After 96 h post-infection, each sample was harvested and diluted at the TCID50/mL end-point on Vero-76. CTRL+ refers to virus-infected cells treated with ivermectin at 12 μM [20], while CTRL− indicates virus-infected cells. **** p < 0.0001; ** p < 0.0020; ns: not significant.
Figure 8
Figure 8
Molecular simulation of SARS-CoV-2 FP interacting with the S protein (PDB 6XM4) obtained by the HPEPDOCK server. The different color codes of the peptide, represented as balls, refer to the different binding free energy.
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