Research Article | Therapeutics and Prevention

Stapled Peptides Based on Human Angiotensin-Converting Enzyme 2 (ACE2) Potently Inhibit SARS-CoV-2 Infection In Vitro

Francesca Curreli, Sofia M. B. Victor, Shahad Ahmed, Aleksandra Drelich, Xiaohe Tong, Chien-Te K. Tseng, Christopher D. Hillyer, Asim K. Debnath
Nancy C. Reich, Editor
DOI: 10.1128/mBio.02451-20

Article Figures & Data

Figures

  • FIG 1
    FIG 1

    X-ray structure showing binding of SARS-CoV-2 RBD (violet) with ACE2 receptor (green). The detailed interactions of Helix-1 of ACE2 with SARS-CoV-2 RBD are indicated in the illustration on the right.

  • FIG 2
    FIG 2

    Design of double-stapled peptides as anti-SARS-CoV-2 inhibitors. The linear peptide, NYBSP-C, and an ACE2-unrelated double-stapled peptide, StRIP16 (29), were used as controls.

  • FIG 3
    FIG 3

    α-Helical propensity measured by CD spectroscopy. (A) CD spectra. (B) Percent helicity of the double-stapled peptides and the linear control peptide.

  • FIG 4
    FIG 4

    Evaluation of binding affinity of peptides to SARS-CoV-2 RBD by SPR. Kinetics fitting curve (sensogram) of SARS-CoV-2 RBD to NYBSP-C, a linear control peptide (A); NYBSP-4, a double-stapled peptide (B); and the binding affinity KD and kinetic parameters kon and koff of NYBSP-C and NYBSP-4 (C).

  • FIG 5
    FIG 5

    The plasma stability of double-stapled peptide NYBSP-4 (red) and control linear peptide NYBSP-C (green). Propantheline bromide (a small-molecule antimuscarinic agent) was used as an assay control.

  • FIG 6
    FIG 6

    Validation of SARS-CoV-2 pseudovirus and ACE2 expression in different cells. (A) Western blot analysis to validate the incorporation of the S spike protein in the SARS-CoV-2 pseudovirus using the SARS spike protein antibody (Novus Biologicals), which targets the spike protein S2. (B) Infection of cells expressing different levels of ACE2, with the same amounts of SARS-CoV-2 pseudovirus. Columns represent the means ± standard deviations (n = 3). (C) Immunoblot analysis of cell lysates to evaluate ACE2 expression. β-Actin was used as a loading control.

  • FIG 7
    FIG 7

    Dose-response plots of the antiviral activity of peptides in a single-cycle assay performed in HT1080/ACE2 and A549/ACE2 cells infected with pseudovirus NL4-3ΔEnv-NanoLuc/SARS-CoV-2. Antiviral activity in HT1080/ACE2 cells (A) and A549/ACE2 cells (B). Data on stapled peptides NYBSP-1, NYBSP-2, NYBSP-3, and NYBSP-4 and control linear peptide NYBSP-C are shown as mean ± SD from three independent experiments.

Tables

  • TABLE 1

    Antiviral activity (IC50) of peptides in single-cycle assay in HT1080/ACE2 and A549/ACE2 cells infected with pseudoviruses (NL4-3ΔEnv-NanoLuc/SARS-CoV-2 and NL4-3.Luc.R-E-/VSV-G) and cytotoxicity (CC50)

    TABLE 1

    • ↵a The reported IC50 and CC50 values represent the means ± standard deviations (n = 3).

    • ↵b Less than 10% toxicity at this dose.

    • ↵c NYBSP-C, a linear peptide from Helix-1, was used as a control.

    • ↵d StRIP16, an ACE2-unrelated Rab8a GTPase-binding double-stapled peptide reported earlier (29), was used as a control.

  • TABLE 2

    Antiviral activity (IC100) of peptides in Vero E6 cells infected with SARS-CoV-2 (US_WA-1/2020)

    TABLE 2

    • ↵a Values indicate the lowest concentration capable of completely preventing virus-induced CPE in 100% of the wells.

    • ↵b NA means the presence of CPE at the highest concentration.

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KEYWORDS

angiotensin-converting enzyme 2 (ACE2)
coronavirus disease 2019 (COVID-19)
severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
hydrocarbon stapling
stapled peptides

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