YL1004 is a SARS-CoV-2 papain-like protease inhibitor with immunomodulatory and antiviral activity in mice

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

Abstract
The evolution of SARS-CoV-2, coupled with its immune evasion mechanisms, underscores the urgent need for antiviral strategies beyond vaccination. The papain-like protease (PLpro) is a dual-functional enzyme essential for viral replication and suppression of host innate immunity. Here, we present the rational design and characterization of YL1004, a tricyclic oral PLpro inhibitor demonstrating robust, cross-variant SARS-CoV-2 antiviral activity and favorable pharmacokinetic properties. YL1004 potently inhibits the enzymatic function of PLpro, disrupts deubiquitination and deISGylation processes, and restores antiviral immune signaling cascades. Notably, YL1004 suppresses the replication of SARS-CoV-2 wild type, Delta and Omicron variants. Additionally, it is also effective against the Mpro E166V recombinant SARS-CoV-2 strain, which confers resistance to nirmatrelvir. In the lethal SARS-CoV-2 infection model using K18-hACE2 mice, YL1004 confers complete protection to animal survival, significantly reduces viral load in nasal turbinate and lung tissues, and alleviates virus-induced pathological tissue damages. Co-crystal structural analysis revealed a distinctive binding mode, enhancing target engagement through expanded hydrophobic interactive interface and additional hydrogen bonding interactions. Collectively, these findings establish YL1004 as a promising therapeutic candidate, harnessing dual antiviral and immunomodulatory mechanisms to combat SARS-CoV-2 and emerging variants.

Introduction
The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 777 million infections and more than 7.08 million deaths as of March 20251. While COVID-19 vaccines have in part mitigated the virus’s capacity in causing severe infections2,3, the emergence of Omicron and its sublineages with significant immune escape has showed substantially reduced sensitivity to therapeutic antibodies and undermined vaccine efficacies4–7. Given SARS-CoV-2’s anticipated long-term circulation and potential, developing alternative antiviral strategies beyond vaccination remains crucial for addressing both current and future threats.

Papain-like protease (PLpro) of SARS-CoV-2 mediates the cleavage of coronavirus polyprotein pp1a and pp1ab at the N-terminal to release Non-structural protein 1-3 (Nsp1-3), which is required for virus transcription and replication. For example, Nsp1 of SARS-CoV-2 has been implicated in shutdown of the host translation machinery, thus suppressing the host innate immune responses while allowing efficient expression of the viral proteins to facilitate virus replication9,10. Interestingly, apart from its proteolytic activity PLpro is a multi-functioned viral protein, which also plays critical roles in deubiquitination and deISGylation11–15. Inhibition of PLpro offers dual therapeutic benefits by simultaneously suppressing viral replication and restoring host innate immunity. Therefore, PLpro represents a promising therapeutic target for the treatment of SARS-CoV-2 infection. Despite successful development of inhibitors targeting other viral components - including RNA-dependent RNA polymerase (RdRp) inhibitors (Remdesivir16, Molnupiravir17, Renmindevir18, Azvudine19) and main protease (Mpro) inhibitors (Nirmatrelvir20, Ensitrelvir21, Simnotrelvir22, and Leritrelvir23), PLpro inhibitor development has proven challenging. Earlier PLpro inhibitors demonstrated either suboptimal antiviral activity or unfavorable pharmacokinetic profiles, leading to clinical trial failures24–33. Recent advances with orally active PLpro inhibitors (Jun1268234, PF-0795747235, GZNL-P3636, WEHI-P837 and Jun1329638) in murine SARS-CoV-2 models are promising.

In this investigation, we report the design and characterization of YL1004, a potent and highly selective SARS-CoV-2 PLpro inhibitor featuring a tricyclic scaffold and favorable pharmacokinetic properties. YL1004 demonstrated potent antiviral efficacy against all evaluated SARS-CoV-2 variants (Delta, Omicron JN.1, and Omicron KP.3) and nirmatrelvir-resistant SARS-CoV-2 mutant. Mechanistically, YL1004 directly targets PLpro and effectively neutralizes PLpro-dependent immune evasion, underscoring its strong antiviral potential and selectivity. In JN.1-infected K18-hACE2 transgenic mice, oral treatment with YL1004 significantly reduced viral burdens and also ameliorated virus-induced pathologies in the nasal turbinate and lung tissues of infected mice. Strikingly, therapeutic treatment with YL1004 conferred full protection to the infected mice against lethal SARS-CoV-2 Delta challenge. Overall, these findings position YL1004 as a promising clinical candidate for developing next-generation antiviral therapies targeting SARS-CoV-2.

Results
Discovery of PLpro inhibitors with a tricyclic scaffold
To identify novel hit compounds for PLpro-targeted drug design, we first screened an in-house chemical library of ~35,000 compounds using a fluorescence resonance energy transfer (FRET) assay. The library consists of structurally diverse small molecules derived from our previous antiviral discovery efforts, as well as compounds with known activity against other targets such as proteases and kinases that were developed in house. These molecules include both covalent and noncovalent scaffolds with functionalities suitable for engaging cysteine proteases. Compounds that inhibited PLpro activity by more than 70% were considered primary hits. Five compounds demonstrated PLpro inhibitory activity with 50% inhibition concentration (IC50) values below 50 μM (Supplementary Fig. 4). Among these, Hit-1 (1a), featuring a unique tricyclic scaffold, emerged as the most promising lead compound with an IC50 of ~26.82 μM.

To guide structural optimization, we compared 1a with reported PLpro inhibitors24–33. Interestingly, molecular superposition revealed partial overlap between the tricyclic core of 1a (blue) and the benzamide group of classical PLpro inhibitor GRL061724 (pink) (Fig. 1a, b)39. Leveraging this observation, we matched up 1a with GRL0617 via a fragment merging strategy to design and synthesize enantiomeric compounds 2a and 2b (Fig. 1a). The (S)-enantiomer 2b exhibited significantly higher potency (IC50 = 98.6 nM) than its (R)-counterpart 2a (IC50 = 5.77 μM), demonstrating a 50-fold activity difference (Fig. 1c). Differential scanning fluorimetry (DSF) assays further confirmed direct binding to PLpro, with ΔTm values of 3.45 °C for 2a and 10.04 °C for 2b (Fig. 1d).

Notably, the tricyclic phenylamino group in 2b differed structurally from the phenylmethyl group in GRL0617. To this end, we synthesized compound 2d, which differs from GRL0617 only by the replacement of the methyl group with an amino group on the benzene ring. Enzymatic evaluation revealed a substantial decrease in activity for 2d (IC50 = 10.97 ± 0.33 μM) relative to GRL0617. Correspondingly, differential scanning fluorimetry (DSF) indicated reduced thermal stabilization (ΔTm = 2.67 ± 0.33 °C), suggesting weaker binding affinity. This loss of activity may be attributed to the absence of both the N-methylpiperazinyl group and the (S)-tricyclic core, which appear essential for properly orienting the anilino moiety to form hydrogen bonds with PLpro. To further probe the role of the N-methylpiperazine substitution, we prepared analog 2c by introducing this group into the 2d scaffold. Compound 2c showed improved potency (IC50 = 337.7 nM) and a greater thermal shift (ΔTm = 9.13 °C) compared to 2d, yet its activity remained 3.4-fold lower than that of the (S)-enantiomer 2b. The significant activity difference between 2a and 2b further highlights the importance of the tricyclic scaffold’s stereochemistry for PLpro inhibition. Together, these results underscore the synergistic contribution of the (S)-tricyclic core and the anilino modification to inhibitor binding (Fig. 1a, c, d).

Subsequent optimization focused on modifying the R1, R2, and R3 groups of the tricyclic core (Fig. 1e), yielding 47 derivatives. A complete list of the designed tricyclic PLpro inhibitors is shown in Supplementary Fig. 1–3, with representative examples in Table 1. Initial R1 modifications while fixing R2/R3 produced five analogs (2e-i; Supplementary Fig. 1). Notably, the geminal-cyclopropyl-substituted compound 2i showed a 5-fold potency improvement (IC50 = 18.4 nM) over 2b. However, compound 2i exhibited moderate cytotoxicity in VeroE6 cells (CC50 = 74.06 μM), suboptimal oral PK (AUC(0-t) = 9291 μg/L·h at 100 mg/kg), and CYP3A4 inhibition (IC50 = 1.87 μM), prompting further optimization. Next, R2 optimization with fixed R1 (cyclopropyl) and R3 generated nine analogs (3a–i; Supplementary Fig. 1). Unfortunately, none of these improved PLpro inhibition. Finally, R3 modifications while retaining R1/R2 yielded 34 compounds (4a–l, 5a–h, Supplementary Fig. 2; 6a–n; Supplementary Fig. 3), with 24 achieving IC50 ≤ 30 nM. Among them, compound 4b emerged as the most potent derivative, showing a 121-fold potency increase (IC50 = 12.9 nM) over GRL0617. Nevertheless, 4b retained cytotoxicity (CC50 = 38.99 μM) and CYP inhibition (CYP3A4 IC50 = 1.67 μM; CYP2C9 IC50 = 0.685 μM), likely due to metabolic toxicity from the naphthalene 4-methyl group.

Based on the molecular superposition results (Fig. 1b), which indicated that the naphthalene 4-position points toward the solvent-exposed region, we hypothesized that chemical modification at this site would minimally affect the compound’s inhibitory activity against PLpro. This hypothesis was corroborated by our structure-activity relationship (SAR) data, which demonstrated that derivatives modified at this position generally maintained IC50 values against PLpro below 50 nM. Consequently, we introduced diverse aromatic and heterocyclic substituents at this position to further optimize pharmacokinetic properties. Several derivatives (2i, 4e, 4k, 4l, 6k, 6f, 6g, 6h, 6i) achieved balanced activity, reduced cytotoxicity, and improved CYP profiles (Table 1). These compounds would undergo further evaluation to identify a clinical development candidate.

Selection of YL1004 as the candidate compound for further studies
Building on structure-activity relationship (SAR) insights and preliminary safety profiles, we systematically evaluated nine prioritized derivatives (2i, 4e, 4k, 4l, 6k, 6f, 6g, 6h, 6i) alongside the positive control Jun12682. MTT assays across four cell lines (VeroE6, BEAS-2B, HUVEC, and 293T) revealed that compound 6f (designated as YL1004) exhibited the most favorable safety profile, demonstrating significantly reduced cytotoxicity in VeroE6 cells (CC50 = 226.90 μM) compared to Jun12682 (CC50 = 59.34 μM) (Supplementary Table. 2). Pharmacokinetic (PK) studies in Balb/c mice following oral administration (100 mg/kg) further highlighted YL1004’s advantages, with an oral AUC of 79,818 h·ng·mL⁻¹ with 61.2% bioavailability (F), outperforming all other derivatives (Supplementary Table. 3). Additionally, cross-species pharmacokinetic evaluation in rats showed that YL1004 maintained favorable PK properties, attaining an oral AUC of 14,723 h·ng·mL⁻¹ at 10 mg/kg with 62.9% bioavailability (Supplementary Table. 4). Enhanced target engagement was further confirmed by differential scanning fluorimetry (DSF), where YL1004 induced a dose-dependent increase in PLpro thermal denaturation temperature (ΔTm = 18.07 °C at equivalent concentrations), surpassing Jun12682 (Supplementary Fig. 5). Consistent with the DSF findings, tight binding kinetic analysis using the Morrison equation yielded a Ki of 2.3 ± 0.3 nM for YL1004, further substantiating its high inhibitory potency (Supplementary Table. 5).

Comprehensive profiling of YL1004 highlighted its favorable drug-like properties: promising metabolic stability in human liver microsomes (in vitro clearance = 20.07 mL/min/kg, T1/2 = 57.75 min; Supplementary Table. 6); moderate plasma protein binding (89.57%; Supplementary Table. 7); optimal solubility (134.22 μg/mL); and superior permeability (Papp = 8.17 × 10-6 cm/s in MDCK monolayers) (Supplementary Table. 8). Safety assessments further demonstrated YL1004’s high selectivity, showing very weak or no activity against 377 human kinases (<50% inhibition at 10 μM; Supplementary Table. 9), negligible effects on major cytochrome P450 isoforms (CYP1A2, CYP2C8, CYP2C9, CYP2D6, and CYP3A4; IC50 > 30 μM; Supplementary Table. 10), and no significant inhibition of the hERG channel (IC50 > 50 μM). Collectively, these findings underscore YL1004’s low risk of drug-drug interactions and cardiotoxicity.

In summary, YL1004 integrates target selectivity, robust safety, favorable pharmacokinetics, and balanced drug-like properties, positioning it as a promising therapeutic clinical candidate for further in-depth studies.

Co-crystal structure of SARS-CoV-2 PLpro in complex with YL1004
To obtain the co-crystal structure of PLpro in complex with YL1004, we generated a catalytically inactive mutant (PLpro-Cys111Ser) and determined the complex structure at 2.7 Å resolution (Fig. 2a-d). The SARS-CoV-2 PLpro consists of an N-terminal ubiquitin-like (Ubl) domain and a catalytic domain comprising thumb, palm, and finger subdomains (Fig. 2b). The catalytic triad (Cys111, His272, and Asp286) is positioned at the palm-thumb interface. YL1004 binds within the pocket formed by the flexible blocking loop 2 (BL2) and S3/S4 protein subsites (Fig. 2c). The N-methylpiperazinyl moiety of YL1004 forms a 2.9 Å hydrogen bond with the side chain of Glu167, while the aniline group establishes a 3.1 Å hydrogen bond with Tyr264 (Fig. 2d). Additionally, the piperazinyl-benzotetrahydropyran moiety participates in extensive hydrophobic interactions with Leu162, Gly163, Asp164, and Tyr268. The central amide group makes hydrogen bonds with Gln269 (2.9 Å) and Asp164 (2.6 Å), respectively. The cyclopropyl group forms hydrophobic interactions with the side chains of Pro248, Tyr264 and Thr301. Furthermore, the naphthyl ring, which is sandwiched between Met208, Pro247, Pro248 and Tyr268, forms a T-shaped π-π stacking interaction with Tyr268 and hydrophobic contacts with residues Met208, Pro247 and Pro248 (Fig. 2d). The tetrahydropyran moiety also contributes to hydrophobic interactions with residue Pro248.

The binding mode of YL1004 differs from that of several reported representative PLpro inhibitors, including GRL061724 (Fig. 2e, f), Jun1268234, PF-0795747235, GZNL-P3536, and WEHI-P437 (Supplementary Fig. 6a–d). Taking GRL0617 as an example, the strategic amino group substitution on the benzamide moiety of YL1004 forms additional hydrogen bonds with Tyr264, thereby significantly enhancing its binding affinity for PLpro (Fig. 2e). Although both compounds utilize the central amide for hydrogen bonding with Asp164 and Gln269, YL1004 exhibits shorter bond distances, suggesting enhanced stabilization (Fig. 2f). Furthermore, the methyl-to-cyclopropyl substitution in YL1004 enhances hydrophobic interactions. The extended tetrahydropyran moiety enhances hydrophobic interactions with Pro248 and promotes an upward shift (1.2 Å) of the naphthyl ring, facilitating additional hydrophobic interactions with Met208 (Fig. 2g).

YL1004 antagonizes PLpro-mediated innate immune suppression
Coronavirus PLpro is a multifunctional viral protease that hydrolyses viral polyprotein precursors. Recent studies have revealed that PLpro also acts as an immunoregulatory enzyme that exerts deubiquitinating (DUB) and deISGylating activities, removing ubiquitin and ISG15 conjugates from key signaling mediators such as IRF3 to suppress type I interferon and NF-κB signaling and facilitate viral immune evasion40,41. We investigated whether YL1004 could offset these immunosuppressive effects by specifically inhibiting PLpro.

To measure the effects of YL1004 on the DUB and deISGylating activities of PLpro, we first used Ub-AMC and ISG15-AMC as fluorogenic substrates in FRET-based enzyme kinetic assays. YL1004 significantly reduced PLpro-catalyzed hydrolysis with IC50 values of 22.9 nM and 43.6 nM against Ub-AMC and ISG15-AMC, respectively, surpassing the potency of the reference compound Jun12682 (IC50 = 87.0 nM and 124.0 nM) (Fig. 3a). Consistent with the IC50 values of YL1004, tight binding kinetic analysis using the Morrison equation yielded Ki values of 4.2 nM for deubiquitination and 39.3 nM for deISGylation, respectively (Supplementary Table. 11). To verify these findings in cells, HEK293T cells were co-transfected with plasmids expressing PLpro and ubiquitin. As shown in Fig. 3b, PLpro reduced the level of ubiquitinated proteins, whereas YL1004 restored ubiquitination in a dose-dependent manner, demonstrating that YL1004 inhibits PLpro-mediated deubiquitination. Remarkably, 1 µM YL1004 was more potent than 10 µM Jun12682 in suppressing the DUB activity of PLpro (Fig. 3b). In parallel, A549 cells stimulated with interferon-α (IFN-α) to induce ISGylation showed reduced ISG15-conjugated proteins upon PLpro expression. YL1004 co-treatment reversed this effect in a dose-dependent manner, indicating potent inhibition of PLpro-driven deISGylation (Fig. 3c).

Given PLpro’s structural homology with human deubiquitinating enzymes (DUBs), we also assessed the selectivity of YL1004. FRET assays demonstrated that, at concentrations up to 50 μM, YL1004 suppressed the activities of common host DUBs by less than 50%, indicating a high degree of specificity (Fig. 3d). In a parallel investigation using the active-site probe HA-Ub–vinyl sulfone (VS), the positive control N-ethylmaleimide (NEM) broadly blocked DUB labeling in HEK293T cells, whereas YL1004 selectively interfered only with PLpro labeling, showing negligible impact on other DUBs (Fig. 3e). Moreover, in HEK293T cells lacking PLpro expression, YL1004 treatment did not alter IFN-α–induced ISGylation, reinforcing the notion that YL1004 does not interfere with host ubiquitination or ISGylation processes (Fig. 3c).

To elucidate the structural basis for YL1004’s selectivity, we performed structural alignments between PLpro and ten human DUBs. The calculated Cα RMSD values ranged from 9.95 to 31.52 Å (based on all Cα atoms, Supplementary Fig. 7), indicating substantial overall structural divergence. With respect to the substrate-binding pockets, UCHL1 adopts a configuration completely distinct from that PLpro (Supplementary Fig. 7b, c). Of note, among the tested DUBs, USP2 possesses a substrate-binding pocket most closely resembling that of PLpro (Supplementary Fig. 7a), however, a loop spanning residues 503–516 would cause steric clashes with the naphthyl ring and the tetrahydropyran moiety of YL1004 (Supplementary Fig. 7d, e). This structural incompatibility provides a mechanistic explanation for the lack of USP2 inhibition by YL1004. Overall, these findings could provide structural insight into the selectivity of YL1004 toward human USPs.

SARS-CoV-2 PLpro was implicated in antagonizing the host type I interferon and NF-κB pathways14. To this end, we further performed luciferase-based reporter assays to characterize the efficacies of YL1004 on the activation of IFN and NF-κB signalling with the expression of PLpro. Our luciferase reporter assays demonstrated that PLpro suppressed TNF-α–induced NF-κB activation, IFN-α–induced ISRE activation, and poly(I:C)–induced IFNB1/IRF3 promoter activity, whereas YL1004 counteracted these suppressions and restored the activation of the evaluated signaling pathways by targeting PLpro (Fig. 3f–i). To more comprehensively elucidate the underlying mechanism of YL1004 treatment, we performed transcriptomic profiling in PLpro-transfected cells with or without YL1004 treatment. Poly(I:C) was used as a surrogate for viral RNAs to stimulate innate immune responses. Our data indicated that PLpro expression broadly repressed genes involved in innate immunity (e.g., IFI6, ISG20, TNFRSF10D), autophagy (ULK4, SESN1), and mitochondrial stress response (FDXR, RASSF1), suggesting a concerted suppression of antiviral defenses (Fig. 4a). GO enrichment confirmed inhibition of immune-related pathways, including NF-κB and interleukin-7 signaling (Fig. 4b), consistent with functional immune suppression. YL1004 reversed these effects, restoring genes linked to immune activation (IFITM10, NFKBIB, CD86), oxidative stress control (GCLM, FDXR), and autophagy (DRAM1, MAP2K5). Enriched terms included “interleukin-1β production”, “NF-κB activation”, and “immunoglobulin response” (Fig. 4c). These findings indicate that YL1004 reactivates suppressed antiviral programs at transcriptional level.

Together, our findings indicate that YL1004 effectively antagonizes PLpro-dependent innate immune suppression, therefore further potentiating its antiviral efficacies. Further evaluation of its drug-like properties and efficacy is warranted to confirm its promise as an antiviral agent.

YL1004 protects against multiple SARS-CoV-2 variants and the nirmatrelvir-resistant SARS-CoV-2 mutant
To examine the in vitro antiviral potency of YL1004, we infected Calu3 with wildtype SARS-CoV-2 and variants (Delta, Omicron JN.1 and Omicron KP.3). Given the potency of YL1004 shown in enzymatic assays, we decided to perform 4-fold serial dilution starting from 10 μM. Our data suggested YL1004 dose-dependently decreased the subgenomic RNA of the viral envelope gene (sgE RNA) in Calu3 against wildtype and all tested SARS-CoV-2 variants. For wildtype, Delta and JN.1, YL1004 decreased viral gene copies by over 1 log at 2.5 μM (Fig. 5a). To better describe the in vitro antiviral efficacy of YL1004, we performed side-by-side comparison with Jun12682 we found YL1004 suppressed the replication of SARS-CoV-2 more efficiently at 2.5 and 10 μM when compared to Jun12682 (Supplementary Fig. 8). Additionally, we also examined its efficacy against the recombinant SARS-CoV-2 carrying the E166V substitution in Nsp5, which predominantly contributes to nirmatrelvir-resistance42–44. Our data showed that YL1004 inhibited the nirmatrelvir-resistant NSP5-E166V-rSARS-CoV-2 as effectively as that against Omicron KP.3. To examine the efficacy of YL1004 in reducing infectious viral progenies, we performed plaque reduction assays against the matching SARS-CoV-2 strains. In keeping with our findings with viral gene copy quantification, YL1004 efficiently decreased the infectious viral titers in SARS-CoV-2-infected Calu3, with the EC50 ranging from 0.08 μM to 0.50 μM (Fig. 5b). Based on the corresponding cytotoxicity values obtained in the same Calu3 cellular background (CC50 = 296.33 μM), the calculated selectivity indices (CC50/EC50) of YL1004 ranged from approximately 102 to 103 across tested viral variants, confirming a wide therapeutic window and favorable cellular safety profile (Supplementary Fig. 9).

Importantly, the EC50 of YL1004 against NSP5-E166V-rSARS-CoV-2 was 1.37 μM, suggesting its antiviral efficacy against the nirmatrelvir-resistant virus was largely not affected. In sharp contrast, E166V mutation caused up to 288-fold increase in the EC50 in vitro when treated with nirmatrelvir45. In conclusion, our in vitro data demonstrates that YL1004 potently protects against multiple SARS-CoV-2 variants and that YL1004 maintains its strong antiviral efficacy against the nirmatrelvir-resistant mutant virus.

YL1004 rescued infected mice from lethal SARS-CoV-2 challenge
To explore the in vivo antiviral potency of YL1004 against severe SARS-CoV-2 infection, we used the K18-hACE2 transgenic mouse model, which supports lethal SARS-CoV-2 challenge46,47. We first examined whether the in vivo pharmacokinetic exposure of YL1004 was sufficient to support antiviral activity. As shown in Supplementary Fig. 10, following oral administration of YL1004 at 100 mg/kg, plasma concentrations remained above the in vitro EC50 and EC90 values for more than 24 h and approximately 16 h, respectively. These data indicate that the in vivo antiviral efficacy of YL1004 is supported by sufficient systemic exposure. We next intranasally challenged the transgenic mice with 10000 PFU SARS-CoV-2 Omicron JN.1 and harvested the nasal turbinate and lung tissues at 3 days post infection (dpi.) for quantification of viral sgE RNA copies. Mice in the treatment groups were orally treated twice per day with 100 mg/kg of drug. We found that YL1004 treatment decreased viral loads in the nasal turbinates by 3.6-fold (P = 0.0457) when compared to vehicle group (Fig. 6a). Notably, YL1004 decreased viral gene copies by 26.1-fold (P = 0.0049) in the infected lung tissues, which is more potent than Jun12682 (4.6-fold decrease, P = 0.0132) (Fig. 6a). Consistently, YL1004 also lowered the infectious viral titers in the nasal turbinate lung tissues by 21- (P = 0.0107) and 13.4-fold (P = 0.0119), respectively (Fig. 6b). In addition to the virological assessments, we further evaluated whether YL1004 treatment might rescue mice from lethal SARS-CoV-2 challenge. Therefore, we infected the transgenic mice with the sublethal dose of SARS-CoV-2 Delta variant (1250 PFU/mouse). Oral treatment with YL1004 or vehicle was initiated at 1 h post virus challenge and was given twice per day. The infected mice treated with YL1004 experienced less body weight loss when compared to both vehicle (P = 0.0298) and Jun12682 (P = 0.0032) (Fig. 6c). In the survival analysis, only one out of ten mice (1/10, 10% survival) in the control group survived, which is in stark contrast with mice in the experiment group that all challenged subjects (10/10, 100% survival, P < 0.0001) were rescued by YL1004 therapeutic treatment at 14 dpi. (Fig. 6d). We continued to examine the expression of viral antigen in lung tissues of the infected transgenic mice. While multi-focal patches of viral nucleocapsid proteins were detected by immunohistochemistry staining (indicated with black arrows) in the control mice, viral antigen was rarely detected in the YL1004-treated mouse lungs (Fig. 6e). Besides, pathological findings including inflammatory infiltrations, alveolar septa thickening hemorrhage, alveolar hyperinflation and alveoli collapse were extensively identified in the control mouse lungs (Fig. 6f). In comparison, virus-imposed pathological changes were significantly alleviated in the YL1004-treated mice and only mild alveolar deformation was found (Fig. 6f). Together, we conclude that YL1004 demonstrates potent in vivo antiviral efficacies as evidenced by the marked reduction of viral burdens, viral antigen expression and virus-induced pathological lesions in target organ tissues. Importantly, YL1004 is capable of rescuing infected animals from lethal SARS-CoV-2 challenge.

Methods
Ethical Statement
All procedures related to animal in pharmacokinetic (PK) study and in vivo toxicity study were performed according to the guidelines approved by the Institutional Animal Care and Use Committee of West China Hospital, Sichuan University (20211063A). The use of animals in antiviral study complied with all relevant ethical regulations and was approved by the Committee on the Use of Live Animals in Teaching and Research of The University of Hong Kong (24–128). The animals were kept in cages with individual ventilation under 65% humidity and an ambient temperature of 21 °C–23 °C and a 12 h–12 h day–night cycle for housing and husbandry. All donors of the virus strains involved in this paper have provided written consent and the work was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster. All infectious experiments involving MERS-CoV, SARS-CoV-2 and SARS-CoV-1 followed the approved standard operating procedures of the Biosafety Level 3 facility at the Department of Microbiology, Hong Kong University.

Compounds, cell lines, and viruses
The compounds (1a, 2a-h, 3a-i, 4a-l, 5a-h, 6a-n) shown in Fig. 1 and Fig. 2 were synthesized in our internal facilities. And the detailed synthesis methods and characterization data of these are listed in the supplementary materials (Supplementary Table. 1). MDCK (Cell Bank, Chinese Academy of Sciences, Shanghai, SCSP-5258), HUVEC (CCTCC, Wuhan, GDC0635), BEAS-2B (Cell Bank, Chinese Academy of Sciences, Shanghai, GNHu27), VeroE6 (ATCC, CRL-1586), and HEK293T (Cell Bank, Chinese Academy of Sciences, Shanghai, GNHu17) cells were maintained in Dulbecco’s Modified Eagle medium (DMEM, Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 unit/mL penicillin and 100 μg/mL streptomycin. Calu3 cells obtained from ATCC were maintained in DMEM/F12 supplemented with 10% FBS, 1% P/S. VeroE6-TMPRSS2 cells were obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank and cultured also in 10% FBS, 1% P/S DMEM and 1 mM (1%) sodium pyruvate.

WTSARS-CoV-2 HKU-001a (GenBank: MT230904), B.1.617.2/Delta (GenBank: OM212471), B.1.1.529/Omicron JN.1 (GISAID: EPL_ISL_18841631) and KP.3 (GISAID: EPI_ISL_19535068) were isolated from patients with laboratory confirmed COVID-19 in Hong Kong48. Recombinant SARS-CoV-2 with nirmatrelvir resistance was constructed as we previously reported49. Briefly, the cDNA from ancestral SARS-CoV-2 HKU001a was assembled into the pSMART-BAC vector (E2621S, NEB, USA), followed by site-directed mutation of E166V in NSP5 which was confirmed by Sanger sequencing. The recombinant virus was rescued by transfection of the infectious clone into VeroE6-TMPRSS2 cells using Lipofectamine 3000 (L3000015, Thermo Fisher Scientific). All viruses were cultured in VeroE6-TMPRSS2 cells and titrated with plaque assays.

Protein Expression and Purification
The gene encoding SARS-CoV-2 (MN908947) papain-like protease (PLpro; ORF1ab residues 1564-1877) with an N-terminal His tag and TEV protease cleavage site was synthesized by General Biosystems following E. coli codon optimization. Using the recombinant plasmid as a template, site-directed mutagenesis PCR was performed with primers 5’-agataataatagttatctggcaaccgcactgctgaccctg-3’ and 5’-tgccagataactattattatctgcccacttaatactggtc-3’ (mutated codons shown in italics) to mutate the catalytic cysteine to serine (PLpro-Cys111Ser). The PCR product was digested with DpnI (TakaRa) and transformed into E. coli DH5α competent cells (Novagen). Single colonies were selected, cultured, and sequenced for validation.

Subsequently, the PLpro or PLpro-Cys111Ser recombinant plasmid was transformed into E. coli BL21(DE3) competent cells (Novagen). Cells were cultured until OD600 reached ~0.8, followed by induction with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 18 °C for 18 h. Cells were harvested by centrifugation at 7656 xg, and the pellets were resuspended in Buffer A (20 mM HEPES pH 7.5, 10 mM imidazole, 500 mM NaCl, 5 mM DTT and 10% glycerol). After sonication on ice, lysates were centrifuged at 38,759 xg (4 °C) to collect the supernatant. Target proteins were enriched via a single Ni-NTA resin column and eluted with Buffer B (20 mM HEPES pH 7.5, 500 mM imidazole, 500 mM NaCl, 5 mM DTT and 10% glycerol). The His tag was removed by TEV protease digestion, and the solution was exchanged into Buffer C (20 mM HEPES pH 7.5, 150 mM NaCl and 5 mM DTT). A second single Ni-NTA resin column purification step yielded tag-free PLpro or PLpro-Cys111Ser. Final purification was achieved by gel filtration (Superdex 75 Increase 10/300 GL, GE Healthcare) in Buffer C. The protein purity of PLpro or PLpro-Cys111Ser was confirmed by SDS-PAGE.

Crystallization and Structure Determination
The PLpro-Cys111Ser (~ 10 mg/mL) was incubated with compound YL1004 at a 1:10 molar ratio overnight at 4 °C. Crystals were grown at 4 °C using the sitting-drop vapor diffusion method with 1 μL protein-ligand mixture + 1 μL reservoir solution. Initial crystals were observed under condition No. 7 from the Wizard Classic 1: 10% (w/v) PEG 8000, 100 mM MES/Sodium hydroxide pH 6.0, 200 mM Zinc acetate. High-quality crystals were obtained through optimized condition: 6% (w/v) PEG 8000, 100 mM MES/Sodium hydroxide pH 6.0, 200 mM zinc acetate and 20% PEG 400. These crystals were subsequently harvested and flash-cooled in liquid nitrogen.

Diffraction data for the PLpro-Cys111Ser-YL1004 complex was collected at beamline BL19U1 of the Shanghai Synchrotron Radiation Facility (SSRF) using a wavelength of 0.97861 Å. The data were processed with XDS1 and scaled using CCP4 Aimless50. The crystal structure was solved by molecular replacement with CCP4 Molrep51, using the PLpro structure (PDB: 7YBG) as the search model. Iterative refinement was performed with Phenix.refine52 and CCP4 Refmac53, followed by manual model adjustments in Coot54. Supplementary Table. 13 presents the diffraction data sets and statistics of final refinement.

Enzyme Activity Assays
FRET-based assay for PLpro and other DUBs enzymatic activity characterization. The reaction buffer contained 20 mM HEPES (pH 7.5). For viral polyprotein cleavage, 22.5 μL PLpro (200 nM final) was preincubated with 2.5 μL compounds in black 96-well plates (WHB) for 10 min at RT. Reaction initiated with 25 μL Z-RLRGG-AMC substrate (20 μM final). Fluorescence (Ex360/Em460) monitored using CLARIOstar microplate reader (BMG Labtech). Initial linear-phase readings (7–8 cycles) analyzed by simple linear regression in GraphPad 10.0 to calculate V0. Inhibition (%) normalized to DMSO control and IC50 determined via dose-response modeling.

In the primary screen, each compound was tested at a final concentration of 30 μM. Inhibition was normalized to DMSO controls, and compounds showing more than 70% inhibition were defined as primary hits. To ensure hit specificity and chemical stability, known PAINS were excluded. Compounds containing reactive or unstable moieties, such as free aldehydes, quinones, or redox active groups were manually reviewed and deprioritized. Autofluorescent compounds were identified by measuring compound only emission signals and excluded when interference was observed. Selected compounds were further analyzed by LC–MS to confirm structural stability under assay conditions (Supplementary Table. 14).

For deISGylation and deubiquitination, 8.82 μL PLpro or other DUBs (indicated concentration) preincubated with 0.18 μL compounds in black 384-well plates (Corning) for 10 min at RT. Reaction initiated with 9 μL ISG-AMC or Ub-AMC (0.25 μM final). Fluorescence monitored with 20 initial linear-phase cycles. V0 and IC50 calculated as above.

Differential scanning fluorimetry assay
PLpro (2 μM final) was mixed with compounds in white 8-strip PCR tubes using 20 mM HEPES (pH 7.5) buffer (10 μL total volume). Reactions contained 1 μL 10000× Orange dye (Sigma). Fluorescence was monitored during 1.5 °C/min heating (20 °C–95 °C) using a BioRad CFX96 RT-PCR system. Melting temperature (Tm) and thermal shift (ΔTm) were calculated via Boltzmann model in GraphPad 10.0.

Cytotoxicity assay
VeroE6/BEAS-2B/HUVEC cells were seeded in clear 96-well plates (100 μL/well) and cultured overnight. Serial compound dilutions (500 μM start, 2-fold steps) in 100 μL medium were added, with DMSO controls and blank medium. After 72 h treatment, 20 μL MTT (5 mg/mL in saline; Sigma) was added per well. Plates were incubated 1–2 h at 37 °C until DMSO controls showed purple formazan precipitation. After dissolving formazan with 50 μL DMSO/well, absorbance at 560 nm was measured using CLARIOstar Plus. CC50 values were calculated via dose-response modeling in GraphPad 8.0.

PK properties in vivo
Male Balb/c mice (6–8 weeks, 18–25 g) and SD (Sprague Dawley) rats (age: 7-11 weeks) were received test compounds prepared in 5% DMSO/ 5% HS-15/ 20% PEG300/ 70% saline (Supplementary Table. 3 and 4). Blood samples collected in K2-EDTA tubes at designated times were centrifuged to obtain plasma. Compound concentrations determined by LC-MS/MS were used for PK parameter calculation (Phoenix WinNonlin 7.0).

In vivo toxicity study
YL1004 was evaluated in Balb/c mice (6-8 weeks, 16-25 g, 50% male/female) formulated in 5% DMSO/ 3% HS-15/ 20% PEG300/ 75% sterile water. Acute toxicity: single 250 mg/kg oral dose; repeat-dose: 100 mg/kg twice daily for 14 days. Animals were monitored for 28 days (dosing + post-treatment) for weight loss, appetite, activity, and behavior.

Western Blot Analysis
For deubiquitination activity assessment, HEK293T cells were transfected with plasmids encoding Ub-HA and Flag-PLpro (or empty pcDNA3.1 vector) and treated with compounds for 36 h. Immunoblotting utilized primary antibodies: HA-tag (Cell Signaling Technology, #3724, 1:1000, rabbit), DYKDDDDK Tag (Cell Signaling Technology, #14793, 1:1000, rabbit), and GAPDH (Proteintech, 60004-1-Ig, 1:1000, mouse).

DeISGylation activity was evaluated in IFN-α (Sigma, SRP4595)-stimulated A549 cells treated with PLpro and/or compounds for 48 h. Proteins were resolved on 10% SDS-PAGE gels and probed with ISG15 (Thermo Fisher, #703131, 1:1000, rabbit), PLpro (Life Sensors, #AB602, 1:250, chicken), and GAPDH (Proteintech, 60004-1-Ig, 1:1000, mouse).

For HA-Ub-VS DUB labeling, HEK293T cell lysates (150 mM NaCl, 0.1% NP40, 50 mM Tris-HCl, 1 mM DTT, pH 8.0) were incubated with HA-Ub-VS probe in reaction buffer (150 mM NaCl, 50 mM Tris-HCl, 1 mM DTT, pH 7.5) alongside PLpro and/or compounds. Samples were analyzed by 10% SDS-PAGE, followed by immunodetection with HA-tag (Cell Signaling Technology, #3724, 1:1000, rabbit), PLpro (Life Sensors, #AB602, 1:250, chicken), and GAPDH antibodies.

Luciferase Reporter Assay
HEK293T cells were co-transfected with plasmids encoding NF-κB-Luc/Renilla/Flag-PLpro, ISRE-Luc/Renilla/Flag-PLpro, or IRF3-Luc/IFNB1-Luc/Renilla/Flag-PLpro. Transfected cells were compound-treated and stimulated with TNF-α (MCE HY-P7058), IFN-α (Sigma SRP4595), or poly(I:C) (MCE HY-107202), respectively. Dual-luciferase activity was quantified using the Dual Luciferase Reporter Assay Kit on a CLARIOstar plate reader (BMG Labtech).

Transcriptomics
HEK293T cells were transfected with plasmids encoding Flag-PLpro (or vector), treated with the indicated compounds, and stimulated with poly(I:C) (MCE, HY-107202) according to the experimental design. Cells were harvested 18–24 h after treatment for RNA extraction. Total RNA was extracted using TRIzol Reagent (Thermo Fisher, cat. no. 15596026) and treated with DNase I (NEB, cat. no. M0303L). RNA purity and concentration were assessed using NanoDrop OneC and Qubit 3.0 (Qubit RNA BR Assay kit, Thermo Fisher, cat. no. Q10210), respectively, and RNA integrity was evaluated using LabChip GX Touch (Revvity). RNA-seq library preparation, high-throughput sequencing (DNBSEQ-T7, PE150), and data analysis were performed by Seqhealth Technology Co., Ltd. (Wuhan, China) using the KC Digital mRNA Library Prep Kit with a 12-nt unique molecular identifier to reduce PCR duplication bias.

Evaluation of in vitro antiviral activity
SARS-CoV-2 or rSARS-CoV-2 stock viruses were diluted and premixed with the tested compounds at designated concentrations to infect Calu3 cells at 0.01 MOI. At 2 hpi., the inoculum was removed and replaced with supernatants supplemented with inhibitors. Cells were incubated at 37 °C until sample harvest. At 24 hpi. supernatants were harvested for infectious viral titration with plaque assays. Cell lysates were harvested for quantification of viral gene copies by RT-qPCR with specific probe and primers (Forward: CGATCTCTTGTAGATCTGTTCTC; Reverse: ATATTGCAGCAGTACGCACACA; Probe: FAM-ACACTAGCCATCCTTACTGCGCTTCG-BBQ).

SARS-CoV-2 infection in K18-hACE2 mice
8-to-12-week-old male and female K18-hACE2 transgenic mice anaesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine were intranasally inoculated with 1250 PFU SARS-CoV-2 Delta (for survival study) or 1000 PFU SARS-CoV-2 Omicron JN.1 (for sample harvest). For oral delivery, all drugs were formulated with 10% DMSO, 10% Kolliphor HS 15, 30% polyethyleneglycol 400 (PEG400) in normal saline and given to the animals at 100 mg/kg twice per day. Nasal turbinate and lung tissues were harvested at 3 dpi. for virological assessment by RT-qPCR and plaque assays. For survival study, body weight and survival of the infected mice will be monitored for 14 days or until death of the animal as we previously reported55.

RNA extraction and RT-qPCR
Viral RNA was extracted from infected cells using QIA symphony RNA Kit (931636, Qiagen, Germany). Viral RNA from mice lung and nasal turbinate samples were extracted with the RNeasy Mini kit (74106, Qiagen). After RNA extraction, RT-qPCR was performed using QuantiNova Probe RT-PCR Kit (208354, Qiagen) or QuantiNova SYBR Green RT-PCR Kit (208154, Qiagen) with the LightCycler 480 Real-Time PCR System (Roche).

Plaque assays
To determine the infectious virus titres, supernatants from infected cells or organ tissues were ten-fold serially diluted and inoculated into monolayered VeroE6-TMPRSS2 cells with 2 h incubation at 37 °C, followed by 1% low-melting point agarose overlay (16520050, Thermo fisher, USA). The cells were further incubated for 48 h (for SARS-CoV-2 wild type and Delta) or 72 h (for Omicron JN.1 and KP.3) before fixation with 4% formaldehyde for visualization with 0.5% crystal violet diluted in 25% ethanol/distilled water.

Histology and immunohistochemistry staining
Harvested lung tissues from infected mice were fixed with 10% neutral buffered formalin with overnight incubation. Fixed tissues were processed, embedded with paraffin and sectioned into tissue slides at 5 µm thickness. SARS-CoV-2 nucleocapsid protein in the tissue slides was detected using in-house rabbit polyclonal anti-SARS-CoV NP antibody, followed by incubation with biotinylated rabbit anti-guineapig IgG (H+L) (ab6770, Abcam, UK) or biotinylated goat anti-rabbit IgG (H+L) secondary antibody (BA-1000, Vector laboratories, USA). Specificity of the in house primary antibody was validated as previously described56. Color development was carried out with VECTASTAIN® ABC-AP Kit and Vector Red substrate kit (AK-5000 and SK-5100, Vector Laboratories) according to the manufacturer’s instructions. The nuclei counterstaining was performed by Gill’s haematoxylin followed by mounting with Vectamount permanent mounting medium. For H&E staining, the tissue sections are stained with Gill’s haematoxylin (H-3401-500, Vector Laboratories) and eosin-Y. All images are acquired by Olympus BX53 light microscope.

Statistical analysis
All statistical analyses were performed using GraphPad Prism versions 7.0 and 8.0. Data in the figures are presented as mean ± SD (standard deviation). The number of biological replicates (n) is indicated in the corresponding figure legends and table footnotes, and each biological replicate was measured in at least duplicate as technical replicates. Statistical comparison between different groups was performed by one-way analysis of variance (ANOVA) or log-rank (Mantel-Cox) test. All t-test performed in this study is two-tailed. Differences were considered statistically significant when P < 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Nan et al. report the discovery of a SARS-CoV-2 PLpro’s orally bioavailable inhibitor, YL1004, which features a novel chemical scaffold and exhibits potent in vitro inhibitory activity, low cellular cytotoxicity, robust in vivo antiviral efficacy, and the ability to restore host antiviral immune responses antagonized by SARS-CoV-2 infection.\nThe study began with high-throughput screening that identified compound 1a. The authors subsequently optimized 1a by merging it with the classical PLpro inhibitor GRL0617, yielding compounds 2a, 2b, and 2c, which displayed improved IC₅₀ values and ΔTm. Structure–activity relationship studies were then performed by systematically modifying the R1, R2, and R3 groups of compound 2b. Through these efforts, YL1004 was identified as a lead compound. In both in vitro and in vivo assays, YL1004 outperformed the current best-in-class inhibitor Jun12682. Furthermore, the co-crystal structure of the PLpro–YL1004 complex was determined, providing mechanistic insights into its mode of action.\nOverall, this manuscript reports the discovery of a novel PLpro inhibitor with clear improvements over current leading compounds, including Jun12682 and PF-07957472. The study is well-designed, integrates multiple complementary approaches, and provides both mechanistic and therapeutic insights. Nevertheless, there remain several issues that should be addressed before the manuscript can be considered for publication.\nMajor Comments:\n1. In Fig. 1a and lines 100–103, the authors describe analog 2c as a derivative of GRL0617 by replacing the phenylmethyl group with a phenylamino group. However, another key difference is the N-methylpiperazinyl group present in 2c but absent in GRL0617. A more rigorous side-by-side comparison with GRL0617 is necessary to clarify the contributions of these modifications.\n2. The manuscript introduces the terms “fragment fusion” and “fragment assembly” (Fig. 1a). In medicinal chemistry, the concept of “fragment” is more commonly associated with fragment-based drug discovery (FBDD), where “fragment merging” is a standard term. The authors should reconsider the terminology for consistency with established usage.\n3. The data presented in Fig. 2 and Extended Data Figs. 1–3 are dense and difficult to follow. Reorganizing these results into well-structured tables would significantly improve clarity. Moreover, the structure–activity relationship (SAR) analysis could be expanded and presented in a format more commonly seen in medicinal chemistry studies.\n4. In the Results and Methods sections, more details about the in-house chemical library screening are required. How was the screening performed, and what were the overall outcomes beyond the top five compounds? Providing this information will enhance reproducibility.\nMinor Comments:\n1. The abbreviation “Nsp1-3” (Page 3, Line 55) should be written out in full at its first occurrence.\n2. In Fig. 2, the unit of AUC is shown as µg/L·h. Please ensure consistent notation across the manuscript, including Supplementary Table 11.\n3. On Page 7, Lines 156–157, the authors state that “The SARS-CoV-2 PLpro comprises four domains: an N-terminal ubiquitin-like (Ubl) domain, followed by thumb, palm, and finger domains.” In fact, thumb, palm, and finger represent three subdomains within the larger ubiquitin-specific protease (USP) domain. This should be corrected.\n4. In Fig. 3c, the electron density should be shown using the 2Fo–Fc map after Refmac refinement, rather than the Fo–Fc map.\n5. In Fig. 4c legend, the authors state that HEK293T cells were treated with or without IFN-β for 48 h. However, both the figure and methods indicate IFN-α was used to induce ISGylation. Please clarify which interferon was employed.\n6. In Figs. 5 and 6, the “P” values in the statistical analysis should be italicized in accordance with journal formatting guidelines.\n7. Line 36: The term “nirmatrelvir-resistant recombinant SARS-CoV-2” should be clarified. Does this refer to the Mpro E166V mutant recombinant virus?\n8. Line 68: Grammar correction needed: “Recent advances with orally active PLpro inhibitors … in murine SARS-CoV-2 models ARE promising.”\n9. References 14 and 39 are duplicates and should be consolidated.\n10. Two recent studies (PMID: 37130168 and PMID: 37130166) have reported the immunoregulatory functions of PLpro. The authors are encouraged to cite and briefly discuss these works in the manuscript.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "This manuscript presents a well-executed study describing the discovery and characterization of YL1004, a novel SARS-CoV-2 PLpro oral antiviral drug candidate. The authors employed an innovative approach that combined high-throughput screening (HTS) with fragment fusion using GRL0617, followed by systematic chemical optimization. The work demonstrates strong translational potential, with YL1004 showing high potency in enzymatic assays. The mechanism of action is confirmed by an X-ray co-crystal structure, and the compound exhibits favorable pharmacological properties, efficacy in cell-based assays, activity against nirmatrelvir-resistant viruses, and strong protection in a SARS-CoV-2-infected mouse model.\nThe manuscript will be suitable for publication in Nature Communications upon minor modifications, once the authors have addressed the following comments:\n1. While recent advances with orally active PLpro inhibitors in murine models such as Jun12682, PF-07957472, and GZNL-P36 are reported, we would suggest including other recent such as WEHI-P8 (DOI: 10.1038/s41467-025-57905-4) and Jun13296 (DOI: 10.1038/s41467-025-56902-x). Note that many of them have either a naphthalene or quinoline moiety. Some comparison in terms of superposition of the co-crystal structures would be of interest to readers, even if it is just in the Supporting Materials.\n2. Enzymatic Assay Methodology - Tight Binding Kinetics: The enzymatic assay method does not refer to any previous article. Is this a new method? Or is it based on a previous one? If tight-binding kinetics were not explicitly considered, this should be addressed, as it may impact the accuracy of reported IC50 values. The Morrison equation may be more appropriate than standard IC50 calculations for tight-binding inhibitors, especially YL1004, both for the protease and deubiquitanse/de-ISGlase activities. Related to this, the concentration of enzyme utilized (200 nM) may also be a bit too high for tight-binding inhibitors, with reports going as low as 50 nM PLpro for enzymatic inhibition assays (see, for example, DOI: 10.1038/s41467-025-56902-x). Note that Morrison equation fitting might be possible to perform even with the same experimental raw data (see https://www.graphpad.com/guides/prism/latest/curve-fitting/reg_morrison.htm).\n3. Rationale for the naphthalene 4-position Chemistry Exploration: While the manuscript describes the chemical optimization process, the strategic rationale for exploring the naphthalene 4-position modifications is not clearly articulated (for instance, the Pfizer inhibitor or Jun13296 have a quinoline 3-position substituent instead). Adding a brief explanation would be interesting. Was this guided by computational modeling, initial SAR data, analysis of the GRL0617 co-crystal structure, or other considerations?\n4. Figure-Related Issues:\n-In figures 1 and 2, the orientation of the compounds is different than in Fig. 3, that also shows the crystal structure with the inhibitor in the same orientation as in 3a. Given that most of the structural papers display the compounds as in Fig. 3, it would make sense to do the same in Figs. 1 and 2.\n-Figure 1: in panel a, please indicate either in the caption or in the panel that DSF was used to obtain the delta Tm values (this is well explained in the text but not here). Please verify the color scheme and ensure consistency between the figure and its legend in 1b, as GRL0617 is displayed in pink, not in yellow.\n-Figure 6: There is an apparent typographical error with \"p1004\" appearing instead of \"YL1004.\" Please correct this throughout the figure and verify all compound names are consistent.\n-Figure 3a - Stereochemistry: it is not clear whether the stereochemical representation of the chemical structure is consistent to Figure 1. YL1004 may be with the S isomer, correct? Please, clarify and edit if needed.\nMinor:\nP. 2 line 36: add “strain” at the end of the sentence.\nP. 2 lines 39-41: What does it mean by “a distinctive multidimensional binding mode”? The sentence is overly verbose. Please, state in a more precise and understandable way.\nP. 6 line 121: change verbal tense to past.\nP. 8 lines 180-183: This sentence is too complicated; consider splitting it into two.", "reviewer_4": "Reviewer report\nManuscript: YL1004 is a SARS CoV 2 papain like protease inhibitor with immunomodulatory and antiviral activity in mice\nKey results\nNan et al. introduce YL1004, a rationally designed, orally active inhibitor of the SARS CoV 2 papain like protease (PLpro). Starting from a fragment fusion concept that blends an in house molecular library hit of a hetrotricyclic with features of GRL0617, the authors work through clear SAR to a lead that engages PLpro structurally, strongly inhibits PLpro activity, restores antiviral signaling in cells, suppresses replication across several SARS CoV 2 variants, and improves outcomes in a stringent mouse model (full survival of mouse sample population. This study represents a rational and comprehensive design and characterization of a novel PLpro inhibitor having very promising activity in mouse models.\nValidity\nOverall, the evidence thoughtful and presented well: medicinal chemistry rationale → on target engagement → cellular mechanism → antiviral efficacy → in vivo benefit. The structural data provide a persuasive on target anchor, a 2.7 Å co crystal structure (PDB 9U7D) that locates YL1004 in the BL2/S3–S4 pocket; the IC50 and EC50 values of a series of lead compounds support grat promise as PLpro inhibitors, sub micromolar antiviral activity across wildtype, Delta, and Omicron sublineages including JN.1 and KP.3; and full survival protection in a Delta challenge where vehicle survival is 10%; and the cell based deubiquitination/deISGylation rescue, together with innate immune reporter recovery, tie the biochemical activity to cellular consequences. Antiviral data align with the target story and extend to a nirmatrelvir resistant virus (NSP5 E166V), which is a thoughtful orthogonal test of relevance.\nSignificance\nPLpro remains an important, underexploited antiviral target because it couples viral polyprotein processing with antagonism of innate immunity. A well characterized, selective, orally bioavailable inhibitor that shows cross variant antiviral activity and survival benefit in vivo is notable. The manuscript’s integrated view—chemistry, mechanism, virology, and animal efficacy—will be useful to both the PLpro community and the broader antiviral field, especially with a deposited structures, data, and assay results not focused on in this manuscript, as well as the clear and transparent design narrative.\nData and methodology\nThe work is generally well designed and well reported. The discovery path is easy to follow (screen → fragment fusion → regional optimization → lead selection → biochemical/biological assays), with enough intermediate disclosure to be instructive without overwhelming the reader. The structural biology and the cellular mechanism assays are appropriate and mutually reinforcing, and the in vivo studies use a recognized lethal model with virology, histology/IHC, body weight, and survival endpoints. Where a little more context would help: linking on study exposure (infected mice) to in cell potency targets, and presenting selectivity indices (CC50/EC50) in the same cellular backgrounds used for antiviral testing.\nAnalytical approach\nAs a non-expert in statistical methodology, I find the analyses chosen for the biochemical, cellular, and animal studies broadly appropriate for the study designs and presented with sufficient transparency to follow the results. The manuscript and figure legends indicate standard group-comparison and survival analyses and summarize data with means and variability, which is adequate for interpreting the findings.\nTo further aid reproducibility, please state consistently what constitutes biological vs. technical replicates across where appropriate, including all relevant figures and in the Supplemental Information. For example, early figure captions reference “three independent experiments” and “three technical replicates,” whereas later legends specify that “each data point represents one biological repeat” and that data come from “two or three independent experiments.” Unifying this terminology in the figure captions and adding a brief definition in the Supplement would make replication straightforward.\nSuggested improvements\n1. Figure 4d could use some clarification, along with the Human DUB assay against YL1004. There could also be additional discussion in the text on why there is selectivity. Is there a rational for this selectivity of YL1004 for PLpro vs. DUB structural homologs? Is there a structural basis for this, residue interactions?\nThe figure is unclear, is the raw DUB inhibition assays only at one YL1004 concentration of many, could they be reported as IC50s?\nThis is not essential to publishing but would help with impact as a major challenge of PLpro drug design is selectivity against human DUB enzyme homologs, accordingly, this warrants some more discussion/rationale.\n2. The term “broad spectrum” is used, consider “cross variant SARS CoV 2 activity,” since non SARS CoV 2 coronaviruses were not tested.\nClarity and context\nThe narrative is clear and accessible with figures adequately supporting the text. The head to head benchmarking with Jun12682 is useful; ensuring the matched conditions for those comparisons are fully described in the Supplemental Information will make those claims easier to evaluate as YL1004 is competing for a new benchmark PLpro inhibitor.\nReferences\nCoverage is broad and current, spanning PLpro biology, earlier chemotypes, recent mouse efficacious PLpro series, and nirmatrelvir resistance. The manuscript is well situated in the literature.\nYour expertise\nMy expertise is in chemistry and biochemistry with recent experience in medicinal chemistry, specifically anti-viral drug design and characterization of PLpro inhibitors.\nArguments for and against publication\nFor: Coherent design to efficacy story with structural validation, cellular mechanism rescue, cross variant antiviral activity (including an E166V recombinant virus), and survival benefit in a lethal mouse model—uncommon to see all results so successful in one PLpro paper.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response to Reviewers’ Comments\nReviewer #1 (Remarks to the Author):\nIn this manuscript, Nan et al. report the discovery of a SARS-CoV-2 PLpro’s orally\nbioavailable inhibitor, YL1004, which features a novel chemical scaffold and exhibits\npotent in vitro inhibitory activity, low cellular cytotoxicity, robust in vivo antiviral\nefficacy, and the ability to restore host antiviral immune responses antagonized by\nSARS-CoV-2 infection.\nThe study began with high-throughput screening that identified compound 1a. The\nauthors subsequently optimized 1a by merging it with the classical PLpro inhibitor\nGRL0617, yielding compounds 2a, 2b, and 2c, which displayed improved IC₅₀ values\nand ΔTm. Structure–activity relationship studies were then performed by\nsystematically modifying the R1, R2, and R3 groups of compound 2b. Through these\nefforts, YL1004 was identified as a lead compound. In both in vitro and in vivo assays,\nYL1004 outperformed the current best-in-class inhibitor Jun12682. Furthermore, the\nco-crystal structure of the PLpro–YL1004 complex was determined, providing\nmechanistic insights into its mode of action.\nOverall, this manuscript reports the discovery of a novel PLpro inhibitor with clear\nimprovements over current leading compounds, including Jun12682 and PF-07957472.\nThe study is well-designed, integrates multiple complementary approaches, and\nprovides both mechanistic and therapeutic insights. Nevertheless, there remain several\nissues that should be addressed before the manuscript can be considered for publication.\nMajor Comments:\n1. In Fig. 1a and lines 100–103, the authors describe analog 2c as a derivative of\nGRL0617 by replacing the phenylmethyl group with a phenylamino group. However,\nanother key difference is the N-methylpiperazinyl group present in 2c but absent in\nGRL0617. A more rigorous side-by-side comparison with GRL0617 is necessary to\nclarify the contributions of these modifications.\nResponse:\nWe sincerely appreciate the reviewer for enthusiastically underscoring the significance\nand novelty of our work and positively assessing the overall quality of our manuscript.\nWe acknowledge this insightful suggestion and agree that a comparative analysis with\nGRL0617 is crucial to clarify the contributions of specific structural modifications. To\nthis end, we synthesized compound 2d, which differs from GRL0617 only by the\nreplacement of the methyl group with an amino group on the benzene ring. Enzymatic\nevaluation revealed a substantial decrease in activity for 2d (IC = 10.97 ± 0.33 μM)\n50\nrelative to GRL0617. Correspondingly, differential scanning fluorimetry (DSF)\nindicated reduced thermal stabilization (ΔTm = 2.67 ± 0.33 °C), suggesting weaker\nbinding affinity. This loss of activity may be attributed to the absence of both the N-\nmethylpiperazinyl group and the (S)-tricyclic core, which appear essential for properly\norienting the anilino moiety to form hydrogen bonds with PLpro. To further probe the\nrole of the N-methylpiperazine substitution, we prepared analog 2c by introducing this\ngroup into the 2d scaffold. Compound 2c showed improved potency (IC = 337.7 nM)\n50\nand a greater thermal shift (ΔTm = 9.13 °C) compared to 2d, yet its activity remained\n3.4-fold lower than that of the (S)-enantiomer 2b (IC = 98.6 nM, ΔTm = 10.04 °C).The\n50\nsignificant activity difference between 2a and 2b further highlights the importance of\nthe tricyclic scaffold’s stereochemistry for PLpro inhibition. Together, these results\nunderscore the synergistic contribution of the (S)-tricyclic core and the anilino\nmodification to inhibitor binding (Fig. 1).\nIn summary, these results demonstrate that the (S)-tricyclic architecture and anilino\ngroup are indispensable for anti- PLpro efficacy, affirming the structural novelty of our\ninhibitors.\nFig. 1 | Discovery process of new tricyclic PLpro inhibitors.\na, Chemical structures of Hit-1 (1a), 2a, 2b, 2c 2d and design strategy of tricyclic PLpro\ninhibitors by rational fragment merging. IC values were determined using a FRET-\n50\nbased enzymatic assay measuring viral polyprotein cleavage inhibition, and ΔTₘ values\nwere obtained from differential scanning fluorimetry (DSF). b, 1a (blue) partially\noverlaps with the benzamide group of classical PLpro inhibitor GRL0617 (pink) through\nmolecular superposition. c, Dose-activity curves of 2a, 2b, 2c, 2d and GRL0617 against\nSARS-CoV-2 PLpro in the FRET assay. d, Differential scanning fluorimetry analysis of\nthe effect of 2a, 2b, 2c, 2d and GRL0617 on SARS-CoV-2 PLpro stability. Exposure of\nhydrophobic residues monitored by an increase in relative fluorescence units (RFUs).\nCurves represent the average of three experiments. e, Structural optimization regions\nof compound 2b and the molecular structure and drug properties of YL1004. IC\n50\nvalues were determined by FRET enzymatic assay. Cytotoxicity CC was determined\n50\nin Vero E6 cells with 72-hour incubation. Data in a, c, d, and e are presented as mean\n± SD (n = 3 biological replicates). Images in b were processed by using PyMOL\n(https:// pymol.org).\n2. The manuscript introduces the terms “fragment fusion” and “fragment assembly”\n(Fig. 1a). In medicinal chemistry, the concept of “fragment” is more commonly\nassociated with fragment-based drug discovery (FBDD), where “fragment merging” is\na standard term. The authors should reconsider the terminology for consistency with\nestablished usage.\nResponse:\nWe appreciate the reviewer’s professional suggestions and have standardized the\nterminology throughout the manuscript to “fragment-based drug discovery (FBDD)”\nand “fragment merging”.\n3. The data presented in Fig. 2 and Extended Data Figs. 1–3 are dense and difficult to\nfollow. Reorganizing these results into well-structured tables would significantly\nimprove clarity. Moreover, the structure–activity relationship (SAR) analysis could be\nexpanded and presented in a format more commonly seen in medicinal chemistry\nstudies.\nResponse:\nWe thank the reviewer for the suggestion to improve the clarity of Fig. 2 and Extended\nData Figs. 1-3. Accordingly, we have reorganized the enzymatic inhibitory activities,\nand preliminary evaluations of drug-like properties for representative tricyclic PLpro\ninhibitors and Jun12682 into a structured format in Table 1. As comprehensive profiling\ndata (including PLpro IC , VeroE6 CC , major cytochrome P450 isoforms, and PK\n50 50\nproperties) are not available for all compounds originally presented in the Extended\nData Figs. 1–3, we have retained the original figures to ensure a complete display of\ntheir chemical structures and associated data (see Supplementary Fig. 1-3). We hope\nthe reviewer will agree that presenting the full chemical dataset in this manner is\nsuitable, as it follows a precedent validated by previous reports on compounds\nJun126861 and Jun132962.\n4. In the Results and Methods sections, more details about the in-house chemical library\nscreening are required. How was the screening performed, and what were the overall\noutcomes beyond the top five compounds? Providing this information will enhance\nreproducibility.\nResponse:\nWe appreciate the reviewer’s insightful comment. The compound library used in this\nstudy primarily consists of structurally diverse small molecules that have been designed\nand synthesized by our research group in previous antiviral discovery programs. The\nlibrary includes both covalent and non-covalent inhibitors with electrophilic or\nhydrogen-bonding functionalities suitable for targeting cysteine proteases such as PLpro.\nThe compounds have molecular weights ranging from 300–600 Da, and cover a wide\nphysicochemical space. In addition, the library exhibits a broad spectrum of lipophilic\nand hydrophilic characteristics, providing insights into structure–activity relationships\nand aiding the identification of promising candidates., providing broad diversity for\nstructure–activity analysis.\nThe high throughput screening was conducted in 96 well plates using a Z-RLRGG-\nAMC fluorogenic enzymatic assay to measure PLpro activity. Each compound was\ntested at a final concentration of 30 µM under assay buffer conditions containing 20\nmM HEPES at pH 7.5. Compounds that inhibited PLpro activity by more than 70% were\ndefined as primary hits. After removing pan assay interference compounds and unstable\nchemotypes, five compounds showed inhibitory activity against PLpro with half\nmaximal inhibitory concentration (IC ) values below 50 µM, as presented in\nthe strongest inhibition (IC = 26.82 ± 1.91 µM) and potential for structure activity\n50\noptimization, and was therefore selected for further development through fragment\nmerging with GRL0617.\nTo improve clarity and reproducibility, we have added this detailed description of the\nscreening process and the overall hit distribution to both the Results section (page 4,\nline 89) and the Methods section (page 21, line 473) of the revised manuscript. We hope\nthis fully addresses the reviewer’s concern.\nMinor Comments:\n1. The abbreviation “Nsp1-3” (Page 3, Line 55) should be written out in full at its first\noccurrence.\nResponse:\nWe thank the reviewer’s comment and have supplemented the full names of Nsp1-3 at\nthe relevant locations (page 3, line 56).\n2. In Fig. 2, the unit of AUC is shown as µg/L·h. Please ensure consistent notation\nacross the manuscript, including Supplementary Table 11.\nResponse:\nWe thank the reviewer’s comment. We have now unified the units throughout the\nmanuscript to ensure consistency in notation.\n3. On Page 7, Lines 156–157, the authors state that “The SARS-CoV-2 PLpro comprises\nfour domains: an N-terminal ubiquitin-like (Ubl) domain, followed by thumb, palm,\nand finger domains.” In fact, thumb, palm, and finger represent three subdomains\nwithin the larger ubiquitin-specific protease (USP) domain. This should be corrected.\nResponse:\nWe thank the reviewer’s comment. We have corrected the sentences accordingly. The\nupdated content is: “…The SARS-CoV-2 PLpro consists of an N-terminal ubiquitin-like\n(Ubl) domain, and a catalytic domain comprising thumb, palm, and finger subdomains\n(Fig. 2b). The catalytic triad (Cys111, His272, and Asp286) is positioned at the palm-\nthumb interface...” (Page 8, Lines 183)\n4. In Fig. 3c, the electron density should be shown using the 2Fo–Fc map after Refmac\nrefinement, rather than the Fo–Fc map.\nResponse:\nWe thank the reviewer’s comment. As recommended, we have updated Fig. 3c to\ndisplay the 2Fo-Fc electron density map (σ = 1.5) instead of the Fo-Fc map (please see\nthe updated Fig. 3c in the the revised manuscript).\n5. In Fig. 4c legend, the authors state that HEK293T cells were treated with or without\nIFN-β for 48 h. However, both the figure and methods indicate IFN-α was used to\ninduce ISGylation. Please clarify which interferon was employed.\nResponse:\nWe thank the reviewer for carefully noting this inconsistency. The mention of “IFN-β”\nin the Fig. 4c legend was a typographical error. We have verified our experimental\nrecords and reagent purchase information, confirming that IFN-α was used in these\nexperiments to induce ISGylation. Accordingly, the figure legend has been corrected in\nthe revised manuscript.\n6. In Figs. 5 and 6, the “P” values in the statistical analysis should be italicized in\naccordance with journal formatting guidelines.\nResponse:\nWe thank the reviewer’s comment and have standardized the italicization of P values\nin statistical analyses in accordance with journal formatting guidelines.\n7. Line 36: The term “nirmatrelvir-resistant recombinant SARS-CoV-2” should be\nclarified. Does this refer to the Mpro E166V mutant recombinant virus?\nResponse:\nWe appreciate the reviewer’s suggestion. The term “nirmatrelvir-resistant recombinant\nSARS-CoV-2” indeed refers to a recombinant SARS-CoV-2 virus carrying the E166V\nmutation in the main protease (Mpro), which confers high-level resistance to\nnirmatrelvir, as reported in previous studies3–6. To improve clarity, we have revised the\ntext on Line 36 to explicitly state “the Mpro E166V recombinant SARS-CoV-2 strain”.\n8. Line 68: Grammar correction needed: “Recent advances with orally active PLpro\ninhibitors … in murine SARS-CoV-2 models ARE promising.”\nResponse:\nWe appreciate the reviewer’s feedback and have implemented grammatical corrections\nas suggested.\n9. References 14 and 39 are duplicates and should be consolidated.\nResponse:\nWe appreciate the reviewer’s feedback and have consolidated the relevant citations as\nsuggested.\n10. Two recent studies (PMID: 37130168 and PMID: 37130166) have reported the\nimmunoregulatory functions of PLpro. The authors are encouraged to cite and briefly\ndiscuss these works in the manuscript.\nResponse: We thank the reviewer for this valuable suggestion. We have now cited and\ndiscussed the two recent studies (PMID: 37130168 and PMID: 37130166), which\nprovide important insights into the immunoregulatory roles of SARS-CoV-2 PLpro. We\nhave incorporated this discussion into the revised manuscript (page 10, lines 208-212)\nto provide additional mechanistic context for the immunosuppressive functions of PLpro\nand to further highlight the therapeutic significance of targeting this viral protease.\nSpecifically, we added the following sentences to the beginning of the section “YL1004\nantagonizes PLpro-mediated innate immune suppression”:\nRecent studies have revealed that PLpro acts as an immunoregulatory enzyme that exerts\ndeubiquitinating (DUB) and deISGylating activities, removing ubiquitin and ISG15\nconjugates from key signaling mediators such as IRF3 to suppress type I interferon and\nNF-κB signaling and facilitate viral immune evasion.\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse:\nWe acknowledge the co-review initiative by Nature Communications and thank the\nearly career researcher involved in reviewing this manuscript for their contributions.\nReviewer #3 (Remarks to the Author):\nThis manuscript presents a well-executed study describing the discovery and\ncharacterization of YL1004, a novel SARS-CoV-2 PLpro oral antiviral drug candidate.\nThe authors employed an innovative approach that combined high-throughput\nscreening (HTS) with fragment fusion using GRL0617, followed by systematic\nchemical optimization. The work demonstrates strong translational potential, with\nYL1004 showing high potency in enzymatic assays. The mechanism of action is\nconfirmed by an X-ray co-crystal structure, and the compound exhibits favorable\npharmacological properties, efficacy in cell-based assays, activity against nirmatrelvir-\nresistant viruses, and strong protection in a SARS-CoV-2-infected mouse model.\nThe manuscript will be suitable for publication in Nature Communications upon minor\nmodifications, once the authors have addressed the following comments:\nResponse:\nWe sincerely appreciate the reviewer for highlighting the translational potential and\nnovelty of our work and positively assessing the overall quality of our manuscript.\n1. While recent advances with orally active PLpro inhibitors in murine models such as\nJun12682, PF-07957472, and GZNL-P36 are reported, we would suggest including\nother recent such as WEHI-P8 (DOI: 10.1038/s41467-025-57905-4) and Jun13296\n(DOI: 10.1038/s41467-025-56902-x). Note that many of them have either a\nnaphthalene or quinoline moiety. Some comparison in terms of superposition of the co-\ncrystal structures would be of interest to readers, even if it is just in the Supporting\nMaterials.\nResponse:\nWe thank the reviewer for this comment. We have cited the mentioned literature\naccordingly. Following the reviewer’s suggestion, we have performed a structural\nsuperposition analysis of the co-crystal structures of recently reported inhibitors,\nincluding Jun12682, PF-07957472, GZNL-P35, and WEHI-P4. This analysis, which\nhighlights common structural features such as the naphthalene orquinoline moieties and\ntheir interaction with the PLpro binding site, has been presented in Supplementary Fig.\n6 (please also see below).\nSupplementary Fig. 6 | Superimposed comparison of PLpro with YL1004 and other\nrepresentative PLpro inhibitors. PLpro is shown in gray cartoon with the active site\nCys111 in yellow. YL1004 (purple sticks) is superimposed with Jun12682 (salmon\nsticks, PDB code: 8UOB, a), PF-07957472 (yellow sticks, PDB code: 9CSY, b),\nGZNL-P35 (cyan sticks, PDB code: 8YX5, c), WEHI-P4 (green sticks, PDB code:\n9CYD, d).\n2. Enzymatic Assay Methodology - Tight Binding Kinetics: The enzymatic assay\nmethod does not refer to any previous article. Is this a new method? Or is it based on a\nprevious one? If tight-binding kinetics were not explicitly considered, this should be\naddressed, as it may impact the accuracy of reported IC50 values. The Morrison\nequation may be more appropriate than standard IC50 calculations for tight-binding\ninhibitors, especially YL1004, both for the protease and deubiquitanse/de-ISGlase\nactivities. Related to this, the concentration of enzyme utilized (200 nM) may also be a\nbit too high for tight-binding inhibitors, with reports going as low as 50 nM PLpro for\nenzymatic inhibition assays (see, for example, DOI: 10.1038/s41467-025-56902-x). Note that Morrison equation fitting might be possible to perform even with the same\nexperimental raw data (see https://www.graphpad.com/guides/prism/latest/curve-\nfitting/reg_morrison.htm).\nResponse:\nWe appreciate the reviewer’s insightful comment. Our enzymatic assay was based on\nthe fluorescence protocol reported by Tan et al. 1, in which Z-RLRGG-AMC was used\nas the substrate for compound screening and IC determination. As the reviewer rightly\n50\npointed out, the Morrison equation is more appropriate for evaluating tight binding\ninhibitors, where the inhibitor concentration is comparable to or lower than the total\nenzyme concentration.\nIn our initial study, we used IC values to allow consistent comparisons across\n50\nstructurally related analogs and positive control compounds under identical assay\nconditions. However, considering that our lead compound YL1004 exhibited an IC of\n50\n17.5 ± 0.6 nM, which is far lower than the PLpro concentration used in the assay (200\nnM), we agree that tight binding kinetics are applicable and should be explicitly\naddressed.\nWe therefore re-analyzed the inhibition data for YL1004 using the Morrison equation.\nThe initial attempt using data from the 200 nM enzyme condition yielded unstable and\npoorly fitting results, suggesting enzyme depletion due to tight binding. We then\nrepeated the assay at a lower enzyme concentration (50 nM) and performed Morrison\nfitting, which resulted in a well-converged Kᵢ value of 2.3 ± 0.3 nM. This confirms that\nYL1004 is a tight-binding, reversible noncovalent inhibitor. The revised manuscript\nnow reports this value in the main text (page 8, line 161-164; Supplementary Table 5).\nWe further extended the kinetic analysis to the deubiquitination and deISGylation\nactivities of PLpro. Since these assays were originally performed at enzyme\nconcentrations of 50 nM, respectively, tight-binding conditions were already satisfied.\nDirect Morrison fitting of the raw data yielded Kᵢ values of 4.2 ± 0.6 nM for Ub-AMC\nand 39.3 ± 12.8 nM for ISG15-AMC substrates. These values have also been added to\nthe revised manuscript (page 10, lines 218-220; Supplementary Table 11),\nfurther\nsupporting the high inhibitory potency of YL1004 across multiple PLpro enzymatic\nfunctions.\n3. Rationale for the naphthalene 4-position Chemistry Exploration: While the\nmanuscript describes the chemical optimization process, the strategic rationale for\nexploring the naphthalene 4-position modifications is not clearly articulated (for\ninstance, the Pfizer inhibitor or Jun13296 have a quinoline 3-position substituent\ninstead). Adding a brief explanation would be interesting. Was this guided by\ncomputational modeling, initial SAR data, analysis of the GRL0617 co-crystal structure,\nor other considerations?\nResponse:\nWe are grateful to the reviewer for raising this point, which allows us to clarify the\nstrategic rationale behind our chemical exploration. Unlike the inhibitors noted by the\nreviewer (e.g., PF-07957472, Jun13296) that utilize the quinoline 3-position, our\napproach focused on the naphthalene 4-position. This decision was primarily guided by\nanalysis of our GRL0617 co-crystal structure and molecular superposition (Fig. 1b),\nwhich revealed that the naphthalene 4-position is oriented toward a solvent-exposed\nregion. we hypothesized that chemical modification at this site would minimally affect\nthe compound’s inhibitory activity against PLpro. This hypothesis was corroborated by\nour structure-activity relationship (SAR) data, which demonstrated that derivatives\nmodified at this position generally maintained IC values against PLpro below 50 nM.\n4-position to enhance pharmacokinetic properties. Accordingly, we have now included\na brief description of this optimization strategy in the revised manuscript to improve\nthe logical flow of this section.\n4. Figure-Related Issues:\n-In figures 1 and 2, the orientation of the compounds is different than in Fig. 3, that also\nshows the crystal structure with the inhibitor in the same orientation as in 3a. Given that\nmost of the structural papers display the compounds as in Fig. 3, it would make sense\nto do the same in Figs. 1 and 2.\nResponse:\nWe thank the reviewer’s comment and have standardized the structural depictions of all\ncompounds throughout the manuscript. After reviewing multiple PLpro inhibitor\nstudies2,7–10, we noted some variations in compound orientation across the literature. To\nemphasize the structural distinctiveness of our tricyclic compounds and their\ncontributions to PLpro activity, we have uniformly adopted the presentation format\nshown in Fig. 1 and Supplementary Fig. 1-3.\n-Figure 1: in panel a, please indicate either in the caption or in the panel that DSF was\nused to obtain the delta Tm values (this is well explained in the text but not here). Please\nverify the color scheme and ensure consistency between the figure and its legend in 1b,\nas GRL0617 is displayed in pink, not in yellow.\nResponse:\nWe thank the reviewer for the valuable comments. We have revised the legend of Figure\n1, panel a (page 34, line 745), to clearly indicate that the ΔTₘ values were determined\nby differential scanning fluorimetry (DSF). In addition, we have verified and corrected\nthe color scheme in panel b to ensure consistency between the figure and its legend—\nGRL0617 is now displayed in pink, consistent with the revised legend (page 5, line 99;\npage 34, line 748).\n-Figure 6: There is an apparent typographical error with \"p1004\" appearing instead of\n\"YL1004.\" Please correct this throughout the figure and verify all compound names are\nconsistent.\nResponse:\nWe appreciate the reviewer’s careful observation. The typographical error in Figure 6\nhas been corrected (page 44, line 824).\n-Figure 3a - Stereochemistry: it is not clear whether the stereochemical representation\nof the chemical structure is consistent to Figure 1. YL1004 may be with the S isomer,\ncorrect? Please, clarify and edit if needed.\nResponse:\nWe thank the reviewer’s comment and we want to clarify that YL1004 adopts the same\n(S)-configuration as compound 2b in Figure 1. To ensure stereochemical clarity\nthroughout the manuscript, we have uniformly annotated the configurations of all\ncompounds.\nMinor:\nP. 2 line 36: add “strain” at the end of the sentence.\nP. 2 lines 39-41: What does it mean by “a distinctive multidimensional binding mode”?\nThe sentence is overly verbose. Please, state in a more precise and understandable way.\nP. 6 line 121: change verbal tense to past.\nP. 8 lines 180-183: This sentence is too complicated; consider splitting it into two.\nResponse:\nWe thank the reviewer’s comment and have corrected the language accordingly.\nSpecific corrections have been made on Page 2, line 36, and Page 2, lines 39–41, as\nwell as on Page 6, line 121. And we have revised the sentence on page 8, lines 180-183,\nby splitting it into two shorter sentences to improve clarity and readability. The revised\ntext now reads: “Coronavirus PLpro is a multifunctional viral protease that hydrolyses\nviral polyprotein precursors. It also exerts deubiquitinating (DUB) and deISGylating\nactivities, which antagonize type I interferon (IFN-I) signaling and dampen the host\ninnate immune response.” (page 10, line 208)\nReviewer #4 (Remarks to the Author):\nReviewer report\nManuscript: YL1004 is a SARS-CoV-2 papain like protease inhibitor with\nimmunomodulatory and antiviral activity in mice\nKey results\nNan et al. introduce YL1004, a rationally designed, orally active inhibitor of the SARS\nCoV 2 papain like protease (PLpro). Starting from a fragment fusion concept that blends\nan in house molecular library hit of a hetrotricyclic with features of GRL0617, the\nauthors work through clear SAR to a lead that engages PLpro structurally, strongly\ninhibits PLpro activity, restores antiviral signaling in cells, suppresses replication across\nseveral SARS CoV 2 variants, and improves outcomes in a stringent mouse model (full\nsurvival of mouse sample population. This study represents a rational and\ncomprehensive design and characterization of a novel PLpro inhibitor having very\npromising activity in mouse models.\nValidity\nOverall, the evidence thoughtful and presented well: medicinal chemistry rationale →\non target engagement → cellular mechanism → antiviral efficacy → in vivo benefit.\nThe structural data provide a persuasive on target anchor, a 2.7 Å co crystal structure\n(PDB 9U7D) that locates YL1004 in the BL2/S3–S4 pocket; the IC50 and EC50 values\nof a series of lead compounds support grat promise as PLpro inhibitors, sub micromolar\nantiviral activity across wildtype, Delta, and Omicron sublineages including JN.1 and\nKP.3; and full survival protection in a Delta challenge where vehicle survival is 10%;\nand the cell based deubiquitination/deISGylation rescue, together with innate immune\nreporter recovery, tie the biochemical activity to cellular consequences. Antiviral data\nalign with the target story and extend to a nirmatrelvir resistant virus (NSP5 E166V),\nwhich is a thoughtful orthogonal test of relevance.\nSignificance\nPLpro remains an important, underexploited antiviral target because it couples viral\npolyprotein processing with antagonism of innate immunity. A well characterized,\nselective, orally bioavailable inhibitor that shows cross variant antiviral activity and\nsurvival benefit in vivo is notable. The manuscript’s integrated view—chemistry,\nmechanism, virology, and animal efficacy—will be useful to both the PLpro community\nand the broader antiviral field, especially with a deposited structures, data, and\nassay results not focused on in this manuscript, as well as the clear and transparent design\nnarrative.\nResponse:\nWe sincerely appreciate the reviewer for highlighting the systematic approach and\nnovelty of our work and positively assessing the overall quality of our manuscript.\nData and methodology\nThe work is generally well designed and well reported. The discovery path is easy to\nfollow (screen → fragment fusion → regional optimization → lead selection →\nbiochemical/biological assays), with enough intermediate disclosure to be instructive\nwithout overwhelming the reader. The structural biology and the cellular mechanism\nassays are appropriate and mutually reinforcing, and the in vivo studies use a\nrecognized lethal model with virology, histology/IHC, body weight, and survival\nendpoints. Where a little more context would help: linking on study exposure (infected\nmice) to in cell potency targets, and presenting selectivity indices (CC50/EC50) in the\nsame cellular backgrounds used for antiviral testing.\nResponse:\nWe thank the reviewer for the positive evaluation of our study design and for the\nconstructive suggestions to further improve the contextual linkage between the cellular\nand in vivo results. To address these points, we have added a quantitative analysis\nlinking the pharmacokinetic exposure of YL1004 in infected mice to its cellular\nantiviral potency. As shown in Supplementary Fig. 10, following oral administration of\nYL1004 at 100 mg/kg, plasma concentrations remained above the in vitro EC and\n90\nthat the in vivo antiviral efficacy of YL1004 is supported by sufficient systemic\nexposure (page 14, lines 303-308).\nSupplementary Fig. 10 | Plasma concentration–time curves for YL1004 in mice.\nThe mean plasma concentration-time curves of YL1004 (p.o. 100 mg/kg) in mice (n =\n3). The EC and EC values of Y180 were calculated from the cell-based antiviral\n50 90\nactivity data obtained with Calu3 cells infected with SARS-CoV-2 Omicron JN.1 (black\ndashed lines). Data are shown as mean ± SD.\nIn addition, we have included the selectivity indices (CC /EC ) of YL1004, calculated\n50 50\nin the same Calu3 cellular backgrounds used for antiviral assays. The selectivity indices\n(CC /EC ) of YL1004 ranged from approximately 102 to 103 across tested viral\n50 50\nvariants, confirming a wide therapeutic window and favorable cellular safety profile\nThese data have been added to Supplementary Fig 9 and referenced in the main text\n(page 13, lines 289-293).\nSupplementary Fig. 9 | In vitro cytotoxicity of YL1004. Cells were treated with 3-\nfold serial diluted YL1004 for 72 hours before cell viability was measured in Calu3.\nCell viability was normalized to mock treatment controls. Data are shown as mean ±\nSD, n = 3 biological replicates.\nAnalytical approach\nAs a non-expert in statistical methodology, I find the analyses chosen for the\nbiochemical, cellular, and animal studies broadly appropriate for the study designs and\npresented with sufficient transparency to follow the results. The manuscript and figure\nlegends indicate standard group-comparison and survival analyses and summarize data\nwith means and variability, which is adequate for interpreting the findings.\nTo further aid reproducibility, please state consistently what constitutes biological vs.\ntechnical replicates across where appropriate, including all relevant figures and in the\nSupplemental Information. For example, early figure captions reference “three\nindependent experiments” and “three technical replicates,” whereas later legends\nspecify that “each data point represents one biological repeat” and that data come from\n“two or three independent experiments.” Unifying this terminology in the figure\ncaptions and adding a brief definition in the Supplement would make replication\nstraightforward.\nResponse:\nWe thank the reviewer for the positive evaluation of our statistical analyses and for the\nconstructive suggestion to clarify the definitions of biological and technical replicates.\nIn the revised manuscript, we have carefully reviewed all figure legends and table\nfootnotes to ensure consistent terminology across the main and supplementary figures.\nSpecifically, we now use the following unified definitions throughout the manuscript:\n• Biological replicates refer to independent experiments performed using distinct\nbiological samples (for example, independently cultured cell batches or\ndifferent animals).\n• Technical replicates refer to repeated measurements of the same biological\nsample under identical experimental conditions (for example, replicate wells in\nthe same assay plate).\nThese definitions have been added to the “Statistical analysis” subsection of the\nMethods (page 26, lines 575-578). All figure legends have been checked or updated to\nspecify the number and type of replicates used for each experiment. These clarifications\nwill facilitate reproducibility and ensure consistent reporting across the study.\nSuggested improvements\n1. Figure 4d could use some clarification, along with the Human DUB assay against\nYL1004. There could also be additional discussion in the text on why there is selectivity.\nIs there a rational for this selectivity of YL1004 for PLpro vs. DUB structural homologs?\nIs there a structural basis for this, residue interactions?\nThe figure is unclear, is the raw DUB inhibition assays only at one YL1004\nconcentration of many, could they be reported as IC50s?\nThis is not essential to publishing but would help with impact as a major challenge of\nPLpro drug design is selectivity against human DUB enzyme homologs, accordingly,\nthis warrants some more discussion/rationale.\nResponse:\nWe appreciate the reviewer’s constructive comments highlighting the importance of\nselectivity in PLpro inhibitor design. In our study, the human deubiquitinase (DUB)\npanel assay was initially conducted at a relatively high single concentration of 50 μM\nto rapidly evaluate the potential off-target inhibition of YL1004. This design allowed\nefficient identification of any cross-reactivity among representative human DUBs. We\nhave clarified this point in both the Methods section and the Fig. 3 legend of the revised\nmanuscript.\nTo elucidate the structural basis for YL1004’s selectivity, we performed structural\nalignments between PLpro and ten human DUBs. The calculated Cα RMSD values\nranged from 9.95 to 31.52 Å (based on all Cα atoms, Supplementary Fig. 7a), indicating\nsubstantial overall structural divergence. With respect to the substrate-binding pockets,\nUCHL1 adopts a configuration completely distinct from that PLpro (Supplementary Fig.\n7b and 7c). Of note, among the tested DUBs, USP2 possesses a substrate-binding\npocket most closely resembling that of PLpro (Supplementary Fig. 7a), however, a loop\nspanning residues 503–516 would cause steric clashes with the naphthyl ring and the\ntetrahydropyran moiety of YL1004 (Supplementary Fig. 7d and 7e). This structural\nincompatibility provides a mechanistic explanation for the lack of USP2 inhibition by\nYL1004. Overall, these findings could provide structural insight into the selectivity of\nYL1004 toward human USPs.\nWe have incorporated these results and discussion into the revised manuscript (page 11,\nlines 238-248) to provide a clear structural basis for the high selectivity of YL1004\ntoward PLpro over human DUB homologs.\nSupplementary Fig. 7 | Structural basis of YL1004 selectivity for SARS-CoV-2 PLpro\nover human DUBs. (a) Root mean square deviation (Cα RMSD) values from the\nstructural alignment of SARS-CoV-2 PLpro with representative human deubiquitinating\nenzymes (DUBs). UCHL5 PDB code: 3A7S, BAP1 PDB code: 8SVF, USP14 PDB\ncode: 6IIK, OTUB1 PDB code: 2XFY, OTUD2 PDB code: 4BOQ, USP28 PDB code:\n6HEJ, USP8 PDB code: 3N3K, USP2 PDB code: 5XVE, UCHL1 PDB code: 8PW1,\nand USP21 PDB code: 3MTN. (b) Surface representation of SARS-CoV-2 PLpro in\ncomplex with YL1004 (purple). The catalytic residues Cys111 and His272 are shown\nin yellow and blue, respectively. (c) Surface view of human UCHL1. The catalytic\nresidues Cys90 and His161 are colored as in (b). (d) Structural superposition of SARS-\nCoV-2 PLpro (wheat) and human USP2 (cyan), with YL1004 shown as a stick model.\nThe loop503–516 in USP2, which clashes with the inhibitor, is highlighted in orange.\n(e) Close-up view of the steric clash between USP2 loop (orange surface) and YL1004.\n2. The term “broad spectrum” is used, consider “cross variant SARS CoV 2 activity,”\nsince non SARS CoV 2 coronaviruses were not tested.\nResponse:\nWe thank the reviewer for this helpful suggestion. We agree that the term “broad-\nspectrum” may imply activity beyond SARS-CoV-2, which was not evaluated in this\nstudy. To avoid ambiguity, we have revised the text throughout the manuscript to use\nthe term “cross-variant SARS-CoV-2 activity” instead of “broad-spectrum,” reflecting\nthat YL1004 was tested against multiple SARS-CoV-2 variants, including Delta,\nOmicron JN.1, and KP.3. The terminology has been updated in both the Abstract and\nResults sections (page 2, line 32; page 15, line 340; page 18, line 398) for consistency\nand clarity.\nClarity and context\nThe narrative is clear and accessible with figures adequately supporting the text. The\nhead to head benchmarking with Jun12682 is useful; ensuring the matched conditions\nfor those comparisons are fully described in the Supplemental Information will make\nthose claims easier to evaluate as YL1004 is competing for a new benchmark PLpro\ninhibitor.\nResponse:\nWe sincerely appreciate the reviewer’s positive feedback.\nReferences\nCoverage is broad and current, spanning PLpro biology, earlier chemotypes, recent\nmouse efficacious PLpro series, and nirmatrelvir resistance. The manuscript is well\nsituated in the literature.\nResponse:\nWe sincerely appreciate the reviewer’s positive feedback.\nYour expertise\nMy expertise is in chemistry and biochemistry with recent experience in medicinal\nchemistry, specifically anti-viral drug design and characterization of PLpro inhibitors.\nArguments for and against publication\nFor: Coherent design to efficacy story with structural validation, cellular mechanism\nrescue, cross variant antiviral activity (including an E166V recombinant virus), and\nsurvival benefit in a lethal mouse model—uncommon to see all results so successful in\none PLpro paper.\nResponse:\nWe sincerely appreciate the reviewer’s recognition of the study’s integrated approach—\nfrom structural validation, cellular mechanism rescue, cross variant antiviral activity to\nin vivo efficacy. This affirmation strongly motivates our continued exploration of PLpro\ninhibitors.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse:\nWe acknowledge the co-review initiative by Nature Communications and thank the\nearly career researcher involved in reviewing this manuscript for their contributions.\nReferences:\n1. Tan, B. et al. Design of a SARS-CoV-2 papain-like protease inhibitor with antiviral\nefficacy in a mouse model. Science 383, 1434–1440 (2024).\n2. Jadhav, P. et al. Design of quinoline SARS-CoV-2 papain-like protease inhibitors as\noral antiviral drug candidates. Nat Commun 16, 1604 (2025).\n3. Hattori, S. et al. Structural and virologic mechanism of the emergence of resistance\nto Mpro inhibitors in SARS-CoV-2. Proc. Natl. Acad. Sci. U.S.A. 121, e2404175121\n(2024).\n4. Iketani, S. et al. Multiple pathways for SARS-CoV-2 resistance to nirmatrelvir.\nNature 613, 558–564 (2023).\n5. Kiso, M. et al. In vitro and in vivo characterization of SARS-CoV-2 strains resistant\nto nirmatrelvir. Nat Commun 14, 3952 (2023).\n6. Duan, Y. et al. Molecular mechanisms of SARS-CoV-2 resistance to nirmatrelvir.\nNature 622, 376–382 (2023).\n7. Garnsey, M. R. et al. Discovery of SARS-CoV-2 papain-like protease (PLpro)\ninhibitors with efficacy in a murine infection model. Science Advances (2024).\n8. Tan, H. et al. Identification of naturally occurring drug-resistant mutations of SARS-\nCoV-2 papain-like protease. Nat Commun 16, 4548 (2025).\n9. Tan, B. et al. Design of a SARS-CoV-2 papain-like protease inhibitor with antiviral\nefficacy in a mouse model. Science 383, 1434–1440 (2024).\n10. Lu, Y. et al. Discovery of orally bioavailable SARS-CoV-2 papain-like protease\ninhibitor as a potential treatment for COVID-19. Nat Commun 15, 10169 (2024).\nResponses to Reviewers’ Comments\nReviewer #1 (Remarks to the Author):\nThe authors have successfully addressed the majority of the questions raised in my\nprevious review.\nResponse:\nWe sincerely thank the reviewer for acknowledging our efforts in addressing the\ncomments.\nMinor Comments:\n1. In the revised manuscript, the mention of \"IFN-\" in the legend for Fig. 3c (Line 787)\nhas not been corrected. Please ensure that similar discrepancies are carefully checked\nand corrected before publication.\nResponse:\nWe thank the reviewer for pointing out this inconsistency. We have carefully reviewed\nthe manuscript and confirm that the interferon used to induce ISGylation in A549 cells\nwas IFN-α, as verified by our experimental records. Accordingly, we have corrected the\nrelevant text in the figure legend for Fig. 3c (Line 813) as well as on page 1, line 229\nand line 239. We appreciate the reviewer’s attention to this detail, which has contributed\nto improving the accuracy and clarity of our manuscript.\n2. Supplementary Fig 6 (Superimposed comparison of PLpro with YL1004 and other\nrepresentative PLpro inhibitors) is a good addition. Please consider adding the 2D\nchemical structures of these inhibitors to the figure to facilitate direct comparison.\nResponse:\nWe appreciate the reviewer’s professional suggestion and have added the 2D chemical\nstructures of these inhibitors to the Figure S6 to facilitate direct comparison.\nSupplementary Fig. 6 | Superimposed comparison of PLpro with YL1004 and other\nrepresentative PLpro inhibitors. PLpro is shown in gray cartoon with the active site\nCys111 in yellow. YL1004 (purple sticks) is superimposed with Jun12682 (salmon\nsticks, PDB code: 8UOB, a), PF-07957472 (yellow sticks, PDB code: 9CSY, b),\nGZNL-P35 (cyan sticks, PDB code: 8YX5, c), WEHI-P4 (green sticks, PDB code:\n9CYD, d). Figure a-d were generated using PyMOL (http://www.pymol.org).\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse:\nWe acknowledge the co-review initiative by Nature Communications and thank the\nearly career researcher involved in reviewing this manuscript for their contributions.\nReviewer #3 (Remarks to the Author):\nThe authors have addressed the points I raised. The manuscript is ready for publication.\nResponse:\nWe sincerely thank the reviewer for the positive assessment and for acknowledging our\nefforts in addressing the comments.\nReviewer #4 (Remarks to the Author):\nThe authors have adequately and thoughtfully addressed all comments from this\nreviewer and appear to have done the same for all reviewer comments. This work is\nsuitable for publication in Nature Communications.\nResponse:\nWe sincerely thank the reviewer for the positive assessment and for acknowledging our\nefforts in addressing the comments.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse:\nWe acknowledge the co-review initiative by Nature Communications and thank the\nearly career researcher involved in reviewing this manuscript for their contributions."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have successfully addressed the majority of the questions raised in my previous review.\nMinor Comments:\n1. In the revised manuscript, the mention of \"IFN- \" in the legend for Fig. 3c (Line 787) has not been corrected. Please ensure that similar discrepancies are carefully checked and corrected before publication.\n2. Supplementary Fig 6 (Superimposed comparison of PLpro with YL1004 and other representative PLpro inhibitors) is a good addition. Please consider adding the 2D chemical structures of these inhibitors to the figure to facilitate direct comparison.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "The authors have addressed the points I raised. The manuscript is ready for publication.", "reviewer_4": "The authors have adequately and thoughtfully addressed all comments from this reviewer and appear to have done the same for all reviewer comments. This work is suitable for publication in Nature Communications.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": ""}]

Decision letter