Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion

Abstract

The recent outbreak of coronavirus disease (COVID-19) caused by SARS-CoV-2 infection in Wuhan, China has posed a serious threat to global public health. To develop specific anti-coronavirus therapeutics and prophylactics, the molecular mechanism that underlies viral infection must first be defined. Therefore, we herein established a SARS-CoV-2 spike (S) protein-mediated cell-cell fusion assay and found that SARS-CoV-2 showed a superior plasma membrane fusion capacity compared to that of SARS-CoV. We solved the X-ray crystal structure of six-helical bundle (6-HB) core of the HR1 and HR2 domains in the SARS-CoV-2 S protein S2 subunit, revealing that several mutated amino acid residues in the HR1 domain may be associated with enhanced interactions with the HR2 domain. We previously developed a pan-coronavirus fusion inhibitor, EK1, which targeted the HR1 domain and could inhibit infection by divergent human coronaviruses tested, including SARS-CoV and MERS-CoV. Here we generated a series of lipopeptides derived from EK1 and found that EK1C4 was the most potent fusion inhibitor against SARS-CoV-2 S protein-mediated membrane fusion and pseudovirus infection with IC50s of 1.3 and 15.8 nM, about 241- and 149-fold more potent than the original EK1 peptide, respectively. EK1C4 was also highly effective against membrane fusion and infection of other human coronavirus pseudoviruses tested, including SARS-CoV and MERS-CoV, as well as SARSr-CoVs, and potently inhibited the replication of 5 live human coronaviruses examined, including SARS-CoV-2. Intranasal application of EK1C4 before or after challenge with HCoV-OC43 protected mice from infection, suggesting that EK1C4 could be used for prevention and treatment of infection by the currently circulating SARS-CoV-2 and other emerging SARSr-CoVs.

Fig. 1. Establishment of SARS-CoV-2 S protein-mediated cell–cell fusion system.

a The emerging timeline for highly pathogenic viruses and the proposed Disease X. b Schematic representation of SARS-CoV-2 S protein. Its S1 subunit contains NTD (14–305 aa), RBD (319–541 aa), and RBM (437–508 aa). Its S2 subunit contains FP (788–806 aa), HR1 (912–984 aa), HR2 (1163–1213 aa), TM (1214–1237 aa) and CP (1238–1273 aa). c The formation of syncytium in Huh-7 cells 24 h after SARS-CoV-2 infection, with scale bar of 200 µm. d Images of SARS-CoV and SARS-CoV-2 S-mediated cell–cell fusion on 293T/ACE2 cells at 2 h (left) and 24 h (right). e SARS-CoV (I–II) and SARS-CoV-2 (III–IV) S-mediated syncytium formation on 293T/ACE2 cells at 48 h. f SARS-CoV (I–II) and SARS-CoV-2 (III–IV) S-mediated syncytium formation on Huh-7 cells at 48 h. Scale bar equals 400 µm in d–f.

Fig. 2. Overall structure of post-fusion 6-HB in SARS-CoV-2.

a Sequence alignment of HR1 and HR2 domains in SARS-CoV and SARS-CoV-2. b Structure of SARS-CoV-2 6-HB is shown in cartoon representation with HR1 colored in green and HR2 in cyan. The structural dimensions are indicated in angstroms. c HR1 trimer of SARS-CoV-2 6-HB is shown in electrostatic surface, and HR2 domain is shown in cartoon representation, the important binding residues of which are shown in sticks and labeled. d The superposition of 6-HB structure of SARS-CoV (PDB entry 1WYY), MERS-CoV (PDB entry 4NJL) and SARS-CoV-2 is shown in ribbon. The RMSD between structures is indicated. e The sequence comparison of 6-HB structure of different HCoVs is shown in cartoon representation with different colors for HR1 and HR2. The helical fusion core regions are indicated.

Fig. 3. Interaction between HR1 and HR2 of SARS-CoV-2 and SARS-CoV.

a–d The 6-HB structure of SARS-CoV-2 and SARS-CoV is shown in cartoon representation. The HR1 domain is shown in green for SARS-CoV-2 and forest for SARS-CoV, while the HR2 domain is shown in cyan for SARS-CoV-2 and orange for SARS-CoV. Important residues are shown in sticks and labeled.

Fig. 4. EK1-Lipopeptides showed potent inhibitory activity against SARS-CoV-2 infection.

a Amino acid sequences of the designed peptides EK1, EK1P and EK1C. The dotted lines represent E–K salt-bridge with i to i + 3, or i + 4 arrangement. b SARS-CoV-2 S protein-mediated cell–cell fusion in the presence of EK1-scramble (I), EK1 (II), EK1C (III), and EK1P (IV) at 2.5 μM (scale bar: 400 µm). c Inhibitory activity of EK1-scramble, EK1, EK1C and EK1P against SARS-CoV-2 S-mediated cell–cell fusion. d Design diagram of EK1-lipopeptides with cholesterol modification, including EK1C1-EK1C7. e Inhibitory activity of EK1-lipopeptides on SARS-CoV-2 S-mediated cell–cell fusion. f Inhibitory activity of EK1-lipopeptides on SARS-CoV-2 PsV infection. Experiments were repeated twice, and the data are expressed as means ± SD (error bar).

Fig. 5. EK1C4 broadly and potently inhibited cell–cell fusion and PsV infection mediated by S protein of divergent HCoVs.

a–h Inhibitory activity of EK1C4 in cell–cell fusion mediated by the S proteins of SARS-CoV (a), MERS-CoV (b), HCoV-OC43 (c), HCoV-229E (d), HCoV-NL63 (e), WIV1 (f), Rs3367 (g) and SHC014 (h). i–o Inhibitory activity of EK1C4 in PsV infection assays against SARS-CoV (i), MERS-CoV (j), HCoV-OC43 (k), HCoV-229E (l), NL63 (m), WIV1 (n) and Rs3367 (o). Experiments were repeated twice, and the data are expressed as means ± SD.

Fig. 6. EK1C4 effectively inhibited live-CoVs infection in vitro and in vivo.

a–e Inhibitory activity of EK1C4 on live HCoV replication for SARS-CoV-2 (a), MERS-CoV (b), HCoV-OC43 (c), HCoV-229E (d), and HCoV-NL63 (e). f–g In vivo prophylactic efficacy of EK1C4 against HCoV-OC43 infection in mice. Body weight change (f) and survival curves (g) of mice challenged with HCoV-OC43. h–i In vivo therapeutic efficacy of EK1C4 against HCoV-OC43 infection in mice. Body weight change (h) and survival curves (i) of mice challenged with HCoV-OC43. Experiments were repeated twice, and the data are expressed as means ± SD.