key: cord-307504-cogk5kig
authors: Zhu, Yuanmei; Yu, Danwei; Yan, Hongxia; Chong, Huihui; He, Yuxian
title: Design of potent membrane fusion inhibitors against SARS-CoV-2, an emerging coronavirus with high fusogenic activity
date: 2020-03-28
journal: bioRxiv
DOI: 10.1101/2020.03.26.009233
sha: 
doc_id: 307504
cord_uid: cogk5kig

The coronavirus disease COVID-19, caused by emerging SARS-CoV-2, has posed serious threats to global public health, economic and social stabilities, calling for the prompt development of therapeutics and prophylactics. In this study, we firstly verified that SARS-CoV-2 uses human ACE2 as a cell receptor and its spike (S) protein mediates high membrane fusion activity. Comparing to that of SARS-CoV, the heptad repeat 1 (HR1) sequence in the S2 fusion protein of SARS-CoV-2 possesses markedly increased α-helicity and thermostability, as well as a higher binding affinity with its corresponding heptad repeat 2 (HR1) site. Then, we designed a HR2 sequence-based lipopeptide fusion inhibitor, termed IPB02, which showed highly poent activities in inibibiting the SARS-CoV-2 S protein-mediated cell-cell fusion and pseudovirus infection. IPB02 also inhibited the SARS-CoV pseudovirus efficiently. Moreover, the strcuture and activity relationship (SAR) of IPB02 were characterzized with a panel of truncated lipopeptides, revealing the amino acid motifs critical for its binding and antiviral capacities. Therefore, the presented results have provided important information for understanding the entry pathway of SARS-CoV-2 and the design of antivirals that target the membrane fusion step.

In late December of 2019, a new infectious respiratory disease emerged in Wuhan, China. The 34 pathogen was soon identified as a novel coronavirus (CoV) (1-3), which was initially termed 45 CoVs, a large group of enveloped viruses with a single positive-stranded RNA genome, 46 are genetically classified into four genera: α-, β-, γ-, and δ-CoVs (4, 5). The previously known 47 six CoVs that cause human disease include two α-CoVs (NL63; 229E) and four β-CoVs comprises of S1 and S2 subunits and exists in a metastable prefusion conformation. The S1 52 subunit, which contains a receptor-binding domain (RBD) capable of functional folding 53 independently, is responsible for virus binding to the cell surface receptor. A recent study 54 4 suggested that ACE2-binding affinity of the RBD of SARS-CoV-2 is up to 20-fold higher 55 than that of SARS-CoV, which may contribute to the significantly increased infectivity and 56 transmissibility (6). The receptor-binding deem to trigger large conformational changes in the 57 S complex, which destabilize the prefusion trimer resulting in shedding of the S1 subunit and 58 activate the fusogenic activity of the S2 subunit (9-11). As illustrated in Fig. 1 , the sequence 59 structure of S2 contains an N-terminal fusion peptide (FP), heptad repeat 1 (HR1), heptad 60 repeat 2 (HR2), transmembrane region (TM), and cytoplasmic tail (CT). During the fusion 61 process, the FP is exposed and inserts into the target cell membrane, leading S2 in a 62 prehairpin intermediate that bridges the viral and cell membranes; then, three HR1 segments 63 self-assemble a trimeric coiled-coil and three HR2 segments fold into the grooves on the 64 surface of the HR1 inner core, thereby resulting a six-helical bundle (6-HB) structure that 65 drives the two membranes in close apposition for fusion. 86 In the earlier time point, we would like to experimentally verify whether SARS-CoV-2 uses 87 human ACE2 as a receptor for cell entry, thus we generated its S protein pseudotyped 88 lentiviral particles. The SARS-CoV and vesicular stomatitis virus (VSV-G) pseudoviruses 89 were also prepared for comparison. As shown in Fig. 2A , all of three pseudoviruses 90 efficiently infected 293T cells that stably overexpress ACE2 (293T/ACE2); however, the 91 infectivity SARS-CoV-2 and SARS-CoV dramatically decreased in 239T cells which express 92 a low level of endogenous ACE2. As a virus control, VSV-G pesudovirus entered 239T cells 93 even more efficiently relative its infectivity in 293T/ACE2 cells. 94 We further compared the fusion activity of viral S protein in 293T and 293T/ACE2 cells In both the 239T and 293T/ACE2 target cells, we observed that the S protein of 102 SARS-CoV-2 had a significantly increased fusion activity than the S protein of SARS-CoV. 103 Therefore, we further compared the fusion activities of viral S proteins at different time points.

As shown in Fig. 2C and 2D, the SARS-CoV S protein exhibited had no appreciable fusion 105 activity until the effector cells and target cells were cocultured for five or six hours; in sharp 106 contrast, the SARS-CoV-2 S protein mediated a rapid and robust cell fusion reaction, as 107 indicated by its fusion kinetic curves especially in 293T/ACE2 cells. SARS-CoV, SARS-CoV-2 has a HR1 sequence with nine amino acid substitutions, and of 113 them eight are located within the HR1 core site; whereas, two viruses share a fully identical 114 HR2 sequence (Fig. 3A) . In order to explore the mechanism underlying the highly active 115 fusion activity of the SARS-CoV-2 S protein, we synthesized two peptides corresponding to 116 the HR1 sequence and their secondary structures were determined by circular dichroism (CD) Furthermore, we synthesized a peptide containing the HR2 sequence, termed IPB01, and its 123 interactions with the two HR1 peptides were analyzed by CD spectroscopy. As shown in Fig.   124 3D and E, both the SARS2NP and SARS1NP interacted with IPB01 to form complexes with 125 typical α-helical structures, having the T m values of 75 and 68 o C, respectively. In comparison, 126 the complex formed by SARS2NP and IPB01 was much more stable than the complex 127 between the SARS1NP and IPB01 peptides. Taken together, these results suggested that 128 SARS-CoV-2 might evolve an increased interaction between the HR1 and HR2 domains in 129 the S2 fusion protein thus critically determining its high fusogenic activity. 158 We next sought to determine the antiviral functions of the IPB01 and IPB02 peptides. Firstly, 159 their inhibitory activities on S protein-mediated cell-cell fusion were examined by the 160 DSP-based cell fusion assay as described above. As shown in Fig. 6A and Table 1 (Table 1) . As expected, IPB01 and IPB02 had no inhibitory activity against a 168 control virus (VSV-G), indicating their antiviral specificities. Therefore, we conclude that 169 IPB02 is a highly potent fusion inhibitor of SARS-CoV-2 and SARS-CoV. Differently, IPB08 was a C-terminally truncated inhibitor with IPB02 as a template, but its 183 antiviral function was markedly impaired, underscoring the roles of C-terminal residues in 184 IPB02. On the basis of the results above, it was expected that IPB09 with two terminal 185 truncations was antivirally inactive. Indeed, the CD data suggested that both the N-and 186 10 C-terminal sequences contributed critically to the binding of the inhibitors (Table 1) where its S protein is cleaved by endosomal cysteine proteases cathepsin B and L (CatB/L) 225 and activated (45) . However, SARS-CoV also employs the cellular serine protease TMPRSS2 226 for S protein priming, and especially, TMPRSS2 but not CatB/L is essential for viral entry 227 into primary target cells and for viral spread in the infected host (43, 46-48). It was also found 228 that introducing a furin-recognition site between the S1 and S2 subunits could significantly suggesting that it mainly utilizes a plasma membrane fusion pathway for cell entry. Sequence 232 analyses revealed that SARS-CoV-2 harbors the S1/S2 cleavage site in its S protein, although 233 its roles in S protein-mediated membrane fusion and viral life-cycle need to be characterized. 234 One can speculate that furin-mediated precleavage at the S1/S2 site in infected cells might 

*The antiviral assays were repeated three times, and data are expressed as means ± standard 575 deviations. The CD experiment was repeated two times and representative data are shown.

ND means "not done" owing to the solubility problem of the peptides in PBS.

A new coronavirus associated with 332 human respiratory disease in China

A pneumonia outbreak associated with a new coronavirus of 336 probable bat origin

China Novel Coronavirus I, Research T. 2020. A Novel Coronavirus from 339 Patients with Pneumonia in China

Coronaviruses post-SARS: update on replication and pathogenesis

Structure, Function, and Evolution of Coronavirus Spike Proteins

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

Structure, Function, and 346 Antigenicity of the SARS-CoV-2 Spike Glycoprotein

Receptor Recognition by the Novel Coronavirus from 348

Wuhan: an Analysis Based on Decade-Long Structural Studies of SARS Coronavirus

Tectonic conformational 350 changes of a coronavirus spike glycoprotein promote membrane fusion

Interaction between heptad repeat 1 and 2 regions in spike protein of SARS-associated coronavirus: 354 implications for virus fusogenic mechanism and identification of fusion inhibitors

The coronavirus spike protein is a class I virus 356 fusion protein: structural and functional characterization of the fusion core complex

Synthesized peptide inhibitors of HIV-1 gp41-dependent membrane fusion

Structure-based discovery of Middle East respiratory syndrome coronavirus fusion inhibitor

Hydrocarbon-Stapled Short alpha-Helical Peptides as Promising Middle East Respiratory Syndrome 365

MERS-CoV) Fusion Inhibitors

Severe acute respiratory syndrome coronavirus (SARS-CoV) infection inhibition 368 using spike protein heptad repeat-derived peptides

Heptad repeat-derived peptides block protease-mediated direct entry from the cell surface of 371 severe acute respiratory syndrome coronavirus but not entry via the endosomal pathway

Identification of a minimal peptide derived 374 from heptad repeat (HR) 2 of spike protein of SARS-CoV and combination of HR1-derived peptides as 375 fusion inhibitors

Influence of hydrophobic and electrostatic residues on 377

SARS-coronavirus S2 protein stability: insights into mechanisms of general viral fusion and inhibitor 378 design

A pan-coronavirus fusion inhibitor targeting the HR1 domain of human coronavirus spike

Design and Characterization of Cholesterylated Peptide 383

HIV-1/2 Fusion Inhibitors with Extremely Potent and Long-Lasting Antiviral Activity

lipopeptide HIV fusion inhibitor maintains long-term viral suppression in rhesus macaques

Exceptional potency and structural 389 basis of a T1249-derived lipopeptide fusion inhibitor against HIV-1, HIV-2, and simian 390 immunodeficiency virus

Structural and Functional Characterization of Membrane Fusion 392

Inhibitors with Extremely Potent Activity against HIV-1, HIV-2, and Simian Immunodeficiency Virus

Inhibitors of cathepsin L 454 prevent severe acute respiratory syndrome coronavirus entry

Contributes to Virus Spread and Immunopathology in the Airways of Murine Models after Coronavirus 457 Infection

Simultaneous treatment of human 459 bronchial epithelial cells with serine and cysteine protease inhibitors prevents severe acute respiratory 460 syndrome coronavirus entry

Protease inhibitors targeting coronavirus and filovirus entry

Furin cleavage of the SARS coronavirus spike glycoprotein enhances 465 cell-cell fusion but does not affect virion entry

Functional analysis of potential cleavage 467 sites in the MERS-coronavirus spike protein

Proteolytic processing of

Middle East respiratory syndrome coronavirus spikes expands virus tropism

SARS-CoV-2 Cell Entry Depends on ACE2 473 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

Clinical Isolates of Human Coronavirus 229E Bypass 476 the Endosome for Cell Entry

Wild-type human coronaviruses prefer cell-surface TMPRSS2 478 to endosomal cathepsins for cell entry

Teicoplanin: an alternative drug for the 480 treatment of coronavirus COVID-19?

Teicoplanin potently blocks the cell entry of 2019-nCoV

Remdesivir and 485 chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro

Compounds with therapeutic potential against novel respiratory 2019 coronavirus

Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open-label 492

non-randomized clinical trial

The Tryptophan-Rich Motif of HIV-1 Gp41 Can Interact with 494 the N-Terminal

Structural and functional 497 characterization of HIV-1 cell fusion inhibitor T20