Lipidated macrocyclic peptide S-880008 as a broad-spectrum SARS-CoV-2 fusion inhibitor

Lipidated macrocyclic peptide S-880008 as a broad-spectrum SARS-CoV-2 fusion inhibitor | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Biological Sciences - Article Lipidated macrocyclic peptide S-880008 as a broad-spectrum SARS-CoV-2 fusion inhibitor Takao Sanaki, Yoshimasa Kawaguchi, Yuki Anraku, Yoshifumi Kusumoto, and 20 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5977541/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Coronavirus disease 2019, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a threat to public health and the economy. Although several SARS-CoV-2 vaccines exist, they have failed to elicit effective neutralizing antibody responses against emerging SARS-CoV-2 variants harboring spike protein mutations. Moreover, while certain neutralizing-antibody-based therapies were effective in the early stages of the SARS-CoV-2 pandemic, their performance declined with the emergence of spike protein mutations. Thus, it is essential to develop antiviral agents that inhibit the early stages of viral infection by effectively targeting spike protein mutants. Broad-spectrum anti-SARS-CoV-2 activity relies on the targeting of conserved sites within the spike protein. Herein, we used mRNA display screening and chemical modification to generate S-880008, a lipid-modified macrocyclic peptide. S-880008 exhibited efficacy against a broad range of SARS-CoV-2 (including Omicron) variants and inhibited SARS-CoV-2 fusion rather than attachment via a novel mechanism. Cryo-electron microscopy analysis revealed that, unexpectedly, S-880008 simultaneously recognized the vulnerable sites of both the interface between the receptor-binding domain (RBD) and subdomain 1 (via the macrocyclic peptide portion) and the N-terminal domain of the adjacent protomer (via the acyl chain) to enforce a RBD 3-up conformation. The results of the structure-activity relationship experiments with S-880008 derivatives supported the notion that S-880008 binding inhibited fusion by suppressing protomer dissociation. Crucially, the intranasal administration of S-880008 to mouse-adapted SARS-CoV-2-infected mice significantly reduced the viral titer in lung homogenates and improved survival rates in a dose-dependent manner. Our findings show that S-880008 has the potential to overcome the increasing threat posed by emerging SARS-CoV-2 variants, providing a rationale for the design of a broad range of antiviral fusion inhibitors. Biological sciences/Microbiology/Virology/SARS-CoV-2 Biological sciences/Drug discovery/Medicinal chemistry/Lead optimization Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Biological sciences/Drug discovery/Drug screening Biological sciences/Microbiology/Virology/Viral membrane fusion Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Summary paragraph Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for coronavirus disease 2019, still poses a threat to public health. Although several anti-SARS-CoV-2 neutralizing-antibody-based therapies exist, their efficacy is limited by the fact that mutations found in SARS-CoV-2 variants mainly target the spike protein. To create drugs that work against a wide range of SARS-CoV-2 variants, it is important to target conserved parts of the spike protein. Here, we used the peptide screening and structural optimization approaches to generate the lipid-modified macrocyclic peptide S-880008. We showed that S-880008 was effective against a broad range of SARS-CoV-2 (including Omicron) variants. Cryo-electron microscopy revealed that S-880008 bound simultaneously to the conserved interfaces of the receptor-binding domain, subdomain 1 and the N-terminal domain of the adjacent protomer to prevent the protomer dissociation necessary for viral fusion. This novel binding site is completely distinct from that of the angiotensin-converting enzyme 2, which is targeted by neutralizing antibodies. In accordance, S-880008 inhibited viral fusion but not viral attachment in vitro . Crucially, the intranasal administration of S-880008 significantly reduced the viral titer in lungs of mice infected with mouse-adapted SARS-CoV-2 and improved their survival rates. S-880008 represents a promising new drug for a broad range of SARS-CoV-2 variants. Introduction Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to threaten global public health. Although several vaccines 1,2 and antiviral drugs 3–6 for SARS-CoV-2 exist, early vaccines failed to induce neutralizing antibody responses that can keep pace with the emergence of new SARS-CoV-2 variants. Indeed, the early vaccines developed against the ancestral SARS-CoV-2 strain offer suboptimal protection against the recent Omicron variants 7 . The development of effective neutralizing-antibody-based therapies is also hampered by the rapid mutation rates of new SARS-CoV-2 variants. This suboptimal efficacy prompted the U.S. Food and Drug Administration to revoke the Emergency Use Authorization for early SARS-CoV-2 neutralizing-antibody-based therapies. The SARS-CoV-2 spike protein, and particularly its receptor-binding domain (RBD), is the main target molecule of neutralizing antibodies 8 . Mutations found in SARS-CoV-2 variants primarily affect the surface of the SARS-CoV-2 spike, particularly around the RBD 8 , which reduces the ability of neutralizing antibodies to suppress viral replication. Nevertheless, given that some neutralizing antibodies against the spike protein showed clear efficacies in clinical trials 9,10 , the mechanisms underlying the interaction between neutralizing antibodies and the SARS-CoV-2 spike require further elucidation. The SARS-CoV-2 spike, which is composed of two functional subunits (S1 and S2) that assemble into homotrimers on the viral surface, is essential for viral entry into host cells 11,12 . While S1 binds to angiotensin-converting enzyme 2 (ACE2) 13 , the host cell receptor, S2 is involved in the fusion of the viral and cellular membranes 12,14 . Recently, nanobodies 15–17 and peptides 18,19 that bind to the SARS-CoV-2 spike have been identified. These smaller molecules can neutralize regions of the spike protein, which are otherwise inaccessible to the larger antibodies. Moreover, peptides offer several advantages as therapeutic agents over proteins. These include more cost-effective production, higher levels of stability (e.g., resistant to heat and acidic conditions), and versatile administration routes (e.g., oral 20 and respiratory routes 21 ). However, the lack of clinically validated peptide compounds underscores the necessity for the generation of potent, broad-spectrum antiviral peptides that demonstrate therapeutic efficacy in vivo . Herein, we searched for novel peptides targeting the spike protein that could exhibit broad-spectrum antiviral activity and in vivo efficacy against SARS-CoV-2 variants, including the Omicron variants. Unexpectedly, we found lipidated macrocyclic peptides that deeply protrude vulnerable inter-protomer regions of the spike protein to fix its prefusion state, providing insight into the development of a new class of broad-spectrum fusion inhibitors. Results Discovery of S-880008 using mRNA display technology and chemical modification The Peptide Discovery Platform System (PDPS) was developed by PeptiDream Inc. on the basis of mRNA display technology to specifically select peptide sequences that bound to target molecules from a pool of 10 13 macrocycles containing non-natural amino acids 22,23 . In the present study, peptide genetic codes were reprogrammed to generate a macrocyclic peptide library incorporating non-proteinogenic amino acids (e.g., chloroacetyl-phenylalanine), N-methyl amino acids, and carbamoyl phenylalanine. We hypothesized that the incorporation of these non-proteinogenic amino acids would improve peptide pharmacokinetics and create new and more potent binding modes. In early 2021, when SARS-CoV-2 β and γ strains were prevalent, PDPS was performed against the biotinylated S1 domain (mS1-bio), which consisted of the N-terminal domain (NTD) and RBD harboring the K417N, E484K, and N501Y mutations. During subsequent selection rounds, PDPS selection was performed against biotinylated RBDs (mRBD-bio) harboring the K417N, E484K, and N501Y mutations to comprehensively identify peptides that bound strongly to the surface of the RBDs (Fig. 1a). Multiple rounds of panning were conducted. An additional panning step against the ancestral S1 protein was also included to obtain broad-spectrum antiviral peptides. Next-generation sequencing was used to identify four highly enriched peptides (Fig. 1b, Compounds 1–4). These peptide were synthesized and decorated with lipids via a glutamic acid linker to improve their solubility 24 , stability, and pharmacokinetics 25 (Fig. 1b, S-880008 and Compounds 5–7). The antiviral activity of these cyclic peptides against the ancestral wildtype (WT) SARS-CoV-2 strain was then evaluated (Fig. 1b). Among the lipid-modified peptides, S-880008 showed the highest antiviral activity at single-digit nanomolar concentrations (Fig. 1b, c); therefore, S-880008 was selected for further study. In the cell-based assay, S-880008 exerted broad-spectrum antiviral activity against SARS-CoV-2 variants, including the Omicron strains; the 50% effective concentration (EC 50 ) ranged from 2.66 nM for the m strain to 27.5 nM for the XE strain (Table 1). S-880008 proved to be considerably more potent than remdesivir and nirmatrelvir in vitro . Since the 50% cytotoxic concentration (CC 50 ) values of all the compounds tested were much higher than their EC 50 values (Fig. 1d), we concluded that their cytotoxicity did not impact their antiviral activity. S-880008 effectively reduced the intracellular viral RNA concentration. The 90% effective concentration (EC 90 ) of S-880008 against the WT, BA.1, and BA.2 strains, calculated based on the amount of intracellular viral RNA, was 7.46, 5.46, and 52.2 nM, respectively (Extended Data Table 1). Surface plasmon resonance (SPR) analysis demonstrated the direct binding of S-880008 to the SARS-CoV-2 spike. The binding dissociation constants (Kds) against the WT and BA.2 spikes were 20.4 and 23.3 nM, respectively (Extended Data Fig. 1). These data clearly indicate that S-880008 exerts its antiviral activity by acting on the virus rather than the host cells. Mode of action of S-880008: Inhibition of viral fusion after viral attachment to cells In a time of addition test, S-880008 inhibited viral activity before but not after viral entry into cells (Fig. 2a). Since remdesivir exerted antiviral activity regardless of the timing of addition (Fig. 2a), these data indicate that S-880008 functions similarly to a neutralizing antibody cocktail of casirivimab and imdevimab (CAS + IMD). Therefore, we next investigated the effect of S-880008 on the interaction between the SARS-CoV-2 spike and ACE2. CAS + IMD inhibited the ACE2 binding in concentration-dependent manner, with a 50% inhibitory concentration (IC 50 ) of 23.5 ng/mL (Fig. 2b). By contrast, S-880008 did not inhibit ACE2 binding to the spike protein (IC 50 > 25,000 nM) (Fig. 2b). These data indicate that the inhibitory mechanism of S-880008 is different from that of CAS + IMD. We therefore next investigated the effect of S-880008 on SARS-CoV-2-spike-induced cell-to-cell fusion using cell lines which expressed the SARS-CoV-2 spike in addition to either GFP or mCherry. While the green (GFP) and red (mCherry) fluorescence signals of the vector control groups remained unchanged, the SARS-CoV-2-spike-expressing cells emitted a yellow signal, which indicated cell-to-cell fusion between the GFP- and mCherry-expressing cells (Fig. 2c). Cell-to-cell fusion was dramatically inhibited by both S-880008 and CAS + IMD. The EC 50 values of S-880008 and CAS + IMD were 13.7 nM and 383 ng/mL for the WT strain, and 66.0 nM and 650 ng/mL for the d strain, respectively (Fig. 2d). By contrast, remdesivir, an RdRp inhibitor, did not inhibit cell-to-cell fusion. These data demonstrate that S-880008 inhibits viral fusion rather than viral attachment to cells. We next assessed how combination with remdesivir, nirmatrelvir, or CAS + IMD impacted the antiviral effect of S-880008 in vitro . The combination index values of S-880008 with remdesivir, nirmatrelvir, or CAS + IMD were 1.07, 0.91, and 0.73, respectively. These data indicate that while the combination of S-880008 with remdesivir or nirmatrelvir produced an additive effect, S-880008 synergized with CAS + IMD (Fig. 2e). Cryo-EM analysis of S-880008 binding to SARS-CoV-2 spike proteins We next performed cryo-electron microscopy (Cryo-EM) analysis to better understand the mechanism underlying S-880008 binding to the SARS-CoV-2 spike. The overall structure of the WT spike in complex with S-880008 at 3.02 Å resolution showed a RBD 3-up conformation with C3 symmetry; the spike assumes the same conformation in complex with ACE2 26 (Fig. 3a and Supplementary Fig. 1a–d). The overall maps showed that three S-880008 peptides bound to a single spike trimer. To improve the resolution around the S-880008 binding site, we performed focused refinement, which yielded a local map at 3.6 Å resolution (Fig. 3b, 3c and Supplementary Fig. 1b–d). This map revealed that the characteristic S-880008 binding site was entirely distinct from the ACE2 binding site. Notably, S-880008 recognized the surfaces of the RBD and subdomain 1 (SD1), together with the NTD of an adjacent spike protomer. We speculated that this binding arrangement presumably tightened inter-protomer interactions, which suppressed the major structural rearrangements necessary for subsequent fusion (Fig. 3a, 3b). The fact that the binding pocket for S-880008 was only formed in the RBD-up state implied that S-880008 encouraged the spike to adopt a 3-up conformation, which may be beneficial for ACE2 engagement. This mechanism presumably prevents the dissociation of protomers, which ultimately blocks the fusion process (Extended Data Fig. 2a). S-880008 consists of three parts: a cyclic peptide containing non-canonical amino acids, a glutamic acid (Glu)-linker, and a C-terminal C16 lipid. Although the EM map allowed us to clearly observe the side chains of the cyclic peptide section, we were unable to view the Glu linker, likely due to the high flexibility and repulsion of its side chains and the electron radiation damage it incurred during imaging 27 . We were, however, able to characterize the three stages of S-880008 binding (Fig. 3d, 3e). In the images, the cyclic peptide component of S-880008 assumed an anti-parallel β-sheet structure, which was connected by β-turn motifs such as the cis configuration of N-methyl-L-alanine (7-MeA) or the thioether linker formed by 12-Cys. S-880008 used its β-strand to extend the β-sheet into the β-sandwich core of the NTD (Fig. 3f and Extended Data Fig. 2c). Interestingly, the C16 acyl chain of S-880008 protruded deeply into the tunnel of the β-sandwich structure of the NTD (Fig. 3g). Previous studies have reported the binding of this hydrophobic tunnel to biliverdin and polysorbate 80 28,29 , suggesting that this site could be a promising target for drug discovery 30 . Meanwhile, the carbamoyl group in the 11-F4CON side chain interacted with the backbone atoms of K528, forming a loop (residues 522–528) between the RBD and SD1 (Fig. 3h). Additionally, the aromatic ring of 11-F4CON was located in the hydrophobic environment formed by V362 and a loop (residues 328–336) between the RBD and NTD (P330 and I332). This structural information enabled us to delineate the three key events implicated in the interaction between S-880008 and the SARS-CoV-2 spike: (1) cyclic peptide portion extended the β sheet of the NTD; (2) the C-terminal C16 lipid protruded into the hydrophobic tunnel of the NTD; and (3) the non-canonical amino acids captured the flexible interdomain (NTD-RBD and RBD-SD1) loops. Crucially, the amino acid substitutions found in prevalent SARS-CoV-2 variants are concentrated at the ACE2 binding site of the RBD (outside of the NTD), meaning that they do not affect the S-880008 binding site (Fig. 3c and Extended Data Fig. 2b). Thus, S-880008 exhibits potent and broad antiviral activity toward SARS-CoV-2 variants by targeting vulnerable sites in the spike protein (Table 1). S-880008 exerts its antiviral activity by exploiting the interaction between its 11-F4CON sidechain and the K528 backbone of the SARS-CoV-2 RBD To study the structure-activity relationship of S-880008, we next examined antiviral activities of five derivative compounds and determined the Cryo-EM structures of their spike complexes (Fig. 4a) under the same conditions as used for S-880008 evaluation (Fig. 4b-d, Extended Data Fig. 2d-e, and Supplementary Fig. 2–4). Compounds 8 (lipid portion substituted with C15COOH), 9 (altered lipid modification site [to position 8]), and 10 (altered linker and lipid moiety [C14]) all exhibited similar antiviral activities to S-880008 and bound to the spike 3-up state in the same way as S-880008 (Fig. 4b, 4c). By contrast, compound 11 (MeA to Asp substitution at position 7) had 10-fold lower antiviral activity than S-880008 and caused the spike to adopt multiple (3-up, 2-up, and 1-up) conformations (Fig. 4d). Similarly, compound 12 (MeA to Asp substitution at position 7 and Arg to Glu substitution at position 8) had a significantly lower antiviral activity than S-880008 and caused the spike to adopt the 1-up and 3-down conformations. In these less potent compounds, a density corresponding to an interaction at the RBD-NTD interface of the up protomers was observed on the Cryo-EM maps; this density was absent from the RBD-NTD interface of the down protomers (Fig. 4d and Extended Data Fig. 2e). This result is consistent with the fact that the viral RBD only accommodated S-880008 in the up conformation (Extended Data Fig. 2a). Compounds 11 and 12 contained 7-Asp instead of the 7-MeA present in S-880008, suggesting that the differences in the binding proportions of these compounds are attributable to N-methylation. Thus, the ability of S-880008 to undergo the conformation changes necessary for spike protein binding and subsequent viral neutralization relies on N-methylation. Cryo-EM analysis further identified two hydrogen bonds linking the carbamoyl group in the 11-F4CON side chain of S-880008 to the backbone atoms of K528 (Fig. 3h). A structure-activity relationship analysis was performed to investigate whether the 11-F4CON carbamoyl group contributed significantly to S-880008 antiviral activity. Compounds 13 (no carbamoyl group), 14 (containing a hydroxyl group), and 15 (containing a carboxyl moiety), which all had 11-F4CON modifications, had 32-, 13-, and > 400-fold lower antiviral activities than S-880008, respectively (Fig. 4e). These results confirm the importance of the interaction between the carbamoyl group of 11-F4CON and the backbone atoms of K528 in the RBD for the antiviral activity of S-880008, and imply that S-880008 has the potential to exhibit broad-spectrum antiviral activity. Amino acid substitutions in the SARS-CoV-2 S2 region markedly reduce the antiviral activity of S-880008 To investigate which viral amino acid substitutions markedly reduced the antiviral activity of S-880008, several S-880008-resistant viruses were prepared. Ten amino acid substitutions in the spike region were selected by genotypic analysis, according to the criteria for the selection of characteristic amino acid substitutions after S-880008 treatment (Extended Data Table 2). Subsequently, 10 viruses, each containing a single amino acid substitution, were prepared by reverse genetics 31 . Two of the viruses (containing F562C or L984F) did not grow; thus, the antiviral activity of S-880008 was evaluated against the remaining eight viruses (Extended Data Table 3). The isolated S-880008-resistant virions harbored characteristic amino acid substitutions in the binding sites to S-880008 (i.e., P230A, F329L and P330S), as well as in the central helix of S2 (E988D and Q992H), a region which is important for viral fusion 12,14 (Extended Data Fig. 3). Remdesivir and CAS + IMD showed similar antiviral activities against all eight S-880008-resistant viruses (Extended Data Table 3). By contrast, the antiviral activity of S-880008 against all viruses other than the variant harboring the T1009I substitution was lower than that against the WT strain (Extended Data Table 3). Specifically, the reduction in the antiviral activity against the viruses containing the E988D, L752F, Q992H, and F329L substitutions was > 2,165-, 324-, 27.5-, and 12.7-fold, respectively. In vivo antiviral efficacy of S-880008 in mouse-adapted SARS-CoV-2-infected mice Mouse-adapted SARS-CoV-2 (MA-P10) was prepared as previously described 32–34 to evaluate the in vivo antiviral efficacies of S-880008 and the other antiviral compounds 35,36 . Of note, S-880008 also exhibited in vitro antiviral activity against MA-P10 harboring the Q498H amino acid substitution in the spike region, which was comparable to its activity against the SARS-CoV-2 variants (Table 1). We investigated the effects of human and mouse sera on the antiviral activity. Although human and mouse sera did not affect the antiviral activity of CAS + IMD, they did attenuate the antiviral activity of S-880008. The protein binding-adjusted EC 90 (PA-EC 90 ) of S-880008 was 110 nM for human serum and 170 nM for mouse serum (Extended Data Table 4), while its potency shift was 15.2 and 25.6 for human and mouse sera, respectively (Extended Data Table 5). To examine the inhibitory effects of S-880008 on the replication of SARS-CoV-2 in vivo , the MA-P10 strain was inoculated intranasally into 5-week-old mice. The viral titers in the mouse lung homogenates were then measured at 1 and 2 day(s) post-infection (dpi) (Fig. 5a). Beta-D-N 4 -hydroxycytidine (NHC), an orally bioavailable ribonucleoside analog with broad-spectrum antiviral activity against various RNA viruses 37–39 , was used as a control compound. S-880008 was intranasally administered to mice once or twice a day (q.d. or b.i.d) at 1 dpi. The same total daily dose of S-880008 was used for the q.d. (1 mg/kg) and b.i.d. (0.5 mg/kg) modes of administration. The mice received daily oral NHC doses at 1–5 dpi. In the vehicle-treated group, the viral titers of the lung homogenates at 1 and 2 dpi were 5.13 and 6.80–6.97 log 10 TCID 50 /mL, respectively (Fig. 5b, 5c). The viral titer of the NHC-treated group at 2 dpi was 4.33 log 10 TCID 50 /mL, which was > 2-log lower than that of the vehicle-treated group (Fig. 5c). S-880008 administration reduced the viral titer at 2 dpi in dose-dependent manner under both q.d. and b.i.d. administration conditions (Fig. 5b, 5c). Notably, the viral titers of the mice treated with 1 mg/kg (q.d.) or 0.5 mg/kg (b.i.d.) S-880008 were > 4- or 3-log lower, respectively, than those of the vehicle-treated group. The viral titers of the S-880008-treated mice at 4 dpi also decreased in a dose-dependent manner (Fig. 5d, 5e). Moreover, the viral titers of all the groups at 9 dpi were below the lower limit of detection (Extended Data Fig. 4a, 4b). We next performed a pharmacokinetic-pharmacodynamic (PKPD) analysis by measuring the reduction in viral titer and the concentration of S-880008 in mouse lung homogenates or plasma at 2 dpi (24 hours after S-880008 administration). There was a positive correlation between the reduction in viral titer and the concentration of S-880008 in the mouse lung homogenates and plasma; the coefficient of determination ( r 2 ) was 0.75 (Fig. 5f) and 0.68 (Fig. 5g) for the lungs and plasma, respectively. These data demonstrate that a single administration of S-880008 is sufficient to exert a potent antiviral effect in vivo . To evaluate the protective efficacy of S-880008 against SARS-CoV-2 in a lethal infection model, we next inoculated 10- to 12-month-old adult mice with the MA-P10 strain as previously described 33 (Fig. 5h). All the non-infected mice survived (Extended Data Fig. 4c); moreover, their body weights were not significantly affected by the administration of S-880008 (Extended Data Fig. 4d). By contrast, all the infected mice in the vehicle control group died by 5 dpi (Fig. 5i) and incurred a ~15% reduction in body weight by 3 dpi (Extended Data Fig. 4e). In this setting, NHC treatment increased mouse survival to 80% (Fig. 5i). Crucially, all the mice treated with S-880008 at 0.1 or 1 mg/kg (q.d.) or at 0.5 mg/kg (b.i.d.) survived (Fig. 5i) and had normal body weights (Extended Data Fig. 4e). Although the administration of 0.01 mg/kg (q.d.) S-880008 did not increase mouse survival rates relative to the vehicle control group, it did prolong their survival time (Fig. 5i) and suppressed weight loss (Extended Data Fig. 4e). Furthermore, the viral titer (Extended Data Fig. 4f) and the concentration of interleukin (IL)-6 in the lung homogenates was significantly lower in the S-880008- or NHC-treated mice than in the vehicle-treated animals (Extended Data Fig. 4g). Notably, S-880008 and NHC prevented the increase in whole lung tissue weight, as well as the increase in whole lung tissue weight as a proportion of body weight, induced by SARS-CoV-2 infection (Extended Data Fig. 4h). These data demonstrate that S-880008 inhibits SARS-CoV-2-induced lung inflammation in vivo . Discussion In this study, we identified S-880008, a lipid-modified macrocyclic peptide, as a novel broad-spectrum peptide inhibitor of SARS-CoV-2 fusion. S-880008 was discovered using mRNA display screening and then chemically modified to yield a cyclic peptide containing non-canonical amino acids, a chemically modified Glu-linker, and a C-terminal C16 lipid. We demonstrated that S-880008 inhibited SARS-CoV-2 infection in vitro and in vivo via a unique, fusion-blocking mechanism. The WT spike trimer typically assumes one of three conformations: 3-closed RBD (31%), 1-up RBD (55%), and 2-up RBD (14%) 11 . We found that S-880008 bound to a pocket formed by the RBD, the SD1, and an NTD from an adjacent spike protomer, which forced the RBD to assume a 3-up conformation. Moreover, given that S-880008 does not inhibit the interaction between ACE2 and the SARS-CoV-2 spike, it has potential to synergize with CAS + IMD. To the best of our knowledge, this type of fusion inhibition mechanism, which confers broad antiviral activity by recognizing multiple vulnerable sites of the SARS-CoV-2 spike, has not been previously reported. Macrocyclic peptides capable of binding to a cryptic site at the C-terminal region of the RBD were previously identified by mRNA display screening against the spike protein 18,19 . One of these peptides exhibited broad-spectrum antiviral activity by binding to a site sequestered deep within the RBD 18 . While these peptides were thioether-cyclized, they contained an elongator segment comprised of natural amino acids. Therefore, in the present study, we leveraged the advantages of PDPS 22,23 and reassigned non-proteinogenic amino acids in the codon table. This strategy led to the discovery of S-880008, as a macrocyclic peptide containing 7-MeA and 11-F4CON, which exhibited potent anti-SARS-CoV-2 activity. Cryo-EM analysis revealed that S-880008 bound to the interdomain regions of RBD, SD1, and the NTD of an adjacent protomer. Therefore, we validated PDPS as a promising peptide discovery method for identifying drug candidates that bind to the sequestered sites of dynamic target proteins 40 . In addition, we showed that the potent antiviral activity of S-880008 relied on the interaction between the C16 acyl chain and a hydrophobic and conserved tunnel formed within the NTD. Cryo-EM analysis also highlighted the key contribution of 11-F4CON to the antiviral activity of S-880008. 11-F4CON is a non-canonical amino acid, which contains a carbamoyl group that is connected to the benzene ring of a phenylalanine. Cryo-EM revealed that the carbamoyl group formed two hydrogen bonds with the backbone atoms of K528 (Fig. 3g). The connection between the double hydrogen bond and the antiviral activity of S-880008 was investigated in structure-activity relationship experiments by substituting 11-F4CON with phenylalanine (compound 13), tyrosine (compound 14), or F4COO (compound 15). The antiviral activities of all three compounds were over 10-fold lower than that of S-880008. Compound 13, which lacks the carbamoyl group, was unable to form hydrogen bonds with K528. Compound 14, which has a phenolic hydroxyl group, interacted with the backbone atoms of K528 via a single hydrogen bond. Meanwhile, the repulsion between the carboxyl group of F4COO and the carbonyl moiety of K528 caused a substantial conformational change, which markedly weakened the antiviral activity of compound 15. These structure-activity relationship experiments confirmed that the ability of 11-F4CON to form a double hydrogen bond with the backbone atoms of K528 was important for the antiviral activity of S-880008. Our experiments with the S-880008-resistant viruses identified certain characteristic amino acid substitutions, which affected S-880008 binding. Specifically, P230A, F329L, and P330S were found in the S-880008 binding site, while E988D and Q992H were identified in the central helix of S2, a region that is important for viral fusion 12,14 . The variant viruses harboring E988D or Q992H were especially resistant to S-880008. Substitutions within the S2 region of SARS-CoV-2 (e.g., N764K, D796Y, Q954H, and N969K) enable Omicron variants to evade recognition by neutralizing antibodies targeting the RBD and NTD 41 . These S2 mutations, alongside other mutations present in Omicron subvariants, appear to increase the antigenic heterogeneity of the spike protein by keeping RBD in the “down” conformation 41 . These data suggest that the E988D or Q992H mutations affect not only viral fusion but also the binding of S-880008 to its preferred binding site. Given that S-880008 inhibited cell-to-cell fusion by binding to the SARS-CoV-2 spike, these data also support the notion that S-880008 inhibits viral fusion after viral attachment to cells. A recent study investigating S protein dynamics revealed that the mobility of the S protein RBD is higher than previously assumed, and that this RBD mobility plays a significant role in cell entry 42 . Consequently, this report supports the finding that S-880008 inhibits membrane fusion by reducing RBD mobility. The in vivo efficacy and PKPD results at 2 dpi were similar for the q.d. and b.i.d. modes of S-880008 administration, indicating that the trough concentration of S-880008 is important for its in vivo efficacy. Furthermore, PKPD analysis of MA-P10-infected mice confirmed the positive correlation between the in vivo efficacy of S-880008 and its concentration in lung homogenates and in the plasma. These data suggest that the plasma concentration can be used as a surrogate marker for the concentration of S-880008 in lung tissue. This strategy would negate the need to perform invasive procedures, such as the collection of bronchoalveolar lavage fluid, in human studies. Since the potency shift of S-880008 in human serum was lower than that in mouse serum, S-880008 may have a higher antiviral activity in humans than in mice, assuming that its pharmacokinetics in the two organisms are identical. We found that S-880008 reduced the viral titer more than NHC at 2 dpi in the non-lethal mouse model. Meanwhile, NHC reduced the viral titer more than S-880008 at 4 dpi in both the non-lethal and lethal mouse models. However, S-880008 was more effective than NHC at preventing weight loss and improving survival rates (100% vs. 80%, respectively) in the lethal mouse model. The outcomes of these lethal mouse model experiments highlight the importance of reducing viral titers in the early stages of infection. In summary, our findings demonstrate that S-880008 uses a unique mechanism, which enables it to simultaneously recognize cryptic and conserved sites of the virus to inhibit fusion. As such, it has the potential to overcome the increasing threat posed by emerging SARS-CoV-2 variants and become a promising antiviral drug. Declarations Acknowledgments We acknowledge the National Institute of Infectious Diseases, Japan for providing SARS-CoV-2 variants. We thank Sachi Takahara, Shihono Teruya, Shigeru Miki, and Fumika Takagi of Shionogi & Co., Ltd. for their contribution to the pharmacological experiments involving S-880008. We are grateful to all of our colleagues at Shionogi & Co., Ltd. who participated in the COVID-19 antiviral projects and to Shionogi Techno-Advance Research Co., Ltd. for their technical support with the organic synthesis of the relevant compounds and their assistance with the pharmacological experiments. We are grateful to PeptiStar Inc. for assisting us with the organic synthesis of S-880008. We thank all the members of the Japanese Consortium on Structural Virology (JX-Vir) for their technical support with the structural analysis. We thank Prof. Yoshiharu Matsuura (Osaka University, Osaka, Japan) for providing the materials used in the reverse genetics experiments. Finally, we thank Edanz (https://jp.edanz.com/ac) for editing the English text of a draft of this manuscript. This work was supported by the Japan Agency for Medical Research and Development (AMED) (grant JP20fk0108509h0001 awarded to H. Mikamiyama and H.S.; grants JP21wm0125008 and JP243fa627005 awarded to H.S.; grant JP21wm0225003 awarded to H.S.; grant JP22ama121037 awarded to K. Maenaka; grant JP243fa627005 awarded to K. Maenaka, A.S. and H.S.; grant JP243fa627009 awarded to T. Hashiguchi; and grant JP24jf0126002 awarded to T. Hashiguchi); Japan Science and Technology Agency (JST) Moonshot R&D (grant JPMJMS2025 awarded to Y.O.); the World-leading Innovative and Smart Education (WISE) Program from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan (grant 1801 awarded to H.S.); the MEXT/JSPS KAKENHI (grant JP20H05873 awarded to K. Maenaka and grant JPJSCCA20240006 awarded to T. Hashiguchi); the Cooperative Research Program (Joint Usage/Research Center program) of Institute for Life and Medical Sciences, Kyoto University (awarded to K. Maenaka); Hokkaido University, Global Facility Center, Pharma Science Open Unit (awarded to K. Maenaka); Hokkaido University COVID-19 Research Support Program (awarded to A.S.); COVID-19 Drug and Vaccine Development Donation (awarded to A.S.); Takeda Science Foundation (awarded to K. Maenaka); and the Naito foundation (awarded to T. Hashiguchi). Author contributions T. Sanaki, Y. Kawaguchi, Y. Kusumoto, A.S., H. Mikamiyama, and T. Shishido conceived the project and designed the experiments. T. Sanaki, K.T., A.Y., Y.M., T.M., S.A., and A.S. performed the cell culture experiments. S. Toba, K.T., Y.N., K. Mayumi, H. Morita, T. Haruna, and M.I. performed the animal experiments. Y.A., S. Kita, T. Hashiguchi, and K. Maenaka performed the Cryo-EM analysis. Y. Kawaguchi performed PDPS screening. Y. Kusumoto and H. Mikamiyama synthesized the antiviral compounds. Y.O., T. Hashiguchi, H.S., A.S., H. Mikamiyama, and K. Maenaka obtained funding. M.S., Y.O., and H.S. provided resources. T. Sanaki, Y. Kawaguchi, Y.A., Y. Kusumoto, H. Mikamiyama, and K. Maenaka wrote the initial draft. All the authors contributed to the preparation of the final version of the manuscript. T. Sanaki and T. Shishido were responsible for finalizing and submitting the manuscript. Yoshimasa Kawaguchi, Yuki Anraku, Yoshifumi Kusumoto, Shinsuke Toba, and Akihiko Sato contributed equally to this study. Please address all correspondence to Takao Sanaki and Takao Shishido. Competing interest declaration T. Sanaki, Y. Kusumoto, A.S., S. Toba, K.T., A.Y., Y.M., T.M., S.A., Y.N., H. Morita, K. Mayumi, T. Haruna, M.I., H. Mikamiyama, and T. Shishido are full-time employees of Shionogi & Co., Ltd., and a few people have stocks. H.S. and K. Maenaka have received research funding support from Shionogi & Co., Ltd. M.S. and Y.O. have received fees for speaker bureaus from Shionogi & Co., Ltd. Y. Kawaguchi, Y.A., S. Kita, and T. Hashiguchi declare no conflict of interest. Data availability The atomic coordinates and Cryo-EM maps of the following structures have been deposited in the Protein Data Bank (www.rcsb.org) and Electron Microscopy Data Bank (www.ebi.ac.uk/emdb/): the spike protein in complex with S-880008 (8KBN, EMD-37073), RBD/SD1/NTD/S-880008 (8KBO, EMD-37074), the spike protein in complex with compound 8 (8KBP, EMD-37075), RBD/SD1/NTD/compound 8 (8KBQ, EMD-37076), the spike protein in complex with compound 9 (8KBR, EMD-37077), RBD/SD1/NTD/compound 9 (8KBS, EMD-37078), the spike protein in complex with compound 10 (8KBT, EMD-37079), RBD/SD1/NTD/compound 10 (8KBU, EMD-37080), the spike protein 3-up in complex with compound 11 (EMD-37081), the spike protein 2-up in complex with compound 11 (EMD-37082), the spike protein 1-up in complex with compound 11 (EMD-37083), the spike protein 1-up in complex with compound 12 (EMD-37085), and the spike protein 3-down in the presence of compound 12 (EMD-37084). References Polack, F. P. et al. 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Commun. 8 , 1–13 (2017). Kumar, S. et al. Mutations in S2 subunit of SARS-CoV-2 Omicron spike strongly influence its conformation, fusogenicity, and neutralization sensitivity. J. Virol. 97 , e0092223 (2023). Yajima, H. et al. Structural basis for receptor-binding domain mobility of the spike in SARS-CoV-2 BA.2.86 and JN.1. Nat. Commun. 15 , 1–14 (2024). Tables Table 1. Antiviral activities (EC 50 ) of several compounds against SARS-CoV-2 variants SARS-CoV-2 variant EC 50 value a S-880008 Remdesivir Nirmatrelvir Nirmatrelvir + CP-100356 b CAS + IMD c hCoV-19/Japan/TY/WK-521/2020 WT 5.99 ± 1.17 2560 ± 250 5030 ± 630 63.0 ± 11.9 37.8 ± 3.31 hCoV-19/Japan/TY/WK-521/2020 MA-P10 d 4.39 ± 1.31 5014 ± 1880 6266 ± 2507 45.7 ± 3.72 25.5 ± 3.85 hCoV-19/Japan/QK002/2020 a 15.3 ± 2.26 2060 ± 280 5250 ± 510 72.9 ± 2.56 34.8 ± 0.91 hCoV-19/Japan/TY8-612/2021 b 4.71 ± 0.33 2850 ± 210 8620 ± 1990 113 ± 12.9 53.3 ± 10.3 hCoV-19/Japan/TY7-501/2021 g 6.43 ± 1.00 2080 ± 190 8730 ± 2890 92.3 ± 19.2 32.8 ± 4.94 hCoV-19/Japan/TY11-927-P1/2021 d 11.3 ± 5.13 2500 ± 320 6070 ± 1300 95.1 ± 14.9 41.7 ± 7.37 hCoV-19/Japan/TY33-456/2021 l 5.48 ± 0.81 3025 ± 455 5399 ± 2054 41.3 ± 11.6 10.5 ± 0.98 hCoV-19/Japan/TY28-444/2021 q 2.85 ± 0.43 889 ± 153 6383 ± 2492 48.4 ± 16.7 12.5 ± 3.00 hCoV-19/Japan/TY26-717/2021 m 2.66 ± 0.26 3627 ± 622 7716 ± 3703 54.8 ± 8.05 23.5 ± 5.59 hCoV-19/Japan/TY38-873/2021 BA.1 7.27 ± 0.33 971 ± 44.1 3960 ± 930 49.8 ± 15.1 >2000 hCoV-19/Japan/TY38-871/2021 BA.1.1 3.22 ± 1.39 800 ± 53.3 5915 ± 2207 42.5 ± 3.38 >2000 hCoV-19/Japan/TY40-385/2022 BA.2 16.2 ± 4.35 1254 ± 310 4831 ± 1529 42.3 ± 8.47 >2000 hCoV-19/Japan/TY41-716/2022 BA.2.75 13.1 ± 0.61 997 ± 259 4661 ± 1193 47.2 ± 17.0 >2000 hCoV-19/Japan/TY41-686/2022 XE 27.5 ± 8.52 965 ± 270 5550 ± 823 53.1 ± 19.4 >2000 hCoV-19/Japan/TY41-703/2022 BA.4 13.8 ± 3.62 618 ± 56.0 2090 ± 41.5 28.4 ± 6.96 >2000 hCoV-19/Japan/TY41-702/2022 BA.5 18.2 ± 2.25 796 ± 81.7 3690 ± 713 32.8 ± 5.01 >2000 a: S‑880008, remdesivir, and nirmatrelvir: nM, CAS + IMD: ng/mL b: CP-100356 monohydrochloride was added at a final concentration of 1 mM. c: CAS + IMD = antibody cocktail (casirivimab and imdevimab) d: MA-P10 = mouse-adapted strain of SARS-CoV-2 generated at passage 10 Data are expressed as the mean ± standard deviation of three independent experiments. Additional Declarations Yes there is potential Competing Interest. T. Sanaki, Y. Kusumoto, A.S., S. Toba, K.T., A.Y., Y.M., T.M., S.A., Y.N., H. Morita, K. Mayumi, T. Haruna, M.I., H. Mikamiyama, and T. Shishido are full-time employees of Shionogi & Co., Ltd., and a few people have stocks. H.S. and K. Maenaka have received research funding support from Shionogi & Co., Ltd. M.S. and Y.O. have received fees for speaker bureaus from Shionogi & Co., Ltd. Y. Kawaguchi, Y.A., S. Kita, and T. Hashiguchi declare no conflict of interest. Supplementary Files 20250206SupplementaryInformation.docx Materials and Methods ExtendedDatas.docx Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Takao","middleName":"","lastName":"Shishido","suffix":""},{"id":415334657,"identity":"7f0d5b4d-5cf8-4b0d-88d3-cfa842d0e8d7","order_by":23,"name":"Katsumi Maenaka","email":"","orcid":"https://orcid.org/0000-0002-5459-521X","institution":"Hokkaido University","correspondingAuthor":false,"prefix":"","firstName":"Katsumi","middleName":"","lastName":"Maenaka","suffix":""}],"badges":[],"createdAt":"2025-02-07 04:05:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5977541/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5977541/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":76302947,"identity":"2d7f900c-a7f9-4761-af87-e8b138fc3fcd","added_by":"auto","created_at":"2025-02-14 14:19:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":689891,"visible":true,"origin":"","legend":"\u003cp\u003eThe strategy used to discover S-880008.\u003c/p\u003e\n\u003cp\u003ea. Peptide screening strategy. b. Amino acid sequences of the peptides identified through mRNA display screening and their antiviral activities against the WT SARS-CoV-2 strain. c. The structure of S-880008. d. The cytotoxicities (CC\u003csub\u003e50\u003c/sub\u003e) of several compounds were tested in Vero E6/TMPRSS2 cells. Cells and compounds were incubated for 3−4 days. The concentrations of S-880008, remdesivir, and nirmatrelvir are presented is nM. The concentration of CAS + IMD is presented in ng/mL. CP-100356 monohydrochloride was added at a final concentration of 1 mM. Data were expressed as the mean ± standard deviation of three independent experiments (a, d). The antiviral activity of S-880008 was referred in Table 1. CAS + IMD, antibody cocktail (casirivimab and imdevimab).\u003c/p\u003e","description":"","filename":"Picture1.png","url":"https://assets-eu.researchsquare.com/files/rs-5977541/v1/aa720bbb69c14e623d22d17e.png"},{"id":76302941,"identity":"6945a152-b4ba-46ee-88a2-0d3a0eec64a9","added_by":"auto","created_at":"2025-02-14 14:19:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":412529,"visible":true,"origin":"","legend":"\u003cp\u003ea. S-880008 inhibited viral entry into cells. SARS-CoV-2 was incubated in the presence or absence of each compound for 1 h (pre-treatment); these viral solutions were then used to treat VeroE6/TMPRSS2 cells for 1 h. After removing the supernatant, the cells were incubated with viral assay medium, in the presence or absence of each compound for 6 h (post-treatment). The inhibitory effect of each compound (EC\u003csub\u003e90\u003c/sub\u003e) was quantified by measuring the amount of intracellular viral RNA. b. Investigating the inhibitory effect of each compound on ACE2 binding to the SARS-CoV-2 spike. Each compound was incubated with the spike protein, before biotinylated human ACE2 protein and HRP-conjugated streptavidin were added. The inhibitory effect of each compound (IC\u003csub\u003e50\u003c/sub\u003e) was evaluated by quantifying the absorbance at 450 nm after the addition of TMB solution as the final step. c, d. The inhibitory effect on cell fusion induced by the expression of SARS-CoV-2 spike. VeroE6/TMPRSS2/GFP cells and VeroE6/TMPRSS2/mCherry cells were transfected with pCMV-Myc-C (negative control), pCMV-Myc-C-Spro (WT), or pCMV-Myc-C-Spro (d) and then treated with each compound. Fluorescence images were acquired at 48 (WT) or 32 (d) h after plasmid transfection. c. Compound concentration: S-880008; 250 nM for WT, 1,000 nM for d. CAS + IMD; 5,000 ng/mL, remdesivir; 50,000 nM. Scale bar; 200 μm. d. The inhibitory effects of the compounds (EC\u003csub\u003e50\u003c/sub\u003e) were evaluated by quantifying the area of the co-localized region. e. Evaluating the effect of combining S-880008 with remdesivir, nirmatrelvir, or CAS + IMD. VeroE6/TMPRSS2 cells were treated with S-880008 and remdesivir or S-880008 and nirmatrelvir and simultaneously infected with SARS-CoV-2; the cells were then incubated for 3 days. S-880008, CAS + IMD and SARS-CoV-2 were preincubated for 1 hour; this mixture was then incubated with the cells for 3 days. Data were expressed as the mean ± standard deviation of three independent experiments (a, b, d, e). CAS + IMD (a, b, c, d) and remdesivir (a, c, d) were used as entry and non-entry inhibitors, respectively. CAS + IMD, antibody cocktail (casirivimab and imdevimab); CI, combination index; HRP, horseradish peroxidase; TMB, 3,3′,5,5′-tetramethylbenzidine.\u003c/p\u003e\n\u003cp\u003eMode of action of S-880008 \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Picture2.png","url":"https://assets-eu.researchsquare.com/files/rs-5977541/v1/7dff55d1844c0aa348cd67d7.png"},{"id":76302942,"identity":"388656bf-6ba0-49c4-bd99-bf3d6fb246a2","added_by":"auto","created_at":"2025-02-14 14:19:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":985114,"visible":true,"origin":"","legend":"\u003cp\u003eCryo-EM structure of the SARS-CoV-2 spike in complex with S-880008.\u003c/p\u003e\n\u003cp\u003ea. Overall structure of the SARS-CoV-2 spike trimer in complex with S-880008. The spike protomers is colored pink, green, or blue; the S-880008 is colored orange. b. The local refinement map of the spike RBD, SD1, and NTD in complex with S-880008. c. S-880008 exhibited antiviral activity against numerous SARS-CoV-2 variants (listed in Table 1); the amino acid substitutions present in the variants are shown as spheres. d. The overall structure of S-880008 while bound to the spike protein. e. A schematic illustration of spike amino acid residues surrounding S-880008; the image shows the structural formula of S-880008, while the one-letter code and sequence number are used to label the amino acid residues of the spike protein. The hydrogen bonds are depicted as straight dotted lines. The black contour lines indicate the contact areas between the S-880008 and the spike protein residues. (1), (2), and (3) correspond to the close-up view shown in Fig. 2f, 2g, and 2h, respectively. f–h. Detailed images of the interaction between S-880008 and the spike protein. f. The β strand structure of S-880008 and its interaction with the NTD of the spike protein. g. The interaction between the C16 acyl chain of S-880008 and the hydrophobic tunnel of the spike NTD. h. The interaction between the 11-F4CON side chain of S-880008 and the spike SD1. F4CON, L-4-carbamoylphenylalanine; MeA, N-methyl-L-alanine; NTD, N-terminal domain; RBD, receptor-binding domain; SD1, subdomain 1; *, residues that are conjugated via amide bonds between the ε-amino group of a lysine and the carboxyl group of a glutamic acid.\u003c/p\u003e","description":"","filename":"Picture3.png","url":"https://assets-eu.researchsquare.com/files/rs-5977541/v1/85a6080097bdf389f22cf179.png"},{"id":76302945,"identity":"0f1f00a8-d78a-4d86-9802-68c38b359c59","added_by":"auto","created_at":"2025-02-14 14:19:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1238431,"visible":true,"origin":"","legend":"\u003cp\u003eCryo-EM structure of the SARS-CoV-2 spike in complex with S-880008 derivatives.\u003c/p\u003e\n\u003cp\u003ea. The characteristics of S-880008 and its derivative compounds. b–d. Cryo-EM maps and structures of the derivative compounds in complex with the spike protein. b. Compound 8. c. Compound 9. d. Compound 11. d. The conformation of the spike protein in the presence of compound 11 (left) and the enlarged view of the compounds 11 binding pocket (left), with the RBD in the up (top) and down (bottom) conformation. e. Structure-activity relationship between the type of amino acid present at position 11 in S-880008 and antiviral activity. Antiviral activity (a, e) was expressed as the mean ± standard deviation of three independent experiments. The antiviral activity of S-880008 against a range of SARS-CoV-2 variants is presented in Table 1. F4COO, L-4-carboxylphenylalanine; NTD, N-terminal domain; OEG, 8-amino-3,6-dioxaoctanoic acid; RBD, receptor-binding domain; SD1, subdomain 1.\u003c/p\u003e","description":"","filename":"Picture4.png","url":"https://assets-eu.researchsquare.com/files/rs-5977541/v1/20e19d48cfb6a2d56844db7e.png"},{"id":76302944,"identity":"f3867fd4-b8f7-45e4-906a-de95e114b902","added_by":"auto","created_at":"2025-02-14 14:19:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":729986,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of the \u003cem\u003ein vivo \u003c/em\u003eantiviral efficacy of S-880008 in a mouse model of SARS-CoV-2 infection.\u003c/p\u003e\n\u003cp\u003ea. The protocol used for the non-lethal mouse model experiments. b–e. Viral titer in mouse lung homogenates (n = 5/group) at 2 (b, c) and 4 (d, e) dpi after treatment with vehicle, S-880008, or NHC. Vehicle and S-880008 were intranasally administered q.d. (b, d) and b.i.d. (c, e) only at 1 dpi. The total daily dose of S-880008 was the same, regardless of. administration mode (i.e., q.d. and b.i.d). NHC was administered orally b.i.d. on 1–5 dpi. f, g. PKPD analysis of the correlation between a reduction in viral titer and the S-880008 concentration in mouse lung homogenates (f) and mouse plasma (g) at 2 dpi. h. The protocol used for the lethal mouse model experiments. i. The survival time of SARS-CoV-2-infected mice was measured over a 14-day period after treatment with vehicle, S-880008, or NHC (n = 10/group). b–e. Data are expressed as the mean ± standard deviation were analyzed in comparison with the vehicle control group using a one-way ANOVA, followed by a Dunnett’s post-hoc test; ns, not significant; **, p \u0026lt; 0.01; ****, p \u0026lt; 0.0001. i. Data were analyzed in comparison to the vehicle control group using the log-rank test; ****, p \u0026lt; 0.0001. b.i.d., bis in die (twice a day); NHC, beta-D-N4-hydroxycytidine; PKPD, pharmacokinetic-pharmacodynamic; q.d., quaque die (once a day); \u003cem\u003er\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e, coefficient of determination; TCID\u003csub\u003e50\u003c/sub\u003e, 50% tissue culture infective dose.\u003c/p\u003e","description":"","filename":"Picture5.png","url":"https://assets-eu.researchsquare.com/files/rs-5977541/v1/bc23d61161f346b7cffe67d1.png"},{"id":90029353,"identity":"54c34adb-7c2f-4a9c-aad9-f1c2b771b894","added_by":"auto","created_at":"2025-08-27 14:44:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5302164,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5977541/v1/3874bd59-34df-4d80-b0b3-15c3dc13048e.pdf"},{"id":76302943,"identity":"39303795-e3ab-4155-97dc-38627d0bbe58","added_by":"auto","created_at":"2025-02-14 14:19:01","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6133855,"visible":true,"origin":"","legend":"Materials and Methods","description":"","filename":"20250206SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5977541/v1/bf7e05f2b5626e097d9461f0.docx"},{"id":76302946,"identity":"b51111d2-cabc-4636-b5ff-b8a325518b61","added_by":"auto","created_at":"2025-02-14 14:19:01","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10912947,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedDatas.docx","url":"https://assets-eu.researchsquare.com/files/rs-5977541/v1/d4bf17972c32d8c42904b168.docx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nT. Sanaki, Y. Kusumoto, A.S., S. Toba, K.T., A.Y., Y.M., T.M., S.A., Y.N., H. Morita, K. Mayumi, T. Haruna, M.I., H. Mikamiyama, and T. Shishido are full-time employees of Shionogi \u0026 Co., Ltd., and a few people have stocks. H.S. and K. Maenaka have received research funding support from Shionogi \u0026 Co., Ltd. M.S. and Y.O. have received fees for speaker bureaus from Shionogi \u0026 Co., Ltd. Y. Kawaguchi, Y.A., S. Kita, and T. Hashiguchi declare no conflict of interest.","formattedTitle":"Lipidated macrocyclic peptide S-880008 as a broad-spectrum SARS-CoV-2 fusion inhibitor","fulltext":[{"header":"Summary paragraph","content":"\u003cp\u003eSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for coronavirus disease 2019, still poses a threat to public health. Although several anti-SARS-CoV-2 neutralizing-antibody-based therapies exist, their efficacy is limited by the fact that mutations found in SARS-CoV-2 variants mainly target the spike protein. To create drugs that work against a wide range of SARS-CoV-2 variants, it is important to target conserved parts of the spike protein. Here, we used the peptide screening and structural optimization approaches to generate the lipid-modified macrocyclic peptide S-880008. We showed that S-880008 was effective against a broad range of SARS-CoV-2 (including Omicron) variants. Cryo-electron microscopy revealed that S-880008 bound simultaneously to the conserved interfaces of the receptor-binding domain, subdomain 1 and the N-terminal domain of the adjacent protomer to prevent the protomer dissociation necessary for viral fusion. This novel binding site is completely distinct from that of the angiotensin-converting enzyme 2, which is targeted by neutralizing antibodies. In accordance, S-880008 inhibited viral fusion but not viral attachment \u003cem\u003ein vitro\u003c/em\u003e. Crucially, the intranasal administration of S-880008 significantly reduced the viral titer in lungs of mice infected with mouse-adapted SARS-CoV-2 and improved their survival rates. S-880008 represents a promising new drug for a broad range of SARS-CoV-2 variants.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eCoronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to threaten global public health.\u0026nbsp;Although several vaccines\u003csup\u003e1,2\u003c/sup\u003e and antiviral drugs\u003csup\u003e3\u0026ndash;6\u003c/sup\u003e for SARS-CoV-2 exist, early vaccines failed to induce neutralizing antibody responses that can keep pace with the emergence of new SARS-CoV-2 variants. Indeed, the early vaccines developed against the ancestral SARS-CoV-2 strain offer suboptimal protection against the recent Omicron variants\u003csup\u003e7\u003c/sup\u003e. The development of effective neutralizing-antibody-based therapies is also hampered by the rapid mutation rates of new SARS-CoV-2 variants. This suboptimal efficacy prompted the U.S. Food and Drug Administration to revoke the Emergency Use Authorization for early SARS-CoV-2 neutralizing-antibody-based therapies. The SARS-CoV-2 spike protein, and particularly its receptor-binding domain (RBD), is the main target molecule of neutralizing antibodies\u003csup\u003e8\u003c/sup\u003e. Mutations found in SARS-CoV-2 variants primarily affect the surface of the SARS-CoV-2 spike, particularly around the RBD\u003csup\u003e8\u003c/sup\u003e, which reduces the ability of neutralizing antibodies to suppress viral replication. Nevertheless, given that some neutralizing antibodies against the spike protein showed clear efficacies in clinical trials\u003csup\u003e9,10\u003c/sup\u003e, the mechanisms underlying the interaction between neutralizing antibodies and the SARS-CoV-2 spike require further elucidation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe SARS-CoV-2 spike, which is composed of two functional subunits (S1 and S2) that assemble into homotrimers on the viral surface, is essential for viral entry into host cells\u003csup\u003e11,12\u003c/sup\u003e. While S1 binds to angiotensin-converting enzyme 2 (ACE2)\u003csup\u003e13\u003c/sup\u003e, the host cell receptor, S2 is involved in the fusion of the viral and cellular membranes\u003csup\u003e12,14\u003c/sup\u003e. Recently,\u0026nbsp;nanobodies\u003csup\u003e15\u0026ndash;17\u003c/sup\u003e and peptides\u003csup\u003e18,19\u003c/sup\u003e that bind to the SARS-CoV-2 spike have been identified. These smaller molecules can neutralize regions of the spike protein, which are otherwise inaccessible to the larger antibodies. Moreover, peptides offer several advantages as therapeutic agents over proteins. These include more cost-effective production, higher levels of stability (e.g., resistant to heat and acidic conditions), and versatile administration routes (e.g., oral\u003csup\u003e20\u003c/sup\u003e and respiratory routes\u003csup\u003e21\u003c/sup\u003e). However, the lack of clinically validated peptide compounds underscores the necessity for the generation of potent, broad-spectrum antiviral peptides that demonstrate therapeutic efficacy \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eHerein, we searched for novel peptides targeting the spike protein that could exhibit broad-spectrum antiviral activity and \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003eefficacy against SARS-CoV-2 variants, including the Omicron variants.\u003cem\u003e\u0026nbsp;\u003c/em\u003eUnexpectedly, we found lipidated macrocyclic peptides that deeply protrude vulnerable inter-protomer regions of the spike protein to fix its prefusion state, providing insight into the development of a new class of broad-spectrum fusion inhibitors.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eDiscovery of S-880008 using mRNA display technology and chemical modification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Peptide Discovery Platform System (PDPS) was developed by PeptiDream Inc. on the basis of mRNA display technology to specifically select peptide sequences that bound to target molecules from a pool of 10\u003csup\u003e13\u003c/sup\u003e macrocycles containing non-natural amino acids\u003csup\u003e22,23\u003c/sup\u003e. In the present study, peptide genetic codes were reprogrammed to generate a macrocyclic peptide library incorporating non-proteinogenic amino acids (e.g., chloroacetyl-phenylalanine), N-methyl amino acids, and carbamoyl phenylalanine. We hypothesized that the incorporation of these non-proteinogenic amino acids would improve peptide pharmacokinetics and create new and more potent binding modes. In early 2021, when SARS-CoV-2 \u0026beta; and \u0026gamma; strains were prevalent, PDPS was performed against the biotinylated S1 domain (mS1-bio), which consisted of the N-terminal domain (NTD) and RBD harboring the K417N, E484K, and N501Y mutations. During subsequent selection rounds, PDPS selection was performed against biotinylated RBDs (mRBD-bio) harboring the K417N, E484K, and N501Y mutations to comprehensively identify peptides that bound strongly to the surface of the RBDs (Fig. 1a). Multiple rounds of panning were conducted. An additional panning step against the\u0026nbsp;ancestral\u0026nbsp;S1 protein was also included to obtain broad-spectrum antiviral peptides. Next-generation sequencing was used to identify four highly enriched peptides (Fig. 1b, Compounds 1\u0026ndash;4). These peptide were synthesized and decorated with lipids via a glutamic acid linker to improve their solubility\u003csup\u003e24\u003c/sup\u003e,\u0026nbsp;stability, and pharmacokinetics\u003csup\u003e25\u003c/sup\u003e (Fig. 1b, S-880008 and Compounds 5\u0026ndash;7). The antiviral activity of these cyclic peptides against the ancestral wildtype (WT) SARS-CoV-2 strain was then evaluated (Fig. 1b). Among the lipid-modified peptides, S-880008 showed the highest antiviral activity at single-digit nanomolar concentrations (Fig. 1b, c); therefore, S-880008 was selected for further study.\u003c/p\u003e\n\u003cp\u003eIn the cell-based assay, S-880008 exerted broad-spectrum antiviral activity against SARS-CoV-2 variants, including the Omicron strains; the 50% effective concentration (EC\u003csub\u003e50\u003c/sub\u003e) ranged from 2.66 nM for the m strain to 27.5 nM for the XE strain (Table 1). S-880008 proved to be considerably more potent than remdesivir and nirmatrelvir \u003cem\u003ein vitro\u003c/em\u003e. Since the 50% cytotoxic concentration (CC\u003csub\u003e50\u003c/sub\u003e) values of all the compounds tested were much higher than their EC\u003csub\u003e50\u003c/sub\u003e values (Fig. 1d), we concluded that their cytotoxicity did not impact their antiviral activity. S-880008 effectively reduced the intracellular viral RNA concentration. The 90% effective concentration (EC\u003csub\u003e90\u003c/sub\u003e) of S-880008 against the WT, BA.1, and BA.2 strains, calculated based on the amount of intracellular viral RNA, was 7.46, 5.46, and 52.2 nM, respectively (Extended Data Table 1). Surface plasmon resonance (SPR) analysis demonstrated the direct binding of S-880008 to the SARS-CoV-2 spike. The binding dissociation constants (Kds) against the WT and BA.2 spikes were 20.4 and 23.3 nM, respectively (Extended Data Fig. 1). These data clearly indicate that S-880008 exerts its antiviral activity by acting on the virus rather than the host cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMode of action of S-880008: Inhibition of viral fusion after viral attachment to cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn a time of addition test, S-880008 inhibited viral activity before but not after viral entry into cells (Fig. 2a). Since remdesivir exerted antiviral activity regardless of the timing of addition (Fig. 2a), these data indicate that S-880008 functions similarly to a neutralizing antibody cocktail of casirivimab and imdevimab (CAS + IMD). Therefore, we next investigated the effect of S-880008 on the interaction between the SARS-CoV-2 spike and ACE2. CAS + IMD inhibited the ACE2 binding in concentration-dependent manner, with a 50% inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) of 23.5 ng/mL (Fig. 2b). By contrast, S-880008 did not inhibit ACE2 binding to the spike protein (IC\u003csub\u003e50\u003c/sub\u003e \u0026gt; 25,000 nM) (Fig. 2b). These data indicate that the inhibitory mechanism of S-880008 is different from that of CAS + IMD. We therefore next investigated the effect of S-880008 on SARS-CoV-2-spike-induced cell-to-cell fusion using cell lines which expressed the SARS-CoV-2 spike in addition to either GFP or mCherry. While the green (GFP) and red (mCherry) fluorescence signals of the vector control groups remained unchanged, the SARS-CoV-2-spike-expressing cells emitted a yellow signal, which indicated cell-to-cell fusion between the GFP- and mCherry-expressing cells (Fig. 2c). Cell-to-cell fusion was dramatically inhibited by both S-880008 and CAS + IMD. The EC\u003csub\u003e50\u003c/sub\u003e values of S-880008 and CAS + IMD were 13.7 nM and 383 ng/mL for the WT strain, and 66.0 nM and 650 ng/mL for the d strain, respectively (Fig. 2d). By contrast, remdesivir, an RdRp inhibitor, did not inhibit cell-to-cell fusion. These data demonstrate that S-880008 inhibits viral fusion rather than viral attachment to cells. We next assessed how combination with remdesivir, nirmatrelvir, or CAS + IMD impacted the antiviral effect of S-880008 \u003cem\u003ein vitro\u003c/em\u003e. The combination index values of S-880008 with remdesivir, nirmatrelvir, or CAS + IMD were 1.07, 0.91, and 0.73, respectively. These data indicate that while the combination of S-880008 with remdesivir or nirmatrelvir produced an additive effect, S-880008 synergized with CAS + IMD (Fig. 2e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCryo-EM\u003c/strong\u003e \u003cstrong\u003eanalysis of\u003c/strong\u003e \u003cstrong\u003eS-880008 binding to SARS-CoV-2 spike proteins\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next performed cryo-electron microscopy (Cryo-EM) analysis to better understand the mechanism underlying S-880008 binding to the SARS-CoV-2 spike. The overall structure of the WT spike in complex with S-880008 at 3.02 \u0026Aring; resolution showed a RBD 3-up conformation with C3 symmetry; the spike assumes the same conformation in complex with ACE2\u003csup\u003e26\u003c/sup\u003e (Fig. 3a and Supplementary Fig. 1a\u0026ndash;d). The overall maps showed that three S-880008 peptides bound to a single spike trimer. To improve the resolution around the S-880008 binding site, we performed focused refinement, which yielded a local map at 3.6 \u0026Aring; resolution (Fig. 3b, 3c and Supplementary Fig. 1b\u0026ndash;d). This map revealed that the characteristic S-880008 binding site was entirely distinct from the ACE2 binding site. Notably, S-880008 recognized the surfaces of the RBD and subdomain 1 (SD1), together with the NTD of an adjacent spike protomer. We speculated that this binding arrangement presumably tightened inter-protomer interactions, which suppressed the major structural rearrangements necessary for subsequent fusion (Fig. 3a, 3b). The fact that the binding pocket for S-880008 was only formed in the RBD-up state implied that S-880008 encouraged the spike to adopt a 3-up conformation, which may be beneficial for ACE2 engagement. This mechanism presumably prevents the dissociation of protomers, which ultimately blocks the fusion process (Extended Data Fig. 2a).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eS-880008 consists of three parts: a cyclic peptide containing non-canonical amino acids, a glutamic acid (Glu)-linker, and a C-terminal C16 lipid. Although the EM map allowed us to clearly observe the side chains of the cyclic peptide section, we were unable to view the Glu linker, likely due to the high flexibility and repulsion of its side chains and the electron radiation damage it incurred during imaging\u003csup\u003e27\u003c/sup\u003e. We were, however, able to characterize the three stages of S-880008 binding (Fig. 3d, 3e). In the images, the cyclic peptide component of S-880008 assumed an anti-parallel \u0026beta;-sheet structure, which was connected by \u0026beta;-turn motifs such as the cis configuration of N-methyl-L-alanine (7-MeA) or the thioether linker formed by 12-Cys. S-880008 used its \u0026beta;-strand to extend the \u0026beta;-sheet into the \u0026beta;-sandwich core of the NTD (Fig. 3f and Extended Data Fig. 2c). Interestingly, the C16 acyl chain of S-880008 protruded deeply into the tunnel of the \u0026beta;-sandwich structure of the NTD (Fig. 3g). Previous studies have reported the binding of this hydrophobic tunnel to biliverdin and polysorbate 80\u003csup\u003e28,29\u003c/sup\u003e, suggesting that this site could be a promising target for drug discovery\u003csup\u003e30\u003c/sup\u003e. Meanwhile, the carbamoyl group in the 11-F4CON side chain interacted with the backbone atoms of K528, forming a loop (residues 522\u0026ndash;528) between the RBD and SD1 (Fig. 3h). Additionally, the aromatic ring of 11-F4CON was located in the hydrophobic environment formed by V362 and a loop (residues 328\u0026ndash;336) between the RBD and NTD (P330 and I332). This structural information enabled us to delineate the three key events implicated in the interaction between S-880008 and the SARS-CoV-2 spike: (1) cyclic peptide portion extended the \u0026beta; sheet of the NTD; (2) the C-terminal C16 lipid protruded into the hydrophobic tunnel of the NTD; and (3) the non-canonical amino acids captured the flexible interdomain (NTD-RBD and RBD-SD1) loops. Crucially, the amino acid substitutions found in prevalent SARS-CoV-2 variants are concentrated at the ACE2 binding site of the RBD (outside of the NTD), meaning that they do not affect the S-880008 binding site (Fig. 3c and Extended Data Fig. 2b). Thus, S-880008 exhibits potent and broad antiviral activity toward SARS-CoV-2 variants by targeting vulnerable sites in the spike protein (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eS-880008 exerts its antiviral activity by exploiting the interaction between its 11-F4CON sidechain and the K528 backbone of the SARS-CoV-2 RBD\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo study the structure-activity relationship of S-880008, we next examined antiviral activities of five derivative compounds and determined the Cryo-EM structures of their spike complexes (Fig. 4a) under the same conditions as used for S-880008 evaluation (Fig. 4b-d, Extended Data Fig. 2d-e, and Supplementary Fig. 2\u0026ndash;4). Compounds 8 (lipid portion substituted with C15COOH), 9 (altered lipid modification site [to position 8]), and 10 (altered linker and lipid moiety [C14]) all exhibited similar antiviral activities to S-880008 and bound to the spike 3-up state in the same way as S-880008 (Fig. 4b, 4c). By contrast, compound 11 (MeA to Asp substitution at position 7) had 10-fold lower antiviral activity than S-880008 and caused the spike to adopt multiple (3-up, 2-up, and 1-up) conformations (Fig. 4d). Similarly, compound 12 (MeA to Asp substitution at position 7 and Arg to Glu substitution at position 8) had a significantly lower antiviral activity than S-880008 and caused the spike to adopt the 1-up and 3-down conformations. In these less potent compounds, a density corresponding to an interaction at the RBD-NTD interface of the up protomers was observed on the Cryo-EM maps; this density was absent from the RBD-NTD interface of the down protomers (Fig. 4d and Extended Data Fig. 2e). This result is consistent with the fact that the viral RBD only accommodated S-880008 in the up conformation (Extended Data Fig. 2a). Compounds 11 and 12 contained 7-Asp instead of the 7-MeA present in S-880008, suggesting that the differences in the binding proportions of these compounds are attributable to N-methylation. Thus, the ability of S-880008 to undergo the conformation changes necessary for spike protein binding and subsequent viral neutralization relies on N-methylation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCryo-EM analysis further identified two hydrogen bonds linking the carbamoyl group in the 11-F4CON side chain of S-880008 to the backbone atoms of K528 (Fig. 3h). A structure-activity relationship analysis was performed to investigate whether the 11-F4CON carbamoyl group contributed significantly to S-880008 antiviral activity. Compounds 13 (no carbamoyl group), 14 (containing a hydroxyl group), and 15 (containing a carboxyl moiety), which all had 11-F4CON modifications, had 32-, 13-, and \u0026gt; 400-fold lower antiviral activities than S-880008, respectively (Fig. 4e). These results confirm the importance of the interaction between the carbamoyl group of 11-F4CON and the backbone atoms of K528 in the RBD for the antiviral activity of S-880008, and imply that S-880008 has the potential to exhibit broad-spectrum antiviral activity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAmino acid substitutions in the SARS-CoV-2 S2 region markedly reduce the antiviral activity of S-880008\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate which viral amino acid substitutions markedly reduced the antiviral activity of S-880008, several S-880008-resistant viruses were prepared. Ten amino acid substitutions in the spike region were selected by genotypic analysis, according to the criteria for the selection of characteristic amino acid substitutions after S-880008 treatment (Extended Data Table 2). Subsequently, 10 viruses, each containing a single amino acid substitution, were prepared by reverse genetics\u003csup\u003e31\u003c/sup\u003e. Two of the viruses (containing F562C or L984F) did not grow; thus, the antiviral activity of S-880008 was evaluated against the remaining eight viruses (Extended Data Table 3). The isolated S-880008-resistant virions harbored characteristic amino acid substitutions in the binding sites to S-880008 (i.e., P230A, F329L and P330S), as well as in the central helix of S2 (E988D and Q992H), a region which is important for viral fusion\u003csup\u003e12,14\u003c/sup\u003e(Extended Data Fig. 3). Remdesivir and CAS + IMD showed similar antiviral activities against all eight S-880008-resistant viruses (Extended Data Table 3). By contrast, the antiviral activity of S-880008 against all viruses other than the variant harboring the T1009I substitution was lower than that against the WT strain (Extended Data Table 3). Specifically, the reduction in the antiviral activity against the viruses containing the E988D, L752F, Q992H, and F329L substitutions was \u0026gt; 2,165-, 324-, 27.5-, and 12.7-fold, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vivo\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;antiviral efficacy of S-880008 in mouse-adapted SARS-CoV-2-infected mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMouse-adapted SARS-CoV-2 (MA-P10) was prepared as previously described\u003csup\u003e32\u0026ndash;34\u003c/sup\u003e to evaluate the \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003eantiviral efficacies of S-880008 and the other antiviral compounds\u003csup\u003e35,36\u003c/sup\u003e. Of note, S-880008 also exhibited \u003cem\u003ein vitro\u003c/em\u003e antiviral activity against MA-P10 harboring the Q498H amino acid substitution in the spike region, which was comparable to its activity against the SARS-CoV-2 variants (Table 1). We investigated the effects of human and mouse sera on the antiviral activity. Although human and mouse sera did not affect the antiviral activity of CAS + IMD, they did attenuate the antiviral activity of S-880008. The protein binding-adjusted EC\u003csub\u003e90\u003c/sub\u003e (PA-EC\u003csub\u003e90\u003c/sub\u003e) of S-880008 was 110 nM for human serum and 170 nM for mouse serum (Extended Data Table 4), while its potency shift was 15.2 and 25.6 for human and mouse sera, respectively (Extended Data Table 5).\u003c/p\u003e\n\u003cp\u003eTo examine the inhibitory effects of S-880008 on the replication of SARS-CoV-2 \u003cem\u003ein vivo\u003c/em\u003e, the MA-P10 strain was inoculated intranasally into 5-week-old mice. The viral titers in the mouse lung homogenates were then measured at 1 and 2 day(s) post-infection (dpi) (Fig. 5a). Beta-D-N\u003csup\u003e4\u003c/sup\u003e-hydroxycytidine (NHC), an orally bioavailable ribonucleoside analog with broad-spectrum antiviral activity against various RNA viruses\u003csup\u003e37\u0026ndash;39\u003c/sup\u003e, was used as a control compound. S-880008 was intranasally administered to mice once or twice a day (q.d. or b.i.d) at 1 dpi. The same total daily dose of S-880008 was used for the q.d. (1 mg/kg) and b.i.d. (0.5 mg/kg) modes of administration. The mice received daily oral NHC doses at 1\u0026ndash;5 dpi. In the vehicle-treated group, the viral titers of the lung homogenates at 1 and 2 dpi were 5.13 and 6.80\u0026ndash;6.97 log\u003csub\u003e10\u003c/sub\u003e TCID\u003csub\u003e50\u003c/sub\u003e/mL, respectively (Fig. 5b, 5c). The viral titer of the NHC-treated group at 2 dpi was 4.33 log\u003csub\u003e10\u003c/sub\u003e TCID\u003csub\u003e50\u003c/sub\u003e/mL, which was \u0026gt; 2-log lower than that of the vehicle-treated group (Fig. 5c). S-880008 administration reduced the viral titer at 2 dpi in dose-dependent manner under both q.d. and b.i.d. administration conditions (Fig. 5b, 5c). Notably, the viral titers of the mice treated with 1 mg/kg (q.d.) or 0.5 mg/kg (b.i.d.) S-880008 were \u0026gt; 4- or 3-log lower, respectively, than those of the vehicle-treated group. The viral titers of the S-880008-treated mice at 4 dpi also decreased in a dose-dependent manner (Fig. 5d, 5e). Moreover, the viral titers of all the groups at 9 dpi were below the lower limit of detection (Extended Data Fig. 4a, 4b). We next performed a pharmacokinetic-pharmacodynamic (PKPD) analysis by measuring the reduction in viral titer and the concentration of S-880008 in mouse lung homogenates or plasma at 2 dpi (24 hours after S-880008 administration). There was a positive correlation between the reduction in viral titer and the concentration of S-880008 in the mouse lung homogenates and plasma; the coefficient of determination (\u003cem\u003er\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e) was 0.75 (Fig. 5f) and 0.68 (Fig. 5g) for the lungs and plasma, respectively. These data demonstrate that a single administration of S-880008 is sufficient to exert a potent antiviral effect \u003cem\u003ein vivo\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo evaluate the protective efficacy of S-880008 against SARS-CoV-2 in a lethal infection model, we next inoculated 10- to 12-month-old adult mice with the MA-P10 strain as previously described\u003csup\u003e33\u003c/sup\u003e (Fig. 5h). All the non-infected mice survived (Extended Data Fig. 4c); moreover, their body weights were not significantly affected by the administration of S-880008 (Extended Data Fig. 4d). By contrast, all the infected mice in the vehicle control group died by 5 dpi (Fig. 5i) and incurred a ~15% reduction in body weight by 3 dpi (Extended Data Fig. 4e). In this setting, NHC treatment increased mouse survival to 80% (Fig. 5i). Crucially, all the mice treated with S-880008 at 0.1 or 1 mg/kg (q.d.) or at 0.5 mg/kg (b.i.d.) survived (Fig. 5i) and had normal body weights (Extended Data Fig. 4e). Although the administration of 0.01 mg/kg (q.d.) S-880008 did not increase mouse survival rates relative to the vehicle control group, it did prolong their survival time (Fig. 5i) and suppressed weight loss (Extended Data Fig. 4e). Furthermore, the viral titer (Extended Data Fig. 4f) and the concentration of interleukin (IL)-6 in the lung homogenates was significantly lower in the S-880008- or NHC-treated mice than in the vehicle-treated animals (Extended Data Fig. 4g). Notably, S-880008 and NHC prevented the increase in whole lung tissue weight, as well as the increase in whole lung tissue weight as a proportion of body weight, induced by SARS-CoV-2 infection (Extended Data Fig. 4h). These data demonstrate that S-880008 inhibits SARS-CoV-2-induced lung inflammation \u003cem\u003ein vivo\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we identified S-880008, a lipid-modified macrocyclic peptide, as a novel broad-spectrum peptide inhibitor of SARS-CoV-2 fusion. S-880008 was discovered using mRNA display screening and then chemically modified to yield a cyclic peptide containing non-canonical amino acids, a chemically modified Glu-linker, and a C-terminal C16 lipid. We demonstrated that S-880008 inhibited SARS-CoV-2 infection \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e via a unique, fusion-blocking mechanism. The WT spike trimer typically assumes one of three conformations: 3-closed RBD (31%), 1-up RBD (55%), and 2-up RBD (14%)\u003csup\u003e11\u003c/sup\u003e. We found that S-880008 bound to a pocket formed by the RBD, the SD1, and an NTD from an adjacent spike protomer, which forced the RBD to assume a 3-up conformation. Moreover, given that S-880008 does not inhibit the interaction between ACE2 and the SARS-CoV-2 spike, it has potential to synergize with CAS + IMD. To the best of our knowledge, this type of fusion inhibition mechanism, which confers broad antiviral activity by recognizing multiple vulnerable sites of the SARS-CoV-2 spike, has not been previously reported.\u003c/p\u003e\n\u003cp\u003eMacrocyclic peptides capable of binding to a cryptic site at the C-terminal region of the RBD were previously identified by mRNA display screening against the spike protein\u003csup\u003e18,19\u003c/sup\u003e. One of these peptides exhibited broad-spectrum antiviral activity by binding to a site sequestered deep within the RBD\u003csup\u003e18\u003c/sup\u003e. While these peptides were thioether-cyclized, they contained an elongator segment comprised of natural amino acids. Therefore, in the present study, we leveraged the advantages of PDPS\u003csup\u003e22,23\u003c/sup\u003e and reassigned non-proteinogenic amino acids in the codon table. This strategy led to the discovery of S-880008, as a macrocyclic peptide containing 7-MeA and 11-F4CON, which exhibited potent anti-SARS-CoV-2 activity. Cryo-EM analysis revealed that S-880008 bound to the interdomain regions of RBD, SD1, and the NTD of an adjacent protomer. Therefore, we validated PDPS as a promising peptide discovery method for identifying drug candidates that bind to the sequestered sites of dynamic target proteins\u003csup\u003e40\u003c/sup\u003e. In addition, we showed that the potent antiviral activity of S-880008 relied on the interaction between the C16 acyl chain and a hydrophobic and conserved tunnel formed within the NTD.\u003c/p\u003e\n\u003cp\u003eCryo-EM analysis also highlighted the key contribution of 11-F4CON to the antiviral activity of\u0026nbsp;S-880008. 11-F4CON is a non-canonical amino acid, which contains a carbamoyl group that is connected to the benzene ring of a phenylalanine. Cryo-EM revealed that the carbamoyl group formed two hydrogen bonds with the backbone atoms of K528 (Fig. 3g). The connection between the double hydrogen bond and the antiviral activity of S-880008 was investigated in structure-activity relationship experiments by substituting 11-F4CON with phenylalanine (compound 13), tyrosine (compound 14), or F4COO (compound 15). The antiviral activities of all three compounds were over 10-fold lower than that of S-880008. Compound 13, which lacks the carbamoyl group, was unable to form hydrogen bonds with K528. Compound 14, which has a phenolic hydroxyl group, interacted with the backbone atoms of K528 via a single hydrogen bond. Meanwhile, the repulsion between the carboxyl group of F4COO and the carbonyl moiety of K528 caused a substantial conformational change, which markedly weakened the antiviral activity of compound 15. These structure-activity relationship experiments confirmed that the ability of 11-F4CON to form a double hydrogen bond with the backbone atoms of K528 was important for the antiviral activity of S-880008.\u003c/p\u003e\n\u003cp\u003eOur experiments with the S-880008-resistant viruses identified certain characteristic amino acid substitutions, which affected S-880008 binding. Specifically, P230A, F329L,\u0026nbsp;and\u0026nbsp;P330S were found in the S-880008 binding site, while E988D and Q992H were identified in the central helix of S2, a region that is important for viral fusion\u003csup\u003e12,14\u003c/sup\u003e. The variant viruses harboring E988D or Q992H were especially resistant to S-880008. Substitutions within the S2 region of SARS-CoV-2 (e.g., N764K, D796Y, Q954H, and N969K) enable Omicron variants to evade recognition by neutralizing antibodies targeting the RBD and NTD\u003csup\u003e41\u003c/sup\u003e. These S2 mutations, alongside other mutations present in Omicron subvariants, appear to increase the antigenic heterogeneity of the spike protein by keeping RBD in the \u0026ldquo;down\u0026rdquo; conformation\u003csup\u003e41\u003c/sup\u003e.\u0026nbsp;These data suggest that the E988D or Q992H mutations affect not only viral fusion but also the binding of S-880008 to its preferred binding site. Given that S-880008 inhibited cell-to-cell fusion by binding to the SARS-CoV-2 spike, these data also support the notion that S-880008 inhibits viral fusion after viral attachment to cells. A recent study investigating S protein dynamics revealed that the mobility of the S protein RBD is higher than previously assumed, and that this RBD mobility plays a significant role in cell entry\u003csup\u003e42\u003c/sup\u003e. Consequently, this report supports the finding that S-880008 inhibits membrane fusion by reducing RBD mobility.\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e efficacy and PKPD results at 2 dpi were similar for the q.d. and b.i.d. modes of S-880008 administration, indicating that the trough concentration of S-880008 is important for its \u003cem\u003ein vivo\u003c/em\u003e efficacy. Furthermore, PKPD analysis of MA-P10-infected mice confirmed the positive correlation between the \u003cem\u003ein vivo\u003c/em\u003e efficacy of S-880008 and its concentration in lung homogenates and in the plasma. These data suggest that the plasma concentration can be used as a surrogate marker for the concentration of S-880008 in lung tissue. This strategy would negate the need to perform invasive procedures, such as the collection of bronchoalveolar lavage fluid, in human studies. Since the potency shift of S-880008 in human serum was lower than that in mouse serum, S-880008 may have a higher antiviral activity in humans than in mice, assuming that its pharmacokinetics in the two organisms are identical. We found that S-880008 reduced the viral titer more than NHC at 2 dpi in the non-lethal mouse model. Meanwhile, NHC reduced the viral titer more than S-880008 at 4 dpi in both the non-lethal and lethal mouse models. However, S-880008 was more effective than NHC at preventing weight loss and improving survival rates (100% \u003cem\u003evs.\u003c/em\u003e 80%, respectively) in the lethal mouse model. The outcomes of these lethal mouse model experiments highlight the importance of reducing viral titers in the early stages of infection.\u003c/p\u003e\n\u003cp\u003eIn summary, our findings demonstrate that S-880008 uses a unique mechanism, which enables it to simultaneously recognize cryptic and conserved sites of the virus to inhibit fusion. As such, it has the potential to overcome the increasing threat posed by emerging SARS-CoV-2 variants and become a promising antiviral drug.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge the National Institute of Infectious Diseases, Japan for providing SARS-CoV-2 variants. We thank Sachi Takahara, Shihono Teruya, Shigeru Miki, and Fumika Takagi of Shionogi \u0026amp; Co., Ltd. for their contribution to the pharmacological experiments involving S-880008. We are grateful to all of our colleagues at Shionogi \u0026amp; Co., Ltd. who participated in the COVID-19 antiviral projects and to Shionogi Techno-Advance Research Co., Ltd. for their technical support with the organic synthesis of the relevant compounds and their assistance with the pharmacological experiments. We are grateful to PeptiStar Inc. for assisting us with the organic synthesis of S-880008. We thank all the members of the Japanese Consortium on Structural Virology (JX-Vir) for their technical support with the structural analysis. We thank Prof. Yoshiharu Matsuura (Osaka University, Osaka, Japan) for providing the materials used in the reverse genetics experiments. Finally, we thank Edanz (https://jp.edanz.com/ac) for editing the English text of a draft of this manuscript. This work was supported by the Japan Agency for Medical Research and Development (AMED) (grant JP20fk0108509h0001 awarded to H. Mikamiyama and H.S.; grants JP21wm0125008 and JP243fa627005 awarded to H.S.; grant JP21wm0225003 awarded to H.S.; grant JP22ama121037 awarded to K. Maenaka; grant JP243fa627005 awarded to K. Maenaka, A.S. and H.S.; grant JP243fa627009 awarded to T. Hashiguchi; and grant JP24jf0126002 awarded to T. Hashiguchi); Japan Science and Technology Agency (JST) Moonshot R\u0026amp;D (grant JPMJMS2025 awarded to Y.O.); the World-leading Innovative and Smart Education (WISE) Program from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan (grant 1801 awarded to H.S.); the MEXT/JSPS KAKENHI (grant JP20H05873 awarded to K. Maenaka and grant JPJSCCA20240006 awarded to T. Hashiguchi);\u0026nbsp;the Cooperative Research Program (Joint Usage/Research Center program) of Institute for Life and Medical Sciences, Kyoto University (awarded to K. Maenaka); Hokkaido University, Global Facility Center, Pharma Science Open Unit (awarded to K. Maenaka); Hokkaido University COVID-19 Research Support Program (awarded to A.S.); COVID-19 Drug and Vaccine Development Donation (awarded to A.S.); Takeda Science Foundation (awarded to K. Maenaka); and the Naito foundation (awarded to T. Hashiguchi).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eT. Sanaki, Y. Kawaguchi, Y. Kusumoto, A.S., H. Mikamiyama, and T. Shishido conceived the project and designed the experiments. T. Sanaki, K.T., A.Y., Y.M., T.M., S.A., and A.S. performed the cell culture experiments. S. Toba, K.T., Y.N., K. Mayumi, H. Morita, T. Haruna, and M.I. performed the animal experiments. Y.A., S. Kita, T. Hashiguchi, and K. Maenaka performed the Cryo-EM analysis. Y. Kawaguchi performed PDPS screening. Y. Kusumoto and H. Mikamiyama synthesized the antiviral compounds. Y.O., T. Hashiguchi, H.S., A.S., H. Mikamiyama, and K. Maenaka obtained funding. M.S., Y.O., and H.S. provided resources. T. Sanaki, Y. Kawaguchi, Y.A., Y. Kusumoto, H. Mikamiyama, and K. Maenaka wrote the initial draft. All the authors contributed to the preparation of the final version of the manuscript. T. Sanaki and T. Shishido were responsible for finalizing and submitting the manuscript. Yoshimasa Kawaguchi, Yuki Anraku, Yoshifumi Kusumoto, Shinsuke Toba, and Akihiko Sato contributed equally to this study. Please address all correspondence to Takao Sanaki and Takao Shishido.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eT. Sanaki, Y. Kusumoto, A.S., S. Toba, K.T., A.Y., Y.M., T.M., S.A., Y.N., H. Morita, K. Mayumi, T. Haruna, M.I., H. Mikamiyama, and T. Shishido are full-time employees of Shionogi \u0026amp; Co., Ltd., and a few people have stocks.\u0026nbsp;H.S. and K. Maenaka have received research funding support from Shionogi \u0026amp; Co., Ltd. M.S. and Y.O. have received fees for speaker bureaus from Shionogi \u0026amp; Co., Ltd. Y. Kawaguchi, Y.A., S. Kita, and T. Hashiguchi declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe atomic coordinates and Cryo-EM maps of the following structures have been deposited in the Protein Data Bank (www.rcsb.org) and Electron Microscopy Data Bank (www.ebi.ac.uk/emdb/): the spike protein in complex with S-880008 (8KBN, EMD-37073), RBD/SD1/NTD/S-880008 (8KBO, EMD-37074), the spike protein in complex with compound 8 (8KBP, EMD-37075), RBD/SD1/NTD/compound 8 (8KBQ, EMD-37076), the spike protein in complex with compound 9 (8KBR, EMD-37077), RBD/SD1/NTD/compound 9 (8KBS, EMD-37078), the spike protein in complex with compound 10 (8KBT, EMD-37079), RBD/SD1/NTD/compound 10 (8KBU, EMD-37080), the spike protein 3-up in complex with compound 11 (EMD-37081), the spike protein 2-up in complex with compound 11 (EMD-37082), the spike protein 1-up in complex with compound 11 (EMD-37083), the spike protein 1-up in complex with compound 12 (EMD-37085), and the spike protein 3-down in the presence of compound 12 (EMD-37084).\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePolack, F. 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Virol.\u003c/em\u003e \u003cstrong\u003e97\u003c/strong\u003e, e0092223 (2023).\u003c/li\u003e\n\u003cli\u003eYajima, H. \u003cem\u003eet al.\u003c/em\u003e Structural basis for receptor-binding domain mobility of the spike in SARS-CoV-2 BA.2.86 and JN.1. \u003cem\u003eNat. Commun.\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 1\u0026ndash;14 (2024).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1. Antiviral activities (EC\u003csub\u003e50\u003c/sub\u003e) of several compounds against SARS-CoV-2 variants\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"725\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" style=\"width: 255px;\"\u003e\n \u003cp\u003eSARS-CoV-2 variant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"15\" style=\"width: 470px;\"\u003e\n \u003cp\u003eEC\u003csub\u003e50\u0026nbsp;\u003c/sub\u003evalue\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" style=\"width: 94px;\"\u003e\n \u003cp\u003eS-880008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 95px;\"\u003e\n \u003cp\u003eRemdesivir\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 95px;\"\u003e\n \u003cp\u003eNirmatrelvir\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 94px;\"\u003e\n \u003cp\u003eNirmatrelvir\u003cbr\u003e+ CP-100356\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 92px;\"\u003e\n \u003cp\u003eCAS + IMD\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003ehCoV-19/Japan/TY/WK-521/2020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eWT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e5.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e1.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e2560\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e5030\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e630\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e63.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e11.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e37.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34px;\"\u003e\n \u003cp\u003e3.31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003ehCoV-19/Japan/TY/WK-521/2020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eMA-P10\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e4.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e1.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e5014\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e1880\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e6266\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n 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37px;\"\u003e\n \u003cp\u003e2.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e34.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34px;\"\u003e\n \u003cp\u003e0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003ehCoV-19/Japan/TY8-612/2021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e4.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e0.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e2850\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e210\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e8620\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e1990\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e113\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e12.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e53.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34px;\"\u003e\n 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38px;\"\u003e\n \u003cp\u003e11.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e5.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e2500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e320\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e6070\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e1300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e95.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n 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38px;\"\u003e\n \u003cp\u003e3025\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e455\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e5399\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e2054\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e41.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e11.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e10.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n 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\u003cp\u003e6383\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e2492\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e48.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e16.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e12.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34px;\"\u003e\n \u003cp\u003e3.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003ehCoV-19/Japan/TY26-717/2021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003em\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e2.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e0.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e3627\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e622\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e7716\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e3703\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e54.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e8.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e23.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34px;\"\u003e\n \u003cp\u003e5.59\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003ehCoV-19/Japan/TY38-873/2021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eBA.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e7.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e0.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e971\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e44.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e3960\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e930\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e49.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e15.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u0026gt;2000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003ehCoV-19/Japan/TY38-871/2021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eBA.1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e3.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e1.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e53.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e5915\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e2207\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e42.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e3.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u0026gt;2000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003ehCoV-19/Japan/TY40-385/2022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eBA.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e16.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e4.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e1254\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e310\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e4831\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e1529\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e42.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e8.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u0026gt;2000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003ehCoV-19/Japan/TY41-716/2022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eBA.2.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e13.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e0.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e997\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e259\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e4661\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e1193\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e47.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e17.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u0026gt;2000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003ehCoV-19/Japan/TY41-686/2022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eXE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e27.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e8.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e965\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e270\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e5550\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e823\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e53.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e19.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u0026gt;2000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003ehCoV-19/Japan/TY41-703/2022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eBA.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e13.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e3.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e618\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e56.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e2090\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e41.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e28.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e6.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u0026gt;2000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003ehCoV-19/Japan/TY41-702/2022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eBA.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e18.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e2.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e796\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e81.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38px;\"\u003e\n \u003cp\u003e3690\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e713\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e32.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e5.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u0026gt;2000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003ea: S‑880008, remdesivir, and nirmatrelvir: nM, CAS + IMD: ng/mL\u003c/p\u003e\n\u003cp\u003eb: CP-100356 monohydrochloride was added at a final concentration of 1\u0026nbsp;mM.\u003c/p\u003e\n\u003cp\u003ec: CAS + IMD = antibody cocktail (casirivimab and imdevimab)\u003c/p\u003e\n\u003cp\u003ed: MA-P10 = mouse-adapted strain of SARS-CoV-2 generated at passage 10\u003c/p\u003e\n\u003cp\u003eData are expressed as the mean \u0026plusmn; standard deviation of three independent experiments.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5977541/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5977541/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Coronavirus disease 2019, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a threat to public health and the economy. Although several SARS-CoV-2 vaccines exist, they have failed to elicit effective neutralizing antibody responses against emerging SARS-CoV-2 variants harboring spike protein mutations. Moreover, while certain neutralizing-antibody-based therapies were effective in the early stages of the SARS-CoV-2 pandemic, their performance declined with the emergence of spike protein mutations. Thus, it is essential to develop antiviral agents that inhibit the early stages of viral infection by effectively targeting spike protein mutants. Broad-spectrum anti-SARS-CoV-2 activity relies on the targeting of conserved sites within the spike protein. Herein, we used mRNA display screening and chemical modification to generate S-880008, a lipid-modified macrocyclic peptide. S-880008 exhibited efficacy against a broad range of SARS-CoV-2 (including Omicron) variants and inhibited SARS-CoV-2 fusion rather than attachment via a novel mechanism. Cryo-electron microscopy analysis revealed that, unexpectedly, S-880008 simultaneously recognized the vulnerable sites of both the interface between the receptor-binding domain (RBD) and subdomain 1 (via the macrocyclic peptide portion) and the N-terminal domain of the adjacent protomer (via the acyl chain) to enforce a RBD 3-up conformation. The results of the structure-activity relationship experiments with S-880008 derivatives supported the notion that S-880008 binding inhibited fusion by suppressing protomer dissociation. Crucially, the intranasal administration of S-880008 to mouse-adapted SARS-CoV-2-infected mice significantly reduced the viral titer in lung homogenates and improved survival rates in a dose-dependent manner. Our findings show that S-880008 has the potential to overcome the increasing threat posed by emerging SARS-CoV-2 variants, providing a rationale for the design of a broad range of antiviral fusion inhibitors.","manuscriptTitle":"Lipidated macrocyclic peptide S-880008 as a broad-spectrum SARS-CoV-2 fusion inhibitor","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-14 14:18:56","doi":"10.21203/rs.3.rs-5977541/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"003ad9ef-c91c-4c84-b67c-06d6a88f076e","owner":[],"postedDate":"February 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":44289243,"name":"Biological sciences/Microbiology/Virology/SARS-CoV-2"},{"id":44289244,"name":"Biological sciences/Drug discovery/Medicinal chemistry/Lead optimization"},{"id":44289245,"name":"Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy"},{"id":44289246,"name":"Biological sciences/Drug discovery/Drug screening"},{"id":44289247,"name":"Biological sciences/Microbiology/Virology/Viral membrane fusion"}],"tags":[],"updatedAt":"2026-01-27T20:21:01+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-14 14:18:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5977541","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5977541","identity":"rs-5977541","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}