Intranasal administration of broad-spectrum macrocyclic peptide inhibitor protects against SARS-CoV-2 Omicron variants

Domain: neuroscience
Published: 2026-01-26
Editorial outcome: major_revision

Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to cause significant morbidity and mortality despite the end of its pandemic phase. The emergence of highly mutated SARS-CoV-2 variants of concern highlights the requirement of broad-spectrum antiviral countermeasures which possess both prophylactic and therapeutic efficacies. Here, we obtain a macrocyclic peptide, 6L3-3P11K, that effectively inhibits a wide range of SARS-CoV-2 variants and subvariants. Structural studies show that 6L3-3P11K forms homotrimers that lock the spike protein (S) trimer into a “closed” conformation by engaging a conserved non-receptor binding motif (non-RBM) of S. This interaction disrupts the binding between S and ACE2 receptor. Structure-guided modifications result in a thermostable and trypsin-resistant macrocyclic peptide, 6L3-1F3P11hR, that exhibits prophylactic and therapeutic effects against SARS-CoV-2 infection in a male hACE2 transgenic mouse model after intranasal administration. Our results provide a drug candidate for the control and prevention of COVID-19 and may stimulate further research on macrocyclic broad-spectrum anti-coronavirus drug development.

Introduction
Coronavirus disease (COVID-19) is an ongoing infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Besides the significant morbidity and mortality associated with acute infection, especially among elderly and immunocompromised patients, post-COVID-19 condition has also been recognized1. Previous studies have shown that use of the SARS-CoV-2 antivirals in the acute phase may reduce the risk of post-COVID-19 condition2,3.

One of the major challenges in combating COVID-19 is the emergence of SARS-CoV-2 variants and subvariants that are immunoevasive to previously developed vaccines and monoclonal antibodies, such as the Omicron variant and subvariants4,5. While the two main types of clinically approved antiviral drugs for COVID-19, namely, RNA-dependent RNA polymerase (RdRp) inhibitors and main protease (Mpro) inhibitors, remain effective against Omicron6, the widespread use of these drugs that target a single viral enzyme may eventually contribute to the emergence of resistant strains7. Recent clinical studies have shown that, although Paxlovid (an Mpro inhibitor) can prevent the progression of COVID-19 from mild symptoms to severe diseases, it is not associated with a significantly shorter time to sustained alleviation of COVID-19 symptoms than placebo8. Moreover, these systemically administered drugs may be associated with major drug interactions or side effects. Therefore, novel antiviral drugs with high potency, distinctive mechanisms of action, and preferably topical administration to reduce systemic side effects are urgently needed.

Over the past two decades, macrocyclic peptides have garnered significant attention as a promising therapeutic strategy due to their uniquely advantageous properties9–11. These include high binding affinity and target specificity, low risk of off-target effects, minimal metabolic toxicity, strong tissue penetration, ease of manufacturing, metabolic stability, and the ability to disrupt protein-protein interactions. Moreover, these attributes can be further enhanced through strategic modifications.

Here, we employed the RaPID system12–15 to conduct de novo screening of macrocyclic peptides targeting the conserved region of the receptor-binding domain (RBD) of SARS-CoV-2. We discovered several promising macrocyclic peptides with strong binding affinities to the RBD. Structural studies demonstrated that the peptides bind to a conserved non-receptor-binding motif (non-RBM) region of RBD, and can form a homotrimer to lock the S trimer into a “closed” conformation, which prevents binding with ACE2. Further studies revealed the efficacy of these macrocyclic peptides to inhibit SARS-CoV-2 infection in vitro and in vivo. Our research has opened a promising avenue for the development of macrocyclic peptides as versatile agents, with potential efficacy against a wide range of SARS-CoV-2 variants and subvariants.

Results
Screening of macrocyclic peptides against the SARS-CoV-2 RBD
To identify macrocyclic peptides capable of binding to the RBD and potentially inhibiting the activity of SARS-CoV-2, we employed the RaPID system and conducted selections targeting the Omicron BA.2 RBD (Supplementary Fig. 1a). To increase the success rate of selection of antiviral peptides, we used the previously identified RBD-targeting macrocyclic peptide (peptide 4), which lacks antiviral activity, to block the site that could lead to the emergence of non-inhibitory peptides16. The specific binders were subsequently recovered, and their cDNAs were amplified via polymerase chain reaction (PCR) to provide input for the subsequent selection round. After five or six rounds of selection, we analyzed the enriched cDNA pools from the libraries using DNA deep sequencing techniques (Supplementary Fig. 1b). After analyzing the hit frequency, we proceeded to select the top 20 macrocyclic peptide sequences from each library and conducted alignments as shown in Supplementary Fig. 1c.

To validate their binding properties and investigate their antiviral activities, we employed the solid-phase peptide synthesis (SPPS) technique to chemically synthesize three representative sequences, 6L1, 6L3, and 5D1, originating from distinct families (Supplementary Fig. 1c). Subsequently, we conducted in vitro experiments to evaluate their antiviral potency against VSV particles pseudotyped with the BA.2 SARS-CoV-2 spike protein. Only 6L3 displayed weak inhibition activity, and both 5D1 and 6L1 exhibited no inhibition activity against the BA.2 pseudovirus (Supplementary Fig. 2a).

Using 6L3 as a starting point, we endeavored to introduce amino acid mutations to enhance its antiviral efficacy. The sequencing results showed that the similar peptide sequences to 6L3 mainly exhibit polymorphism at the 3rd, 6th, and 11th positions. Therefore, we selected these peptide mutants (L3P, T11K, L3PT11K, L3PT11I, L3PT11F, and L3PT6IP7Q) to evaluate the antiviral activities. Notably, 6L3-3P exhibited a significant improvement in antiviral effect, displaying an IC50 (half maximal inhibitory concentration) value of 8 nM (Supplementary Fig. 2b). Additionally, 6L3-11K also demonstrated enhanced antiviral ability, with an IC50 value of 676 nM. With both L3P and T11K substitutions, 6L3-3P11K also displayed strong antiviral activity, with an IC50 of 12 nM. In contrast, other mutant peptides did not possess antiviral activities. These findings underscore the pivotal roles played by L3P and T11K substitutions in augmenting the antiviral activity of 6L3 (Supplementary Fig. 2b). Consequently, we selected 6L3-3P and 6L3-3P11K for further investigation (Supplementary Fig. 2c).

Broad-spectrum antiviral activities of 6L3-3P and 6L3-3P11K peptides
The remarkable efficacy demonstrated by 6L3-3P and 6L3-3P11K against the pseudotyped SARS-CoV-2 BA.2 variant encouraged us to further evaluate their potencies against other SARS-CoV-2 variants. We first conducted assessments of their binding affinities to different RBDs. Both 6L3-3P and 6L3-3P11K could effectively bind to all the tested SARS-CoV-2 RBDs, including Prototype (PT), Delta, and Omicron variants (BA.2, BF.7, BQ.1.1, XBB, XBB.1.5, and BA.2.86) (Fig. 1a, Supplementary Fig. 3a). These two peptides displayed particularly higher affinity to the Omicron variant RBDs, with KD values ranging from 2.36 to 21.7 nM. In contrast, the binding affinities of these two peptides to Prototype and Delta RBDs were decreased dramatically, with KD values spanning from 11.4 to 65.3 μM. Intriguingly, 6L3-3P and 6L3-3P11K also demonstrated binding ability to RBDs of SARS-CoV-related viruses, with KD values ranging from 19.5 to 95.9 μM. Our results indicated that these two macrocyclic peptides may be potential broad-spectrum pan-sarbecovirus antiviral candidates.

We further conducted inhibition assays of these two peptides on pseudotyped SARS-CoV-2 Prototype and VOCs (Alpha, Beta, Gamma, Delta, and Omicron subvariants BA.1, BA.2.75, BA.4/5, BA.4.6, BF.7, BQ.1, BQ.1.1, XBB, XBB.1.5, XBB.1.16, CH.1.1, BA.2.86 and EG.5.1). Consistent with the binding results, both 6L3-3P and 6L3-3P11K demonstrated broad-spectrum antiviral activities, particularly against the Omicron subvariants, with IC50 values ranging from 3 to 86 nM (Fig. 1b, Supplementary Fig. 3b). In contrast, against the Prototype and early VOCs, including Alpha, Beta, Gamma, Delta, and Omicron subvariant BA.1, these two peptides displayed relatively weaker neutralizing abilities, with IC50 values ranging from 452 to 6933 nM (Fig. 1b, Supplementary Fig. 3b). Collectively, these findings indicated that both 6L3-3P and 6L3-3P11K exhibit broad-spectrum antiviral activities against emerging SARS-CoV-2 variants and subvariants.

Inhibition mechanism of 6L3-3P11K
To elucidate the molecular mechanism of how 6L3-related macrocyclic peptides inhibit virus infection, we prepared the S proteins of SARS-CoV-2 Prototype (PT S) and BA.2.75 (BA.2.75 S), and then incubated them with the 6L3-3P11K peptide to obtain the S-peptide complexes. Subsequently, we resolved the cryo-EM structures of PT S/6L3-3P11K and BA.2.75 S/6L3-3P11K at resolutions of 2.6 and 3.1 Å, respectively (Supplementary Figs. 4 and 5, Supplementary Table 1). Interestingly, the binding of 6L3-3P11K makes the PT S trimers keep a “closed” conformation with all three RBD down. Within this “closed” S trimer, three 6L3-3P11K molecules bound together in the top cavity of S trimer, forming a homotrimer through hydrophobic interactions and van der Waals (VDW) forces. Particularly, the amino acids 7P and 1Y of 6L3-3P11K played a crucial role in these trimerization interactions (Fig. 2a, c). A similar “closed” conformation of S trimer was also observed in the BA.2.75 S/6L3-3P11K complex structure, with 6L3-3P11K molecules occupying the same binding cavity on top of S trimer (Fig. 2b, d). The “up” or “down” conformations of the RBDs are inherent dynamic features for the spike protein, and the peptide binding to RBDs did not induce the “closed” conformation. When the RBDs move to the “down” conformation, the trimerization of the peptides can keep the “closed” conformation of the RBDs on the spike protein and prevent the occurrence of the “up” conformation of the RBDs, which is required for ACE2 receptor binding. This hypothesis was further confirmed through competitive binding experiments using the bio-layer interferometry (BLI) method. As anticipated, the BA.2.75 S trimer displayed a strong binding capacity to ACE2. When we incubated the BA.2.75 S protein with the 6L3-3P or 6L3-3P11K for 10 min, the S protein exhibited a significant reduction in the binding capacity. As a negative control, even incubated with 6L3 peptide for 2 h, the S protein still effectively bound to ACE2 (Supplementary Fig. 6). Native SEC-mass spectrometry analysis revealed that 6L3-3P11K exists exclusively as a monomer in the absence of the spike protein binding (Supplementary Fig. 7), and 6L3-3P11K trimerization occurs specifically upon S protein binding. We also analyzed the cryo-EM density of the PT S and BA.2.75 S in the apo state, and performed the quantitative analysis for the unbound spike trimer. The results showed that the proportion of spike trimers in the three-RBD-down conformation increased notably after peptide binding, from 6.6 to ~100% for PT and from 38.3% to 54% for BA.2.75 (Supplementary Figs. 4, 5 and 8). The cryo-EM density of the BA.2.75 S/6L3 complex was also analyzed, showing that most of the S trimers displayed either “three-RBD-up” or “two-RBD-up” conformation (Supplementary Fig. 9). This binding feature corroborates the weaker inhibition activity of 6L3.

The detailed analyses of the interactions between three 6L3-3P11K molecules and three RBDs in the S trimer revealed that each 6L3-3P11K binds to the inner side of one major RBD and also has minor interaction with the adjacent RBD (Fig. 3a, e and Supplementary Fig. 10). Notably, within the major binding pocket, residues 4I, 5L, 8L and 10F of 6L3-3P11K interact with a hydrophobic region in the RBD, comprised of residues F377, G404, V407, V433, I434 and V503 (Fig. 3b, f). This hydrophobic pocket is highly conserved in the RBD of the SARS-CoV-2 S protein. In addition, in both PT S/6L3-3P11K and BA.2.75 S/6L3-3P11K complex structures, residues two-dimensional (2D), 11K, and 13G-NH2 in the peptide form the hydrogen bond interactions with the RBD residues (Fig. 3c, g).

Of note, the 370-loop region (residues 373-377) of RBD in the PT S/6L3-3P11K complex structure exhibited a noteworthy conformational change upon peptide binding (Fig. 3d and Supplementary Fig. 11). In contrast, the BA.2.75 S/6L3-3P11K structure showed minimal conformational alteration (Fig. 3h and Supplementary Fig. 11). This difference may correlate with the relatively lower inhibition activity of 6L3-3P11K against SARS-CoV-2 strains including Prototype and early VOCs, which might result from different conformations of 370-loop (residues 373-377), and also differential residues at positions 375 and 376 (changed from hydrophilic S375 and T376 to hydrophobic F375 and A376) and position 408 (changed from charged R408 to non-charged S408) of RBD in PT and BA.2.75 virus variants (Supplementary Fig. 12).

Collectively, the inhibition of 6L3-3P11K refers to two important steps: the first step is the peptide binding to the RBD, and the second step is the trimerization of the peptide when the RBDs move to the “down” state in the S trimer.

Structure-based modification of 6L3-3P11K peptide
To increase the antiviral activity of 6L3-3P11K peptide, we introduced an amino acid mutation to the peptide based on the molecular interaction revealed by structural analysis. It is worth noting that, as described above, in addition to its interaction with the major RBD, 6L3-3P11K also exhibited minor interactions with adjacent RBD through VDW forces in both PT S/6L3-3P11K and BA.2.75 S/6L3-3P11K complex structures (Supplementary Fig. 10). As depicted in Supplementary Fig. 13, the side chain of 11K of the peptide might be potentially involved in forming strong hydrogen bonds with the adjacent RBD. To acquire assumed hydrogen bonds, we substituted lysine (K) to arginine (R) that has a longer side chain (Fig. 4a). As expected, 6L3-3P11R demonstrated a more potent neutralizing ability in comparison to 6L3-3P11K (Fig. 4d). Given that peptides containing arginine are susceptible to degradation by trypsin-like proteases in human tissues, we tested the resistance of 6L3-3P11R to trypsin enzyme. The result demonstrated that the antiviral activity of 6L3-3P11R dramatically decreased after trypsin treatment, with an IC50 of 2731 nM against pseudotyped BF.7 SARS-CoV-2 (Fig. 4a). To mitigate this drawback, we then synthesized another modified peptide 6L3-3P11hR which has a homoarginine (hR) instead of the arginine (Fig. 4b). As expected, 6L3-3P11hR was resistant to trypsin treatment and retained potent antiviral activity (Fig. 4b, d).

As the trimerization capacity of the peptide is important for the peptide inhibition, we substituted tyrosine (Y) with phenylalanine (F) at position 1 of the 6L3-3P11hR peptide to increase the hydrophobic trimerization interaction. Notably, 6L3-1F3P11hR peptide not only shows more potent antiviral activity against SARS-CoV-2 Omicron variants, but also acquires much better antiviral activity against the Prototype and Delta variants (Fig. 4c, d). The live virus inhibition assay further confirmed that 6L3-1F3P11hR could efficiently inhibit SARS-CoV-2 replication, including early strains (Prototype and Alpha) and recent Omicron strains (BA.2.12.1 and EG.5.1), with significant viral RNA reduction in supernatants observed even at 10 nM (Supplementary Fig. 14).

Inhibition efficacy of 6L3-1F3P11hR in the human nasal organoid model
Previous studies revealed that Omicron subvariants display growth advantages in human nasal tissues17. Therefore, we further used human nasal organoids to test the antiviral activity of two peptides. To recreate the three-dimensional, pseudostratified architecture of nasal epithelia in vivo, primary human nasal epithelial cells were pooled from three healthy donors and differentiated at the air-liquid interface (ALI). Differentiation status was confirmed by stratification of nuclei, the presence of mature cilia at the tissue surface, and goblet cells (Fig. 4e, left). The expression of ACE2 in the ALI was also analyzed (Fig. 4e, right). The results showed that both 6L3-3P11hR and 6L3-1F3P11hR markedly inhibited the replication of BA.2.12.1 and JN.1 viruses in the ALI culture. Notably, similar levels of inhibition were observed at both 10 μM and 100 μM concentrations, indicating that the peptides can effectively inhibit viral replication even at lower concentrations (Fig. 4f).

Chemical stability and pharmacokinetic characteristics of 6L3-1F3P11hR
We also evaluated the stability of the 6L3-1F3P11hR, including temperature (45°C) sensitivity, pH (pH 5.5–10.0) sensitivity, and mouse serum stability. The results showed that 6L3-1F3P11hR did not show significant alteration in chemical structure after exposure to 45°C for weeks or acidic/alkaline conditions (pH 5.5-pH 10.0) (Supplementary Fig. 15a, b). Over 80% of the original drug remained by 24 h after coincubation in mouse serum at 37°C (Supplementary Fig. 15c). These results indicate that 6L3-1F3P11hR exhibited a good chemical stability. We also performed additional NMR experiments and obtained the 1H-NMR spectrum of 6L3-1F3P11hR. The results are consistent with the chemical structure of 6L3-1F3P11hR (Please see the end of Supplementary information).

We then explored the in vivo pharmacokinetic characteristics of 6L3-1F3P11hR by intranasal administration in C57BL/6 mice (Supplementary Fig. 15d). The results showed that the peptide was mainly accumulated in the nasal region, and the peak concentration of the peptide can achieve 1529 ng/ml in the nasal lavage fluid. The exposure (AUC0-t) over 24 h was 4603 h*ng/ml, with a half-life (T1/2) of 6.45 h in the nasal lavage fluid. In contrast, the peptide concentration in the plasma was very low, with a peak concentration of 5 ng/ml. The exposure (AUC0-t) over 8 h was 18 h*ng/ml, with a half-life (T1/2) of 3.08 h in the plasma.

6L3-1F3P11hR reduces viral burden and tissue damage in vivo
To further demonstrate the in vivo efficacy of the macrocyclic peptide, we first explored the therapeutic effect of 6L3-1F3P11hR in SARS-CoV-2-infected K18-hACE2 mice (Fig. 5). The peptide 6L3-1F3P11hR (3 mg/kg per dose) was intranasally given one dose at 30 min after virus challenge and then every 24 h for 3 more days. Nirmatrelvir (100 mg/kg per dose, intragastric administration) was used as the positive control (Fig. 5a). As shown in Fig. 5b, the viral RNA load and infectious virus titer in the lungs of the mice were significantly reduced after treatment with either 6L3-1F3P11hR or nirmatrelvir. Mice treated with either 6L3-1F3P11hR or nirmatrelvir exhibited significantly lower histopathological scores compared to solvent-treated controls (Fig. 5c)18. Histopathological changes seen in solvent-treated control mice, including alveolar inflammation and congestion, alveolar hemorrhage, bronchiole luminal cell debris, endothelium infiltrates, and perivascular or peribronchiolar infiltrates, were scarcely observed in mice that received 6L3-1F3P11hR or nirmatrelvir (Fig. 5d). We further tested the therapeutic efficacy of 6L3-1F3P11hR (6 mg/kg per dose) which was administrated at 8 h post virus challenge, followed by subsequent doses every 24 h for three additional days (Supplementary Fig. 16). The infectious virus titer in the lungs of the mice was also significantly reduced.

We then explored the prophylactic efficacy of 6L3-1F3P11hR. The antiviral effect of 6L3-1F3P11hR was more significant when given prophylactically (3 mg/kg one dose at 30 min prior to virus challenge) (Fig. 6). The SARS-CoV-2 RNA load and infectious virus titer in the nasal turbinate and lung were significantly reduced (Fig. 6b, d). The mice treated with 6L3-1F3P11hR displayed much lower histopathological scores compared to the untreated mice (Fig. 6c). Histopathological changes of the lung were also obviously alleviated in the peptide-treated group (Fig. 6e).

Methods
Cells
HEK293T cells (ATCC CRL-3216), Vero cells (ATCC CCL81), Vero E6-TMPRSS2 cells (JCRB Cat# JCRB1819), and BHK-21 cells (ATCC CCL-10) were grown at 37°C in a humidified incubator with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FBS), 1‰ penicillin and 1‰ streptomycin. Freestyle 293F cells were cultured in SMM 293-TII medium at 37°C in a shaker with 5% CO2.

Viruses
SARS-CoV-2 isolate hCoV-19/USA/NY-MSHSPSP-PV56475/2022 (Lineage BA.2.12.1; Omicron BA.2 variant; NR-56782) and hCoV-19/USA/MSHSPSP-PV96109/2023 (Lineage JN.1, Omicron variant; NR-59694) were obtained from BEI Resources. SARS-CoV-2 Omicron subvariant BA.1.529.2/BA.2 (GISAID: EPI_ISL_9845731) was used in the animal experiments32. SARS-CoV-2 isolates PT HKU-001a (GenBank: MT230904), B.1.1.7/Alpha (GenBank: OM212469), BA.2.12.1 (GISAID: EPI_ISL_13777659), BA.5.2 (GISAID: EPI_ISL_13777658), EG.5.1 (GISAID: EPI_ISL_18461518) were used in the live virus inhibition assay.

Ethics statement
All procedures in this study involving SARS-CoV-2 infection were performed in the biosafety level 3 (BSL-3) and animal biosafety level 3 (ABSL-3) facilities according to approved standard operating procedures. All the procedures in the animal studies were reviewed and approved by the Committee on the Use of Live Animals in Teaching and Research at The University of Hong Kong (CULATR 5370-20) or Laboratory Animal Welfare and Ethics Committee (PK-R-06012024).

Animals
The use of animals at HKU was approved by the HKU Committee on the Use of Live Animals in Teaching and Research. K18-hACE2 mice (aged 8–10 weeks, male) were obtained from the HKU Centre for Comparative Medicine Research. Six to eight-week-old male C57BL/6 mice were purchased from Vital River. The animals were kept in cages with individual ventilation with 65% humidity and ambient temperature ranging between 21 and 23°C with a 12-h-interval day/night cycle for housing and husbandry before transferral to the Biosafety Level 3 facility.

Protein expression
The coding sequences (codon optimized) for the ectodomain of SARS-CoV-2 Prototype S and the Omicron BA.2.75 S subvariant (containing “6P” stabilizing mutations) were fused at their C-termini with a trimeric tag followed by a Strep-II tag. These sequences were cloned into the pcDNA3.1 expression vector. The coding sequences (codon optimized) of Omicron subvariant BA.2 RBD with C-terminal His-tag and Avi-tag were cloned into the pCAGGS vector. The recombinant plasmids were transfected into Freestyle 293F cells to express the RBD, S proteins. Five days later, the supernatants were collected, and soluble proteins were purified by Ni affinity chromatography using a HisTrap EXCEL 5-mL column (GE Healthcare). S protein was further purified via gel filtration chromatography with Superose 6 Increase 10/300 GL column (GE Healthcare) in a buffer composed of 20 mM Tris-Base and 150 mM NaCl (pH 8.0). RBD protein was purified via gel filtration chromatography with a Superdex 200 column (GE Healthcare) in 1× phosphate-buffered saline (PBS). RBD protein was further biotinylated using a Biotin Labeling Kit (Beyotime); biotinylated RBD was used for macrocyclic peptides selection. The coding sequences of RBDs with human IgG Fc-tag (hFc) (codon optimized) were also cloned into the pCAGGS vector, including Prototype, Delta, Omicron subvariants (BA.2, BF.7, BQ.1.1, XBB, and XBB.1.5), as well as three sarbecoviruses (SARS-CoV, RaTG13, and GD/1/2019). The coding sequence of ACE2 with hFc (ACE2-hFc, codon optimized) was also cloned into the pCAGGS vector. The recombinant plasmids were transfected into mammalian 293T cells to express the RBD-hFc and ACE2-hFc. Five days later, the supernatants were collected and used in SPR or BLI assays.

Selection of macrocyclic peptides against RBD
Macrocyclic peptides that target the RBD were screened using the RaPID system as previously reported. Initially, the mRNA library was prepared with the sequence AUG-(NNK)8-12-(GGC-AGC)3-UAG and linked to a puromycin (DNA-PEG-CC-Pu) linker utilizing T4 RNA ligase. This process resulted in the creation of the mRNA-Pu library. The resultant mRNA-Pu library was then subjected to the RF1 (release factor 1) and Met (methionine)-deficient FIT (flexible in vitro translation) system, which contained 50 μM ClAc-L(or D)Tyr-tRNAfMetCAU, with a total volume of 25 μL for the initial selection round. Following a 2 h reaction at 37°C, the solution was incubated at 25°C for 15 min, followed by quenching through the addition of 5 μL of 100 mM EDTA (pH 8.0). The mixture was further incubated at 30°C for 30 min. Subsequently, the library underwent reverse transcription by incubating the mixture at 42°C with the addition of 0.25 mM dNTPs, 2 μM CGS3an13.R39, 25 mM Tris-HCl (pH 8.3), 15 mM Mg(OAc)2, 10 mM KOH, and 50 U M-MLV reverse transcriptase RNase H Minus. After 60 min, an equal volume of blocking solution was added, and the resulting mixture was employed for affinity selection against RBD-immobilized Dynabeads™ M-280 Streptavidin at 4°C for 60 min in the presence of 20 μM peptide 4 (a blocking peptide). The beads were then washed three times with the selection buffer and subsequently heated at 95°C for 5 min with 1× PCR mix to elute cDNAs. Quantification of the resulting cDNAs was performed using the QuantStudio™ real-time PCR system (Applied Biosystems), followed by PCR amplification and transcription into mRNAs to generate the library for the second round of selection. In the subsequent rounds, the translation scale was reduced to 5 μL, and the pre-clear steps were repeated multiple times by adding the resulting mixture to fresh beads to selectively remove the peptides that bound to the magnetic beads (negative selection). The supernatant from the final negative selection was incubated with RBD-immobilized Dynabeads™ M-280 Streptavidin in the presence of 20 μM peptide 4 to selectively bind cDNAs that interacted with the RBD (positive selection). This selection process was iterated for several rounds until a substantial enrichment of cDNAs binding to the RBD was observed. Finally, the enriched sequences were recovered and identified through deep sequencing utilizing the NovaSeq 6000 system (Illumina).

Chemical synthesis of macrocyclic peptides
The synthesis of macrocyclic peptides was conducted following the standard Fmoc SPPS protocol. Initially, 0.5 g of Rink Amide MBHA resin was treated with a 20% piperidine in dimethylformamide (DMF) solution to remove the Fmoc protecting group. Subsequently, a solution containing 0.3 mmol of Fmoc-Gly-OH (0.3 mmol of 1-Hydroxybenzotriazole (HOBT)), and 5% N, N’-diisopropylcarbodiimide (DIC) in 10 mL of DMF was added to the resin, and the mixture was incubated under nitrogen gas bubbling conditions for 1.5 h. The resin was thoroughly washed three times with DMF and dichloromethane (DCM) before deprotecting the Fmoc group using a 20% piperidine in DMF solution. To couple the subsequent amino acids, a freshly prepared solution of 0.9 mmol Fmoc-AA-OH, 0.9 mmol HOBT, and 9% DIC in 10 mL of DMF was employed, and the coupling reaction was conducted for 1 h under nitrogen gas bubbling conditions. These deprotection and coupling steps were repeated iteratively until the desired peptide length was achieved. The peptides were then conjugated to their free N-terminal α-amino group using the bromoacetyl group and subsequently cleaved from the resin. The resulting peptides were precipitated with diethyl ether (Et2O), re-dissolved in dimethylsulfoxide (DMSO), and subjected to thiol-ether cyclization by incubating the solution at pH 8.0 for 1 h. Finally, trifluoroacetic acid (TFA) was added to acidify the solution, and the peptides were purified using RP-HPLC with a mobile phase consisting of a linear gradient of 0.1% TFA aqueous solution and 0.1% TFA in acetonitrile (MeCN; mobile phase B). The characterization of synthesized macrocyclic peptides was conducted by employing ESI-MS, while their purity confirmation was achieved through analytical HPLC analysis.

Surface plasmon resonance (SPR) assay
The binding affinities of 6L3-3P or 6L3-3P11K with RBDs from SARS-CoV-2 and its variants (Delta, Omicron subvariants BA.2, BF.7, BQ.1.1, XBB, XBB.1.5, and BA.2.86), as well as three sarbecoviruses (SARS-CoV, RaTG13, and GD/1/2019), were evaluated by SPR analysis. The experiments were performed using a BIAcore 8000 system (GE Healthcare) with protein A chips (Cytiva Life Sciences) at 25°C in single-cycle mode. All proteins used for kinetic analysis were exchanged into the PBST buffer (2.7 mM KCl, 137 mM NaCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, and 0.5‰ (v/v) Tween 20) with 5% DMSO. The supernatants containing hFc-tagged RBD proteins were captured by the protein A chip at more than 3000 response units. Then, serial dilutions of 6L3-3P or 6L3-3P11K were injected over the chip surface to test binding. The ka, kd, and KD for each pair of interactions were calculated with BIAcore 8K evaluation software (GE Healthcare).

Bio-layer interferometry assay
The supernatants containing ACE2-hFc proteins were captured by the protein A sensors. The BA.2.75 S trimer was incubated with either 6L3-3P or 6L3-3P11K peptides at a molar ratio of 1:5 for 10 min, and incubated with the 6L3 peptide for 2 h. The binding ability of BA.2.75 S or BA.2.75 S/peptide complexes to ACE2 were tested using the Octet RED96 system (FortéBio).

Pseudovirus preparation
The VSV-ΔG-GFP-based pseudoviruses were prepared as previously described33–35. The coding sequence for the spike protein of the tested viruses with 18 amino acids truncated at the C-terminal was synthesized and cloned into the pCAGGS vector, respectively. Briefly, 30 μg of the plasmids were transfected into HEK293T cells. After 24 h, the VSV-ΔG-GFP pseudovirus was added to the cells. Then the cell culture medium was removed and replaced with fresh DMEM containing 10 μg mL−1 of anti-VSV-G antibody (I1-Hybridoma ATCC® CRL-2700™) after 2 h of incubation. After 30 h, the supernatant was collected, centrifuged, and filtered through a 0.45 μm filter to remove cell debris. The pseudoviruses were frozen at −80°C until use. Vero cells or HEK293T cells were used to determine the pseudovirus titers, and BHK-21 cells were used to detect the VSV-ΔG-GFP pseudovirus residue.

Pseudovirus neutralization assay
For the neutralization assay, 1×104 of Vero cells were plated into each well of a 96-well plate 24 h before infection. The peptides were three-fold serially diluted starting from 100 mM with DMEM, then the diluted peptides were incubated with an equal volume of supernatant containing 1000 fluorescence focus units of pseudovirus for 1 h at 37°C. Then, the mixture was added to the Vero cells. Fifteen hours later, the infected cells were measured using the CQ1 Confocal Quantitative Image Cytometer (Yokogawa). The half maximal inhibitory concentration (IC50) was calculated using GraphPad Prism 8.0.

The live virus inhibition assay
6L3-1F3P11hR were serially diluted and mixed with SARS-CoV-2 (Prototype, B.1.1.7/Alpha, BA.2.12.1 and EG.5.1) for 1 h of incubation. The mixtures were then added to Vero E6-TMPRSS2 cells with three repeats and incubated for 3 days. The supernatant samples were collected for RNA extraction and quantitative RT-qPCR.

Culture and differentiation of human primary nasal epithelium at the air-liquid interface
Primary nasal epithelial cells (Epithelix) from three healthy donors were pooled and cultured in hAEC medium (Epithelix) until confluency. The cells were then transferred to 6.5 mm Transwell® inserts with 0.4 µm Pore Polyester Membranes (Corning) in 24-well plates. After reaching confluency in the Transwells, the apical medium was removed, and PneumaCult™-ALI Medium (STEMCELL Technologies) was introduced to induce differentiation. The cells were cultured at the ALI for 28 days to fully differentiate into nasal epithelium. Following differentiation, the nasal ALI cultures were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and stained with anti-ACE2 (Abcam; ab15348), anti-Acetyl-α-Tubulin (Lys40) (D20G3) (Cell Signaling; 5335), anti-MUC5A (Thermo Fisher, MA5-12178), anti-F actin (Invitrogen, A12379), and DAPI to evaluate ACE2 expression, the presence of ciliated cells, the presence of goblet cells, cytoskeleton and nuclear pseudostratification, respectively. Images were captured using a Leica Stellaris 5 LSM microscope and processed into 3D reconstructions via volume rendering using Imaris software.

Production of SARS-CoV-2 variants and infection of nasal ALI
SARS-CoV-2 isolates hCoV-19/USA/NY-MSHSPSP-PV56475/2022 and hCoV-19/USA/MSHSPSP-PV96109/2023 were preincubated with macrocyclic peptides at final concentrations of 10 µM or 100 µM. A 100 µL aliquot of the virus-peptide mixture was then added to the apical chamber of the Transwell insert. Inoculation was conducted at 37°C with an initial viral load of 10,000 TCID50. Following 1 h of incubation, the viral inoculum was removed, and the cells were rinsed with medium. Twenty-four hours post-infection, the cells were homogenized in Trizol-LS (Sigma) for total RNA extraction. Quantification of viral RNA was performed by targeting the ORF1a region36.

In vivo evaluation of 6L3-1F3P11hR
To evaluate the in vivo antiviral activity of 6L3-1F3P11hR, 8 to 10-week-old male K18-hACE2 mice (n=5 per group) were intranasally challenged with 4×103 plaque-forming units of SARS-CoV-2 Omicron BA.2 at day 0. 6L3-1F3P11hR (3 mg/kg per dose, dissolved in PBS with 2% DMSO and 0.25% methylcellulose) was administered intranasally prophylactically (given one dose at 30 min before virus challenge) or therapeutically (given one dose at 30 min after virus challenge and then every 24 h for 3 more days, a total of four doses were administered). Control groups included solvent-treated mice (PBS with 2% DMSO and 0.25% methylcellulose per dose) and nirmatrelvir-treated mice (100 mg/kg per dose, intragastric administration) as negative and positive controls, respectively. At 4 days post-infection (dpi), the mice were sacrificed, and the nasal turbinate and lung were harvested for virological and histological analyses. The therapeutic efficacy of the peptide administered 8 h after virus challenge was also evaluated. 6L3-1F3P11hR (6 mg/kg per dose, dissolved in PBS with 2% DMSO and 0.25% methylcellulose) was administered intranasally at 8 h after virus challenge and then every 24 h for 3 more days; a total of four doses were administered. Control groups included solvent-treated mice (PBS with 2% DMSO and 0.25% methylcellulose per dose) and nirmatrelvir-treated mice (100 mg/kg per dose, intragastric administration) as negative and positive controls, respectively. Solvent and nirmatrelvir were administered using the same method as previously described. The animal experimental scheme figures were created with BioRender.com.

RNA extraction and quantitative RT-qPCR
The RNA extraction and RT-qPCR were performed as previously described37. SARS-CoV-2 genome copies were quantified with sequence-specific probes and primers targeting the Envelope gene. E_Sarbeco_F1, (SARS-CoV-2), forward primer: 5′-CGATCTCTTGTAGATCTGTTCTC-3′ E_Sarbeco_R2 (SARS-CoV-2), reverse primer: 5′-ATATTGCAGCAGTACGCACACA-3′ E_Sarbeco_P1 (FAM) Probe (SARS-CoV-2): 5′-FAM-ACACTAGCCATCCTTACTGCGCTTCG-ZEN-IBHQ-3′

Infectious virus titration by plaque assays
Plaque assays were performed as previously described37. The nasal turbinate and lung harvested from virus-inoculated animals were homogenized and followed by centrifugation. Tissue homogenates were then 10-fold serially diluted with DMEM and incubated with Vero E6 cells for 2 h. After inoculation, cells were washed and overlaid with 1% low-melting agarose containing 1% FBS. Cells were further incubated for 72 h and fixed with 0.5% crystal violet for visualizing plaque formation.

Immunofluorescence staining
Lung tissues from mice were harvested and fixed with formalin. Nucleocapsid of SARS-CoV-2 was detected by rabbit polyclonal anti-SARS-CoV-2 nucleocapsid antibody as previously reported38. The cell nuclei were stained with DAPI (Thermo Fisher Scientific) before the tissue sections were mounted with the Diamond Prolong Antifade mounting buffer (Thermo Fisher Scientific). Images were acquired with the Olympus BX53 light microscope.

Cryo-EM sample preparation and data acquisition
To prepare the cryo-EM sample of S-6L3-3P11K, the SARS-CoV-2 Prototype and Omicron BA.2.75 S protein sample was diluted with purification buffer (20 mM Tris-Base, 150 mM NaCl, pH 8.0), and then mixed with 6L3-3P11K at a molar ratio of protein:peptide of 1:3, respectively. After incubation for 1 h on ice, the complex was applied to a glow-discharged graphene-coated grid (R1.2/1.3), which was blotted for 3 s with a humidity of 100% at 4°C, and then plunged into liquid ethane using an FEI Vitrobot Mark IV. The well-prepared cryogenic specimens were transferred onto an FEI Titan Krios transmission electron microscope for data collection. Cryo-EM micrographs were automatically collected using Serial-EM software using a beam-image shift imaging scheme. Images were recorded with a K3-subunit detector using the super-resolution counting mode at a calibrated magnification of 22,500, corresponding to a pixel size of 1.07 Å. The exposure was performed with a dose rate of 20 e−1 pixel−1 s−1 and an accumulative dose of 60 e−1 Å2 for each micrograph, which was fractionated into 32 frames. The defocus range of this dataset was roughly −1.5 to −2.6 μm.

SARS-CoV-2 Prototype and Omicron BA.2.75 S protein samples were diluted with purification buffer (20 mM Tris-Base, 150 mM NaCl, pH 8.0) and were applied to a glow-discharged quantifoil copper grid (R1.2/1.3), which was blotted for 6 s with a humidity of 100% at 4°C, and then plunged into liquid ethane using an FEI Vitrobot Mark IV. The well-prepared cryogenic specimens were transferred onto an FEI Titan Krios G4 transmission electron microscope for data collection. Cryo-EM micrographs were automatically collected using EPU software at a nominal magnification of 105,000× (calibrated pixel size of 0.825 Å per pixel) with a defocus range of −0.8 to −2.0 μm. The exposure was performed with a dose rate of 12 e−1 pixel−1 s−1 and an accumulative dose of 50 e−1 Å2 for each micrograph, which was fractionated into 40 frames.

Image processing, model building, and structure refinement
The movie frames were aligned using MotionCor239, and the contrast transfer function (CTF) values of each micrograph were determined using CTFFind440. Fifty micrographs were selected for automatic particle picking using Laplacian-of-Gaussian blob detection, and were subjected to 2D classification to generate templates for autopicking against the entire dataset. All subsequent classification and reconstruction procedures were performed using cryoSPARC41. After 2D classification, particles with good qualities were selected for global 3D reconstruction and then subjected to homogeneous refinement. We performed manual model building to improve local fit using COOT42. The stereochemical quality of each model was assessed using MolProbity43. Structural figures were prepared with Pymol (https://pymol.org/) and ChimeraX44.

Effects of temperature, serum, and pH on the chemical stability of the peptide
The effects of temperature, serum, and pH on the stability of the peptide were explored. The peptide was treated with heating at 45°C, mouse serum, and different pH levels, respectively. The peptide was heated at 45°C for different time intervals, i.e., 1, 2, 3, and 4 weeks, and then analyzed by HPLC to determine the stability of the peptide at 45°C. The peptide was incubated with various pH buffers (pH 5.5, 7.5, 8, 9, and 10) at room temperature for 24 h, respectively. Sodium acetate buffer was used for the preparation of a pH 5.5 buffer. PBS was used for preparing pH buffers at 7.5, 8, 9, and 10. Then, the clear supernatant was obtained by centrifuging the sample and further analyzed by HPLC. For the serum stability test, 2.5 μL of 2.5 mM peptide solution was added to 47.5 μL of serum solution prepared by mixing 10 μL of 1× phosphate buffer with 37.5 μL of mouse serum to obtain the reaction mixture (125 μM peptide), and incubated at 37°C. At the specified time points (0, 0.5, 1, 2, 4, 8, and 24 h), a 50 μL aliquot of the peptide-serum mixture was combined with 0.6 μL of 17.5 mM acetaminophen and 50 μL of acetonitrile containing 3% trifluoroacetic acid (TFA). The reaction was cooled to 4°C for 5 min and then added to 100 μL of 20% trichloroacetic acid (TCA). After incubation at 4°C for 5 min, the mixture was centrifuged at 18,700×g for 5 min to precipitate the serum proteins. The supernatant was used to determine the concentration of peptides by HPLC. During HPLC analysis, water (0.1% TFA) and acetonitrile (0.1% TFA) were mixed in a linear gradient (10–85% acetonitrile in 30 min), with a flow rate of 1 mL/min, and the absorbance was detected at 210 nm. The concentrations of the remaining soluble peptides were calculated by peak integration relative to the initial peak integration (0 h).

Pharmacokinetic study
Male C57BL/6 mice were used to assess the pharmacokinetics. C57BL/6 mice were dosed at an age of 6–8 weeks and were provided free access to food and water prior to dosing. Mice were injected intranasally with a dose of 3 mg/kg 6L3-1F3P11hR. Blood and nasal lavage fluid were collected at each time point (2 min, 5 min, 15 min, 30 min, 1, 4, 8, and 24 h). Approximately 0.02 mL of blood was collected. Blood from each sample was transferred into plastic microcentrifuge tubes containing anticoagulant EDTA-K2 and mixed well with the anticoagulant. The blood samples were centrifuged at 4000×g for 5 min at 4°C to obtain plasma. For nasal lavage fluid collection, 1 mL of normal saline was slowly infused via the nasopharynx after locating the nasal opening (two repetitions). Rinse solutions were collected in EP tubes. Then, the samples were immediately frozen in the upright position and stored at −80°C prior to analysis. Concentrations of 6L3-1F3P11hR in the plasma and nasal lavage fluid were analyzed using an LC-MS/MS method. WinNonlin (Phoenix™, version 8.4) software was used for pharmacokinetic calculations.

Peptide oligomerization analysis by LC-MS in solution
To explore the mass of peptides, the standard stock solutions and synthesized sample were diluted to a 0.5 μg/μL (6L3-3P11K) in 50 mM Ammonium acetate (pH 7.48). 10 μL of sample was injected onto an ACQUITY UPLC H-Class PLUS System configured with a BEH SEC 200 Å 1.7 μm, 2.1×150 mm Column (Waters, American). The mobile phase was 50 mM ammonium acetate in water. Chromatographic separation was achieved using an isocratic rate of 0.065 mL/min with a total runtime of 10 min and a 25°C column temperature. MS experiments were performed using a SYNAPT XS Mass Spectrometer with a standard electrospray ionization (ESI) source (Waters, American). The MS parameters were set as follows: positive ESI mode, capillary voltage 2.0 kV, cone voltage 30 V, source heating temperature 125°C, desolvation temperature 250°C, cone gas flow 50 L/h, desolvation gas flow 600 L/h, mass/charge (m/z) range 50–3000, Scan Time 0.5 s. Lock mass data was collected during each acquisition, and the correction was applied during post-acquisition data processing via MassLynx Software v4.1 (Waters, American). Deconvolution of mass spectra was performed using MaxEnt3 in UNIFI software v3.1 (Waters, American). Deconvolution was performed in the 0–12,500 Da range, and a target mass of ~1462 Da. The mass of the peptide was compared to the standard mass to detect any mass modification and get additional information about the characterized compounds.

Statistical analysis
All statistical tests were performed as described in the indicated figure legends using GraphPad Prism software. The number of independent experiments performed is indicated in the relevant figure legends. No samples were excluded from the analysis.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, the authors characterize the macrocyclic peptide 6L3 and its optimized variants, 6L3-3P11K and 6L3-1F3P11hR. These peptides exhibit potent neutralizing activity against a broad range of SARS-CoV-2 variants. Structural analysis reveals that the peptides function as “molecular glue,” binding to cryptic sites on the RBDs of the SARS-CoV spike protein. This interaction stabilizes the RBDs in a closed conformation, thereby preventing the spike protein from binding to the human ACE2 receptor. Additionally, both in vitro and in vivo experiments demonstrate significant prophylactic and therapeutic efficacy against SARS-CoV-2. Overall, this study presents a promising peptide inhibitor with strong therapeutic potential against SARS-CoV-2.\nSpecific Comments:\n1. The structural explanation of the mechanism of the peptides primarily relies on its ability to induce the \"closed\" conformation of the RBDs on the spike protein. However, in its prefusion state, the spike protein can naturally adopt a 3-down conformation. Could the authors provide a quantitative analysis of how the peptides increase the proportion of down RBDs compared to the unbound spike protein?\n2. The trimeric conformation of the peptides appears to play a crucial role in stabilizing the closed state of the three RBDs. Could the authors provide additional details on the intra-peptide interactions at the trimer interface? Furthermore, are these peptides intrinsically trimeric even in the absence of spike protein binding?\nMinor points:\n1. Lines 68-69: “it does not significantly alleviate symptoms of COVID-19 compared with placebo among nonhospitalized adults[10].” differs slightly but importantly from the description in Reference 10, which states, “Nirmatrelvir–ritonavir was not associated with a significantly shorter time to sustained alleviation of Covid-19 symptoms than placebo.” The authors may wish to reword this sentence to ensure it accurately reflects the findings of the cited study.\n2. Lines 125-126. The authors initially selected mutations to improve binding, including L3P, T11F, T11I, T11K, L3PT6IP7Q, and L3PT11K. Providing additional details on the rationale behind these mutation choices would help readers better understand the design strategy.\n3. Line 130: “6L3-11K also demonstrated enhanced antiviral ability, with an IC50 value of 1.03 μM”. However, in Extended Data Fig. 2b, the IC50 is reported as 676 nM.\n4. Figs. 1b and 1d. The number of decimal places should be made consistent across these figures for uniformity.\n5. Fig. 1b and 1d, the peptides exhibit stronger neutralization to Omicron BA.2 and later variants, compared to BA.1 and earlier variants. It would be helpful if the authors could provide an explanation.\n6. Figs. 3b and 3f, The rotamers of 4I and 8L differ when bound to PT and BA.2.75 – why is that?\n7. Did the authors introduce any modifications to the spike protein to enhance stability and expression, such as S2P, HexaPro, or the Foldon domain?\n8. Extended Data Fig. 6. The figure title is \"BA.2.75 S,\" but in Extended Data Table 1, the spike protein appears to be labeled as BA.2 (right two columns).\n9. Extended Data Fig. 6e. The peptide does not appear to fit well into its density. Were the BA.2.75 S/6L3 (all RBDs up and two RBDs up) structures refined? If so, the authors may want to include the refinement statistics in Extended Data Table 1. Otherwise, please provide an explanation and discuss the reliability of the structures presented in Extended Data Fig. 6.\n10. Extended Data Table 1. The symmetry of the \"Two RBDs up\" structure should not be C3, as the protomers are not symmetrical. The final particle image numbers for “BA.2.75 S 6L3-3P11K” and “BA.2 S/6L3 (All RBDs up)” are identical (224,700) in both Extended Data Table 1 and Extended Data Figs. 4 and 6. Is this a coincidence? Should the FSC threshold (0.143) be applied to all four structures?", "reviewer_2": "This study presents macrocyclic peptides as potential therapeutics against SARS-CoV-2. Your work on peptides against Spike offers valuable insights into structure-based inhibition of the spike protein and highlights an innovative approach for antiviral drug development.\nHowever, there are several concerns regarding the presented data, interpretations, and claims that require further clarification and supporting evidence.\n1. The structural models built on the cryo-EM data appear inconsistent. In Figure 3, the side chain of lysine 11 (K11) is shown as not participating in interactions with either the prototype or BA.2 RBDs. However, in Supplementary Figure 9, this residue is depicted in close proximity to Arg408, Gln414, and Thr415 of the RBD.\nAdditionally, both Arg408 in the RBD and Lys11 (K11) in the peptide are positively charged, leading to a steric clash (repulsion). Despite this, the distance between these two residues is reported as 3.2 Å, which requires further clarification.\nMoreover, the entire “Structure-based modification of the 6L3-3P11K peptide” is based on the K11R substitution, which further intensifies the clash between Arg408 and Arg11 in the 6L3-3P11hR peptide.\nFigure 3 Figrue S9\n2. The authors state, “This difference correlates with the relatively lower binding affinity of 6L3-3P11K to PT RBD, as the PT RBD needs to alter the conformation of the 370-loop and overcome a higher energy barrier to bind to the 6L3-3P11K peptide.” However, no evidence is provided to support this claim.\n3. The authors state, “As the trimerization of the peptide is important for peptide inhibition, we substituted tyrosine (Y) with phenylalanine (F) at position 1 of the 6L3-3P11hR peptide to enhance trimerization interactions.” To substantiate the peptide trimerization findings, the oligomerization of these peptides should be evaluated in the absence of RBD. If oligomerization is low or absent, it suggests that the peptides oligomerize upon binding their target, where they likely find a symmetric cavity within the closed conformation of the Spike protein.", "reviewer_3": "Wang et al report the discovery and characterization of macrocyclic peptides that bind to the spike protein of SARS-CoV-2 and thereby exert an antiviral effect in infection models of SARS-CoV-2. The peptides were discovered by an affinity-based screening approach using the innovative RaPID technology. Initial actives were refined by solid phase peptide synthesis, driven by structural information on their binding mode that was disclosed by Cryo-EM. The peptides form trimers and lock the RBD of the spike protein in a locked conformation. They exert activity against pseudotyped viruses in the low nanomolar range and reduce viral load in an organoid model of primary nasal epithelial cells and in a mouse infection model after intranasal administration.\nOverall, the study reports several important findings. It demonstrates how the complex spike protein, a target for monoclonal antibodies, can be inhibited by a mid-sized macrocyclic peptide with high potency. Also the structure-based refinement is a showcase for the optimization of peptide drugs. The relevance of the findings for drug development is somewhat limited by the fact that the peptides probably require an intranasal administration, due to the lack of oral bioavailability.\nSeveral aspects of the study deserve a closer examination to meet the standards of Nat Commun:\n- The traceability of the findings is insufficient, and the methods section is incomplete.\n- Most importantly, the chemical characterization of the peptides is insufficient. The authors provide a general protocol for solid phase peptide synthesis, but information on the individual compounds is missing. At least for the compounds that were investigated closely (6L3-3P11K and others), the authors need to provide quantities and yields of the isolated final product. This is important to assess the scalability of the synthesis. Analytical data are also missing. This includes HPLC, HR-MS and NMR data.\n- I do not find information on how ACE2 has been obtained for Extended Data Fig 5.\n- The authors claim that thermal stability is an advantage of 6L3-1F3P11hR and show the IC50 after heating to 160°C (Extended data Fig 10). There is no information on how this experiment was performed, e.g. on the duration of the heating period. Moreover, a 160°C condition is artificial, and I encourage the authors to demonstrate a chemical and metabolic stability of the drug under standard conditions (pH stability, stability in plasma or ELF matrix, 45°C stability over weeks, etc).\n- Figure 1: There is a discrepancy between very different antiviral activities (e.g. against XBB vs BA2.86) in spite of highly similar binding affinities. How do the authors explain this?\n- Most antiviral data were obtained with pseudotype assays. I missed a ‘standard’ antiviral assay on a single infected cell line (e.g. A549-expressing ACE2/TMPRSS2 or Vero E6) with 1-2 SARS-CoV-2 viruses. Although the organoid assay is more complex and potentially more relevant, it is easier to understand the potency of the inhibitors in such a standard assay format (with appropriate positive controls such as nirmatrelvir).\n- With respect to administration routes, is an iv administration an option? To show whether the compounds qualify as a hit or even lead, information on their pharmacokinetic (iv admin), ADME and physicochemical properties (e.g. solubility) would be relevant.\nMinor points\n- Figure 4e: I believe that the left and right panels were confused in the caption.\n- Figure 1a and 1c are too small and cannot be deciphered after being printed on a high-quality laser printer in DIN A4 format. Same is true for Ext. Data Fig. 1b.\n- Figure 3b: Have the labels for 8L and 5L been confused?\n- line 95: Define RBM\n- Extended Data Fig 2a: Redraw the structures and avoid overlapping atoms.\n- line 130: An IC50 of 1.03 µM is mentioned, but the Figure gives a value of 676 nM.\n- line 131: I suggest rounding to 12 nM.\n- I assume that the ‘prototype’ virus is the original one isolated from Wuhan? Is ‘prototype’ an accepted term?\n- line 204: The ‘correlation’ is in the ‘right’ direction, but can the differences in the loop conformation really account for the 100fold difference in potency?\n- line 261-263: The reference to the panels 5d and 5e has been confused.\n- Discussion: I suggest removing words describing subjective assessments like ‘exciting’ or ‘excellent’\n- line 274: The authors compare their peptides to a previously reported one, but I cannot see any data on a head-to-head comparison.", "reviewer_4": "In this manuscript, Wang M. et al, explored the in vitro and in vivo efficacy of new macrocyclic peptide against different Sarbecoviruses where the peptide showed potent and promising activity against omicron subvariants in different assay systems. Overall, the manuscript is well written and data interpretation is matching the conclusion. However, I have some comments especially for the in vivo studies.\nMajor comments:\nA) The methodology of the in vivo study is lacking important information:\n1. number of animals per each group\n2. if the prophylactic and therapeutic studies are 2 independent experiments or were run together\n3. Nirmatrelvir administration schedule, was it 100 mg/kg once daily or twice daily? was it also given prophylactically and therapeutically in the same time frame as the peptide (the data in Fig 5 mention that but not mentioned in methods)\n4. the information on how histopathology scoring was done is missing (how many parameters were checked and what was the scoring scale)\n5. Is there an ethical approval (project number) for the in vivo work? nothing mentioned in the methods section about ethics.\nB) 30 min after infection is a very short time to consider that the peptide has therapeutic potential (30 min is more like post-exposure prophylaxis rather than therapeutic). Was the therapeutic efficacy of the peptide tested at longer period after infection e.g. 8h or 24h post infection?\nC) is any information known about the stability of the peptide in the nasal cavity of mice?\nD) Figure 5 panels have low resolution, it is also not clear in the legend whether the data presented are median or mean\nOther comments:\nA) was he peptide tested against seasonal coronaviruses e.g. OC43 and 229E?\nB) from the sequence alignment shown in extended Figure 11, is it possible to explain/suggest why the peptide is less active against non-omicron SARS-CoV-2 and other Sarbecoviruses?\nMinor comment: the manuscript needs to be more concise (words count to be reduced)"}, "author_response": "Response: We must clarify that the mechanism of the peptides did not rely on its ability to induce the “closed” conformation of the RBDs on the spike protein, but to lock the “closed” conformation of RBDs. The “up” or “down” conformations of the RBDs are inherent dynamic features for the spike protein, and the peptide binding of RBDs did not induce the “closed” conformation. When the RBDs move to the “down” conformation, the trimerization of the peptides can keep the “closed” conformation of the RBDs on the spike protein and prevent the occurrence of “up” conformation of the RBDs. We have performed the quantitative analysis in the cryo-EM datasets of the unbound PT spike trimer (PDB: 6VXX) and the peptide-binding PT spike trimer, and found that the percentage of spike trimer with 3-down conformation can increase from 47% to 96.4%. Please see Supplementary Table 1 in the paper (Walls AC, Park YJ, Tortorici MA, Wall A, McGuire AT, Veesler D. Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell. 2020 Apr 16;181(2):281-292.e6. doi: 10.1016/j.cell.2020.02.058.) and Extended Data Fig. 4 in our manuscript.\n2. The trimeric conformation of the peptides appears to play a crucial role in stabilizing the closed state of the three RBDs. Could the authors provide additional details on the intra-peptide interactions at the trimer interface? Furthermore, are these peptides intrinsically trimeric even in the absence of spike protein binding?\nResponse: The intra-peptide interactions stabilizing the trimer interface are presented in the Extended Data Fig. 7b and 7e. Structural analysis reveals that residues 1Y, 3P, 4I, and 7P form the most interactions, predominantly mediated by hydrophobic interactions and van der Waals (VDW) forces. We also characterized the oligomerization state of 6L3-3P11K using native SEC-Mass method. The results demonstrated that 6L3-3P11K exists exclusively as a monomer in the absence of the spike protein binding and confirmed that peptide trimerization occurs specifically upon S protein binding. Please see Extended Data Fig. 7.\nMinor points:\n1. Lines 68-69: “it does not significantly alleviate symptoms of COVID-19 compared with placebo among nonhospitalized adults [10].” differs slightly but importantly from the description in Reference 10, which states, “Nirmatrelvir–ritonavir was not associated with a significantly shorter time to sustained alleviation of Covid-19 symptoms than placebo.” The authors may wish to reword this sentence to ensure it accurately reflects the findings of the cited study.\nResponse: Done. We have reworded this sentence as you suggested.\n2. Lines 125-126. The authors initially selected mutations to improve binding, including L3P, T11I, T11K, L3PT11F, L3PT6IP7Q, and L3PT11K. Providing additional details on the rationale behind these mutation choices would help readers better understand the design strategy.\nResponse: Thanks for your instructive comment. We have selected mutations based on the peptide sequences in the library. The sequencing results show that the similar peptide sequences to 6L3 mainly exhibits polymorphism at the 3rd, 6th and 11th positions. Therefore, we selected these peptides mutants (L3P, T11K, L3PT11K, L3PT11I, L3PT11F and L3PT6IP7Q) to evaluate the antiviral activities. Please see lines 112-115 in page 7.\n3. Line 130: “6L3-11K also demonstrated enhanced antiviral ability, with an IC value of 1.03 μM”.\n50\n676 nM which was the mean value of three replicates.\n4. Figs. 1b and 1d. The number of decimal places should be made consistent across these figures for uniformity.\nResponse: Done. The corrections have now been made consistent across these figures for uniformity.\n5. Fig. 1b and 1d, the peptides exhibit stronger neutralization to Omicron BA.2 and later variants, compared to BA.1 and earlier variants. It would be helpful if the authors could provide an explanation.\nResponse: We have tested the binding affinities of 6L3-3P to RBDs from different variants, and the results showed that 6L3-3P displayed much lower binding affinities to RBDs of BA.1 and early variants, in comparison to the Omicron BA.2 and later variants. We assume that the difference in binding affinity would be one of the key factors that contribute to the difference of inhibition capacity. Further structural analysis revealed that the different binding affinities might result from the different conformations of the 370-loop region (residues 373-377) and differential residues at positions 375, 376 and 408 of RBD, in earlier and later variants.Moreover, the “up” or “down” conformations of the RBDs are inherent dynamic features for the spike protein, and the different RBDs dynamic features would also likely contribute to the different antiviral capacity. Collectively, these structural and kinetic differences likely contribute to the different inhibition capacities against the SARS-CoV-2 variants.\n6. Figs. 3b and 3f, the rotamers of 4I and 8L differ when bound to PT and BA.2.75 – why is that?\nResponse: The macrocyclic peptides intrinsically may have structural flexibility and may undergo conformational adjustments upon binding to different protein targets. That’s why we observed the rotamers of 4I and 8L differ when bound to PT and BA.2.75.\n7. Did the authors introduce any modifications to the spike protein to enhance stability and expression, such as S2P, HexaPro, or the Foldon domain?\nResponse: Yes, we have introduced HexaPro and trimeric Foldon domain to the spike protein to enhance stability and expression. We have described it in the method section.\n8. Extended Data Fig. 6. The figure title is \"BA.2.75 S,\" but in Extended Data Table 1, the spike protein appears to be labeled as BA.2 (right two columns).\nResponse: We apologize for this typo error, and we have corrected it to BA.2.75 S.\n9. Extended Data Fig. 6e. The peptide does not appear to fit well into its density. Were the BA.2.75 S/6L3 (all RBDs up and two RBDs up) structures refined? If so, the authors may want to include the refinement statistics in Extended Data Table 1. Otherwise, please provide an explanation and discuss the reliability of the structures presented in Extended Data Fig. 6.\nResponse: For the BA.2.75 S/6L3 complex structure, most of the S trimers displayed either \"three RBDs up\" or \"two RBDs up\" conformation. Only in the \"two RBDs up\" S structure, we can see the density of peptides near the “down” RBD, and we did not refine BA.2.75 S/6L3 structure due to the relatively poor density of the RBD and peptide.\n10. Extended Data Table 1. The symmetry of the \"Two RBDs up\" structure should not be C3, as the protomers are not symmetrical. The final particle image numbers for “BA.2.75 S 6L3-3P11K” and “BA.2 S/6L3 (All RBDs up)” are identical (224,700) in both Extended Data Table 1 and Extended Data Figs. 4 and 6. Is this a coincidence? Should the FSC threshold (0.143) be applied to all four structures?\nResponse: We apologize for the mistake. The symmetry of the \"Two RBDs up\" structure is C1, and we have corrected it. Coincidentally, the final particle image numbers for “BA.2.75 S 6L3-3P11K” and “BA.2.75 S/6L3 (All RBDs up)” are identical (224,700). The FSC threshold used for the four structures is 0.143, and we have revised it in the Extended Data Table 1.\nReviewer #2 (Remarks to the Author):\nThis study presents macrocyclic peptides as potential therapeutics against SARS-CoV-2. Your work on peptides against Spike offers valuable insights into structure-based inhibition of the spike protein and highlights an innovative approach for antiviral drug development.\nHowever, there are several concerns regarding the presented data, interpretations, and claims that require further clarification and supporting evidence.\n1. The structural models built on the cryo-EM data appear inconsistent. In Figure 3, the side chain of lysine 11 (K11) is shown as not participating in interactions with either the prototype or BA.2 RBDs. However, in Supplementary Figure 9, this residue is depicted in close proximity to Arg408, Gln414, and Thr415 of the RBD.\nResponse: We must clarify that the peptide will have major interaction with the major RBD and minor interaction with the adjacent RBD. In Figure 3, we showed the major interaction between the peptide and the major RBD of spike protein, while the K11 of peptide has minor interaction with the adjacent RBD (please see the Extended Data Figure 9 in the revised manuscript). In the Extended Data Fig. 12 in the revised manuscript, we just showed the proximity of K11 to residues of the adjacent RBD and hypothesized that modification of K11 might increase the peptide trimerization through hydrogen bonding with the adjacent RBD. To test this hypothesis, we generated a K11R peptide variant which has a long side chain to explore the possibility.\nAdditionally, both Arg408 in the RBD and Lys11 (K11) in the peptide are positively charged, leading to a steric clash (repulsion). Despite this, the distance between these two residues is reported as 3.2 Å, which requires further clarification. Moreover, the entire “Structure-based modification of the 6L3-3P11K peptide” is based on the K11R substitution, which further intensifies the clash between Arg408 and Arg11 in the 6L3-3P11hR peptide.\nResponse: Based on our experiences with introducing substitution on the cyclic peptide, we found sometimes only one amino acid substitution in the key position will totally change the inhibition activity of the peptide. We have tried the 6L3-3P11Q peptide variant and found that the inhibition activity will dramatically decrease. Therefore, we try to introduce K11R substitution in the peptide, as both Lys (K) and Arg (R) are positively charged residues, but Arg will have a longer side chain to form potential hydrogen bond interactions with the residues Q414 and T415 in the adjacent RBD. The amino acid at position 408 of RBD has polymorphism (R or S respectively) in early SARS-CoV-2 strains and late Omicron strains. If the amino acid at position 408 is S, there will be no electrostatic repulsion. We noticed that there will be electrostatic repulsion between Arg11 of the peptide and Arg408 in the RBD for the early strains and BA.1, but the side chain of Arg408 is flexible and can orient towards another direction to decrease the repulsion. This hypothesis was experimentally validated, as 6L3-3P11R has better inhibition activity than the peptide 6L3-3P11K against the late Omicron strains.\n2. The authors state, “This difference correlates with the relatively lower binding affinity of 6L3-3P11K to PT RBD, as the PT RBD needs to alter the conformation of the 370-loop and overcome a higher energy barrier to bind to the 6L3-3P11K peptide.” However, no evidence is provided to support this claim.\nResponse: As the reviewer points out, we have changed the description as “This difference correlates with the relatively lower binding affinity of 6L3-3P11K to PT RBD, which might result from different conformations of 370-loop (residues 373-377) and differential residues at positions 375&376 (change from hydrophilic S&T to hydrophobic F&A ) and position 408 (change from charged R to non-charged S) of RBD in PT and BA.2.75 virus variants. As shown in the PT-S/6L3-3P11K complex structure, the 370-loop region exhibited a noteworthy conformational change upon peptide binding, while in the BA.2.75 S/6L3-3P11K structure, the 370-loop region did not show obvious conformational change.”\n3. The authors state, “As the trimerization of the peptide is important for peptide inhibition, we substituted tyrosine (Y) with phenylalanine (F) at position 1 of the 6L3-3P11hR peptide to enhance trimerization interactions.” To substantiate the peptide trimerization findings, the oligomerization of these peptides should be evaluated in the absence of RBD. If oligomerization is low or absent, it suggests that the peptides oligomerize upon binding their target, where they likely find a symmetric cavity within the closed conformation of the Spike protein.\nResponse: As recommended by the reviewer, we characterized the oligomerization state of 6L3-3P11K using native SEC-Mass method. The results demonstrated that 6L3-3P11K exists exclusively as a monomer in the absence of spike protein, confirming that the peptides oligomerizes upon binding their target, where they likely find a symmetric cavity within the closed conformation of the spike protein.\nReviewer #3 (Remarks to the Author):\nWang et al report the discovery and characterization of macrocyclic peptides that bind to the spike protein of SARS-CoV-2 and thereby exert an antiviral effect in infection models of SARS-CoV-2. The peptides were discovered by an affinity-based screening approach using the innovative RaPID technology. Initial actives were refined by solid phase peptide synthesis, driven by structural information on their binding mode that was disclosed by Cryo-EM. The peptides form trimers and lock the RBD of the spike protein in a locked conformation. They exert activity against pseudotyped viruses in the low nanomolar range and reduce viral load in an organoid model of primary nasal epithelial cells and in a mouse infection model after intranasal administration.\nOverall, the study reports several important findings. It demonstrates how the complex spike protein, a target for monoclonal antibodies, can be inhibited by a mid-sized macrocyclic peptide with high potency. Also the structure-based refinement is a showcase for the optimization of peptide drugs. The relevance of the findings for drug development is somewhat limited by the fact that the peptides probably require an intranasal administration, due to the lack of oral bioavailability.\nSeveral aspects of the study deserve a closer examination to meet the standards of Nat Commun:\n- The traceability of the findings is insufficient, and the methods section is incomplete.\n- Most importantly, the chemical characterization of the peptides is insufficient. The authors provide a general protocol for solid phase peptide synthesis, but information on the individual compounds is missing. At least for the compounds that were investigated closely (6L3-3P11K and others), the authors need to provide quantities and yields of the isolated final product. This is important to assess the scalability of the synthesis. Analytical data are also missing. This includes HPLC, HR-MS and NMR data.\nResponse: The synthesis of macrocyclic peptides was conducted following the standard Fmoc solid-phase peptide synthesis (SPPS) protocol. The characterization of synthesized macrocyclic peptides was conducted by employing ESI-MS, while their purity confirmation was achieved through analytical HPLC analysis. The HPLC and HR-MS results of the peptides were provided in the supplementary materials. (Please see page 21-26 in the Supplementary information file)\n- I do not find information on how ACE2 has been obtained for Extended Data Fig 5.\nResponse: We apologize for the insufficient information. The plasmid for ACE2 fused with human IgG Fc-tag (ACE2-hFc) was constructed and expressed in the mammalian 293T cells. The supernatant (which contains ACE2-hFc protein) was used in the SPR assay. (Please see lines 408-412 in the Methods section)\n- The authors claim that thermal stability is an advantage of 6L3-1F3P11hR and show the IC50 after heating to 160°C (Extended data Fig 10). There is no information on how this experiment was performed, e.g. on the duration of the heating period. Moreover, a 160°C condition is artificial, and I encourage the authors to demonstrate a chemical and metabolic stability of the drug under standard conditions (pH stability, stability in plasma or ELF matrix, 45°C stability over weeks, etc).\nResponse: We apologize for the insufficient information. The methodology has now been fully documented in the \"Methods\" section. As suggested, stability of the 6L3-1F3P11hR at 45℃, in serum or different pH buffers were tested, and all the results have been incorporated into the revised manuscript (please see lines 249-255 in page 13). The peptide 6L3-1F3P11hR displayed excellent chemical and metabolic stability under these conditions. (Extended Data Fig. 14)\n- Figure 1: There is a discrepancy between very different antiviral activities (e.g. against XBB vs BA2.86) in spite of highly similar binding affinities. How do the authors explain this?\nResponse: The antiviral activity of the peptide correlates to two key points, the peptide binding to RBD and the peptide trimerization within the closed conformation of spike protein. Although the peptides have highly similar binding affinities against different virus variants (e.g. against XBB vs BA2.86), the antiviral activities could differ due to the different dynamic features of RBDs. These “up and down” kinetic differences of RBDs likely contribute to the different antiviral capacity.\n- Most antiviral data were obtained with pseudotype assays. I missed a ‘standard’ antiviral assay on a single infected cell line (e.g. A549-expressing ACE2/TMPRSS2 or Vero E6) with 1-2 SARS-CoV-2 viruses. Although the organoid assay is more complex and potentially more relevant, it is easier to understand the potency of the inhibitors in such a standard assay format (with appropriate positive controls such as nirmatrelvir).\nResponse: We have evaluated the 6L3-1F3P11hR inhibitory activity against live SARS-CoV-2 at cellular level, including early strains (Prototype and Alpha) and recent Omicron strains (BA.2.12.1 and EG.5.1), and the results showed that 6L3-1F3P11hR could efficiently inhibited the replication of different SARS-CoV-2 variants, with significant viral RNA reduction in supernatants observed even at 10 nM. (Extended Data Fig. 13).\n- With respect to administration routes, is an iv administration an option? To show whether the compounds qualify as a hit or even lead, information on their pharmacokinetic (iv admin), ADME and physicochemical properties (e.g. solubility) would be relevant.\nResponse: We have performed pharmacokinetic analysis of 6L3-1F3P11hR peptide following intranasal administration in C57BL/6 mice, and the pharmacokinetic profile revealed distinct kinetics in different organs, with a plasma half-life (T₁/₂) of 3.08 h compared to 6.45 h in nasal lavage fluid. The concentration of 6L3-1F3P11hR peptide is much higher in the nasal region, and 6L3-1F3P11hR remains detectable (nearly 100 ng/ml) in the nasal lavage fluid 24 hours after administration. As SARS-CoV-2 mainly infected the respiratory tract, and therefore intranasal administration route was selected to evaluate the in vivo efficacy of the macrocyclic peptides. In the future, we will try iv administration to evaluate the in vivo efficacy, but structural modifications remain necessary to enhance their systemic retention time.\nMinor points\n- Figure 4e: I believe that the left and right panels were confused in the caption.\nResponse: We are sorry for the mistake in the caption. We have corrected it.\n- Figure 1a and 1c are too small and cannot be deciphered after being printed on a high-quality laser printer in DIN A4 format. Same is true for Ext. Data Fig. 1b.\nResponse: We are sorry for the figure preparation and have modified the Figure 1 and Extended Data Fig. 1 to show the panels more clearly.\n- Figure 3b: Have the labels for 8L and 5L been confused?\nResponse: The figure has been revised to improve the clarity of atomic representations.\n- line 95: Define RBM\nResponse: Done. RBM means receptor-binding motif. Please see line 84 in page 5.\n- Extended Data Fig 2a: Redraw the structures and avoid overlapping atoms.\nResponse: Done. The panel c of figure was redrawn as suggested.\n- line 130: An IC50 of 1.03 µM is mentioned, but the Figure gives a value of 676 nM.\nResponse: We apologized for the mistake, and we have corrected it. The IC should be 676 nM.\n50\n- line 131: I suggest rounding to 12 nM.\nResponse: OK. We have standardized IC values without decimal place.\n50\n- I assume that the ‘prototype’ virus is the original one isolated from Wuhan? Is ‘prototype’ an accepted term?\nResponse: “Prototype” virus was widely used in the research related to COVID-19, and referred to the strain that isolated from Wuhan. (For example, Xu K, et.al., Protective prototype-Beta and Delta-Omicron chimeric RBD-dimer vaccines against SARS-CoV-2. Cell. 2022;185(13):2265-2278.e14. doi: 10.1016/j.cell.2022.04.029.; Xie H et.al., The contributions of vaccination and natural infection to the production of neutralizing antibodies against the SARS-CoV-2 prototype strain and variants. Int J Infect Dis. 2024 Jul:144:107060. doi: 10.1016/j.ijid.2024.107060.)\n- line 204: The ‘correlation’ is in the ‘right’ direction, but can the differences in the loop conformation really account for the 100fold difference in potency?\nResponse: We have carefully examined the peptide binding residues in the PT-S/6L3-3P11K and BA.2.75 S/6L3-3P11K complex structures, and most of the binding residues are highly conserved between these two complex structure except the residues at position 375, 376 and 408 of RBD in PT and BA.2.75 virus variants. We assume that both the difference of the loop conformation and residue substitutions together would be the key factors contributing to the 100-fold difference in potency.\n- line 261-263: The reference to the panels 5d and 5e has been confused.\nResponse: We are sorry for the confused reference, and the mistake has been corrected in the revised Figure 4.\n- Discussion: I suggest removing words describing subjective assessments like ‘exciting’ or ‘excellent’\nResponse: Done. We have removed the words describing subjective assessments like “exciting” or “excellent”.\n- line 274: The authors compare their peptides to a previously reported one, but I cannot see any data on a head-to-head comparison.\nResponse: As reviewer #4 suggested the manuscript needs to be more concise, and therefore the description was removed owing to the absence of head-to-head comparison.\nReviewer #4 (Remarks to the Author):\nIn this manuscript, Wang M. et al, explored the in vitro and in vivo efficacy of new macrocyclic peptide against different Sarbecoviruses where the peptide showed potent and promising activity against omicron subvariants in different assay systems. Overall, the manuscript is well written and data interpretation is matching the conclusion. However, I have some comments especially for the in vivo studies.\nMajor comments:\nA) The methodology of the in vivo study is lacking important information:\n1. number of animals per each group\nResponse: Five animals were used for each group. Please see line 544 in page 28.)\n2. if the prophylactic and therapeutic studies are 2 independent experiments or were run together\nResponse: The prophylactic and therapeutic studies were 2 independent experiments. Please see Fig. 5 and Fig. 6.\n3. Nirmatrelvir administration schedule, was it 100 mg/kg once daily or twice daily? was it also given prophylactically and therapeutically in the same time frame as the peptide (the data in Fig 5 mention that but not mentioned in methods)\nResponse: For therapeutic evaluation, nirmatrelvir (100 mg/kg) was given once daily via intragastric administration, and a total of four doses were administered. For prophylactic evaluation, nirmatrelvir (100 mg/kg) was given once at 0.5 h prior to SARS-CoV-2 challenge, and a total of one dose was administered. Please see Fig. 5 and Fig. 6.\n4. the information on how histopathology scoring was done is missing (how many parameters were checked and what was the scoring scale)\nResponse: Histopathology scoring scale was based on a published paper. (Zhang AJ, Lee AC, Chan JF, Liu F, Li C, Chen Y, Chu H, Lau SY, Wang P, Chan CC, Poon VK, Yuan S, To KK, Chen H, Yuen KY. Coinfection by Severe Acute Respiratory Syndrome Coronavirus 2 and Influenza A(H1N1) pdm09 Virus Enhances the Severity of Pneumonia in Golden Syrian Hamsters. Clin Infect Dis. 2021 Jun 15;72(12):e978-e992. doi: 10.1093/cid/ciaa1747.) We have supplemented this information in the method section. Please see line 274 in page 14.\n5. Is there an ethical approval (project number) for the in vivo work? nothing mentioned in the methods section about ethics.\nResponse: The use of animals was approved by the Committee on the Use of Live Animals in Teaching and Research of The University of Hong Kong under CULATR 5370-20. We have supplemented this information in the methods section. Please see lines 380-381 in page 21.\nB) 30 min after infection is a very short time to consider that the peptide has therapeutic potential (30 min is more like post-exposure prophylaxis rather than therapeutic). Was the therapeutic efficacy of the peptide tested at longer period after infection e.g. 8h or 24h post infection?\nResponse: Thanks for the suggestion. We have performed additional animal studies, and the therapeutic efficacy of 6L3-1F3P11hR peptide (6 mg/kg per dose) was tested at longer period after infection (8 hours post infection). At 4 dpi, the mice were sacrificed, and the lung tissue was harvested for virological analyses. The results revealed that 6L3-1F3P11hR treatment significantly reduced infectious viral titers in the lung tissue. Please see Extended Data Fig. 15, and the method can be found in lines 554-562 in page 29.\nD) Figure 5 panels have low resolution, it is also not clear in the legend whether the data presented are median or mean\nResponse: In response to reviewers' comments, we have updated figures and figure legends. Data are presented as mean ± SD.\nOther comments:\nA) was the peptide tested against seasonal coronaviruses e.g. OC43 and 229E?\nResponse: In response to reviewers' comments, we evaluated the inhibitory effect of 6L3-1F3P11hR on OC43 coronaviruses, and the results demonstrated an extremely weak antiviral activity. Please see the following figure.\nAs the manuscript mainly focus on the SARS-CoV-2, therefore we did not add this data in the revised manuscript.\nB) from the sequence alignment shown in extended Figure 11, is it possible to explain /suggest why the peptide is less active against non-omicron SARS-CoV-2 and other Sarbecoviruses?\nResponse: Sequence alignment reveals distinct variations between the Omicron variant of SARS-CoV-2 and non-Omicron SARS-CoV-2 strains or other sarbecoviruses. We have noticed that the residue difference in the peptide binding region mainly occurred in two sites of RBD, residues at positions 375 and 376 (change from hydrophilic S and T to hydrophobic F and A) and position 408 (change from charged R to non-charged S). In the PT-S/6L3-3P11K complex structure, the hydrophilic residues S and T at positions 375 and 376 will contact the hydrophobic residues 5L and 10F of the peptide, and therefore the hydrophilic residues at positions 375 and 376 will generate an unfavorable environment for the peptide binding. Moreover, the charged residue R at position 408 will also have electrostatic repulsion with the charged 11K of peptide. The residue differences at these two sites could possibly explain why the peptide is less active against non-omicron SARS-CoV-2 and other sarbecoviruses.\nMinor comment: the manuscript needs to be more concise (words count to be reduced)\nResponse: As suggested, we have deleted the unnecessary descriptions throughout the manuscript and make it more concise."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Comment to Response #1: The up and down conformational states of the SARS-CoV-2 spike protein are known to be affected by various factors, including the viral variant, stabilizing proline mutations, sample preparation protocols, and the choice of detergent. The structure referenced here (PDB 6VXX) utilized a spike construct with the 2P mutation, while the current study employs the HexaPro stabilized spike. Therefore, the two datasets are not directly comparable.", "reviewer_3": "Wang et al provide a revised and improved version of their manuscript. Many remarks of mine and of the other reviewers have been addressed adequately, but one issue remains:\n-The analytical characterization of the peptides remains inadequate. High resolution masses need to be given with 4 digits after the comma. Even with the 2 digits given in the SI, many of the measurements do not fall into a 5 ppm error tolerance window. Some show massive deviations. The compounds are pure according to the HPLC traces, but there is no evidence for their structural integrity. This is a fundamental and critical problem.\n-6L311K is not pure according to the MS spectrum. Also the high resolution mass implies that this might be a different compound.\n- I recommend that the authors provide NMR data, at least for the compound that went in vivo.\nOther comments\n- The abstract contains several grammatical errors. In addition, I find the expression ‘extremely thermostable’ inadequate, because one might argue what extreme is (1 month? 50 years?). I would replace it by ‘thermostable’.\n- The authors reply to my question on how ACE2 was obtained: ‘We apologize for the insufficient information. The plasmid for ACE2 fused with human IgG Fc-tag (ACE2-hFc) was constructed and expressed in the mammalian 293T cells. The supernatant (which contains ACE2-hFc protein) was used in the SPR assay. (Please see lines 408-412 in the Methods section)‘\nBut I do not find this information in lines 408-412. Did the authors use 293T or Freesyle 293F cells? Is any evidence of purity and integrity (e.g. an SDS gel) of ACE2 provided?\n- Intranasal administration might be challenging in practice. In the absence of data on systemic exposure, I have some doubt regarding the relevance of the peptides as drugs.", "reviewer_4": "I would like to thank the authors for their thorough and thoughtful revisions. I have carefully reviewed the revised manuscript and the accompanying responses to my previous comments. I am pleased to confirm that all of my concerns have been satisfactorily addressed, and the manuscript has been appropriately adjusted in line with the suggestions.\nI have no further comments and recommend the manuscript for publication in its current form."}, "author_response": "Response: We appreciate the reviewer's comment. We have re-collected cryo-EM data for the same spike constructs in the apo state and performed quantitative analysis for the unbound PT or BA.2.75 spike trimer. The results showed that the proportion of spike trimers in the “three-RBD-down” conformation increased notably after peptide binding, from 6.6 % to ~100% for PT and from 38.3% to ~54% for BA.2.75. Please see lines:181-185, and Extended Data Fig. 4, 5, 8.\nResponse: We appreciate the reviewer's comment, and we apologized for the unclear statement. In our study, we used low-resolution mass measurements for the peptides during our initial screening process, which focuses on identifying the most active peptides. This explains why the reported values have 2 digits and do not meet the 5 ppm error tolerance.\nThe peptides are synthesized using solid-phase peptide synthesis, where Fmoc-protected amino acid building blocks are sequentially added. This involves coupling the amino acids with a coupling reagent, followed by deprotection of the Fmoc group. This coupling and deprotection process is repeated until the desired sequence is achieved. If there is a loss of amino acids during synthesis, it can be easily identified through mass spectrometry. We carefully check both the desired sequence and the synthetic sequence to ensure that each building block is added correctly. Any errors in the sequence can result in incorrect Mass and an inactive peptide.\nTherefore, we performed the high-resolution mass measurements for the 6L3-3P11K and 6L3-1F3P11hR which is used for subsequent cellular and animal tests. The results fall within a 5 ppm error tolerance, indicating the accuracy of the peptide sequence. Please see Extended Data Fig. 7.\nResponse: As the reviewer suggested, we performed additional NMR experiment and obtained the 1H-NMR spectrum of 6L3-1F3P11hR. The results are consistent with the chemical structure of 6L3-1F3P11hR. Please see the 1H NMR data and 1H NMR spectra of peptide 6L3-1F3P11hR.\n1H NMR data of peptide 6L3-1F3P11hR\n1H NMR (600 MHz, d-DMSO): δ 8.76 (brs, 1H, NH), 8.64 (s, 1H, NH), 7.71-8.48 (m, 8H, 8 × NH), 7.46 (s, 1H, NH), 7.01-7.41 (m, 15H, NH, 2 × NH , 10 × Ar-H), 5.24 (s, 1H, C-OH), 4.46-4.89 (m, 2 4H, 4 × CH), 4.28-4.43 (m, 3H, 3 × CH), 4.24 (t, 1H, J = 7.5 Hz, CH), 4.05-4.29 (m, 3H, 3 × CH), 3.89-4.04 (m, 1H, O-CH), 3.73 (brs, 1H, NH), 3.57-3.71 (m, 4H, 2 × CH ), 3.45-3.55 (m, 2H, CH ), 2 2 3.24-3.26 (m, 2H, CH ), 2.96-3.16 (m, 4H, 2 × CH ), 2.62-2.95 (m, 5H, 2 × CH , CH), 2.27-2.49 2 2 2 (m, 2H, CH ), 1.99-2.22 (m, 2H, CH ), 1.74-1.95 (m, 7H, O-C-CH , 2 × C-CH ), 1.53-1.73 (m, 6H, 2 2 3 2 3 × C-CH ), 1.27-1.52 (m, 7H, 3 × C-CH , CH), 1.00-1.20 (m, 4H, CH , CH), 0.88 (d, 3H, J = 5.6 2 2 3 Hz, CH ), 0.76-0.81 (m, 9H, 3 × CH ), 0.65 (d, 3H, J = 5.2 Hz, CH ), 0.53-0.62 (m, 2H, CH ). 3 3 3 2\n1H NMR spectra of peptide 6L3-1F3P11hR\nResponse: Done. It has been corrected.\nResponse: We apologize for the insufficient information. The coding sequence of ACE2 with hFc (ACE2-hFc, codon optimized) was also cloned into the pCAGGS vector. The recombinant plasmids were transfected into mammalian 293T cells to express the ACE2-hFc. Five days later, the supernatants were collected. (Please see lines 416-420 in the Methods section).\nWestern Blot assay was conducted to verify the expression and purity of ACE2-hFc protein. 3 μL supernatant (which contains ACE2-hFc protein) was mixed with loading buffer (+DTT), boiled and detected by 12% SDS-PAGE gel. Proteins were transferred in PVDF (Merck Millipore, USA), blocked with 5% skim milk at room temperature for 2h, and incubated with Goat anti-human IgG Fc (HRP) for 1h at room temperature. Brands were visualized by ECL chemiluminescence reaction using Tanon 5200 imaging system (Tanon, China).\nThe result showed that the ACE2-hFc protein demonstrated favorable purity and structural integrity. Please see the following figure. As the manuscript already includes many figures, therefore we did not add this data in the revised manuscript.\nResponse: Nasal spray offers a convenient and non-invasive method of administration and would enhance patient compliance and broaden the drug’s applicability. There are several nasal spray drugs which are being developed to prevent or treat COVID-19, and some of them have entered clinical trials. For example, HH120-NS02 (clinical trial numbers: NCT05765279, NCT05787418), MY-586 COVID-19 Neutralizing Antibody Nasal Spray (clinical trial number: NCT05977101), MIR19® Inhalation Solution (clinical trial number: NCT05783206), and A8G6 COVID-19 Neutralization Antibody Combined with Nasal Spray (clinical trial number: NCT06127498). Therefore, we believe the macrocyclic peptide could be developed to similar preventive and therapeutic products.\nResponse: We appreciate the reviewer's constructive comments."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The new experiments validate the conclusions. Here are a few minor suggestions:\nPlease include the number of particles or the corresponding percentages in Extended Data Fig. 8f as in Extended Data Fig. 8fc or Extended Data Figs. 4 and 5.\nIn Extended Data Figs. 4, 5, 8, and 9, please indicate and highlight which cluster(s) were selected for heterogeneous refinement. The current color scheme of the maps is somewhat confusing; consider using the same color for all junk classes and distinct colors to represent maps in different conformations.", "reviewer_3": "The authors have improved the manuscript further and adressed most of my comments.\nThe characterization of the compounds is not profound, but acceptable.\nThe quality of the ACE2 protein is probably ok, but I am not convinced of the provided data, because Western blots are inadequate to prove purity. Because this is a smaller methodological detail, and because there is no reason to expect 'bad' surprises, I find the manuscript is acceptable for publication now."}, "author_response": "Response: Done, please see the revised Supplementary Fig. 8 as follows.\nResponse: Done. Selected clusters for heterogeneous refinement are highlighted in distinct colors, while the junk classes are rendered in gray. Please see the revised Supplementary Fig. 4, 5, 8 and 9.\nResponse: Thanks. We appreciate the reviewer's constructive comments."}]

Decision letter