Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public review):
Summary:
Since dimerization is essential for SARS-CoV-2 Mpro enzymatic activity, the authors investigated how different classes of inhibitors, including peptidomimetic inhibitors (PF-07321332, PF-00835231, GC376, boceprevir), non-peptidomimetic inhibitors (carmofur, ebselen, and its analog MR6-31-2), and allosteric inhibitors (AT7519 and pelitinib), influence the Mpro monomer-dimer equilibrium using native mass spectrometry. Further analyses with isotope labeling, HDX-MS, and MD simulations examined subunit exchange and conformational dynamics. Distinct inhibitory mechanisms were identified: peptidomimetic inhibitors stabilized dimerization and suppressed subunit exchange and structural flexibility, whereas ebselen covalently bound to a newly identified site at C300, disrupting dimerization and increasing conformational dynamics. This study provides detailed mechanistic evidence of how Mpro inhibitors modulate dimerization and structural dynamics. The newly identified covalently binding site C300 represents novelty as a druggable allosteric hotspot.
Strengths:
This manuscript investigates how different classes of inhibitors modulate SARS-CoV-2 main protease dimerization and structural dynamics, and identifies a newly observed covalent binding site for ebselen.
Weaknesses:
The major concern is the absence of mutagenesis data to support the proposed inhibitory mechanisms, particularly regarding the role of the inhibitor binding site.
We thank the reviewer for the recognition and comments. We agree that mutagenesis is critical for validating the proposed role of C300. We therefore generated the C300S and C300F mutants and characterized their oligomeric states and proteolytic activities. C300S was designed to remove the reactive thiol group while minimally affecting Mpro structure and dimerization. C300F was introduced to mimic the steric perturbation associated with C300 modification and assess its impact on Mpro dimerization. Native PAGE showed that WT and C300S Mpro predominantly formed dimers, whereas C300F was mainly monomeric. Consistently, C300S retained approximately 70% of WT activity, whereas C300F retained only approximately 10%. Because C300F itself strongly disrupted dimerization, C300S was used as the principal mutant to evaluate the specific contribution of the C300 thiol to ebselen action. Native MS showed that ebselen could still bind to both monomeric and dimeric C300S Mpro but did not markedly shift its monomer-dimer equilibrium toward the monomeric state. In parallel, ebselen reduced WT activity to approximately 53% of the untreated control, whereas C300S retained approximately 78% activity at the same 1:3 Mpro-to-ebselen molar ratio. These results provide experimental support for the contribution of C300 to ebselen-induced dimer destabilization and functional inhibition, while the residual binding and inhibition observed for C300S suggest the involvement of additional C300-independent interactions. The corresponding revisions have been made to Methods (Lines 627–648), and Results (Lines 397–453) of the manuscript, together with the newly added figures (Figures S14–S16).
Reviewer #2 (Public review):
Summary:
This is a mechanistic study that provides new insights into the inhibition of SARS-CoV-2 Mpro.
Strengths
The identification of dimer interface stabilization/destabilization as distinct inhibitory mechanisms and the discovery of C300 as a potential allosteric site for ebselen are important contributions to the field. The experimental approach is modern, multi-faceted, and generally well-executed.
We thank the reviewer for the positive comments and recognition of our study.
Weaknesses:
The primary weaknesses relate to linking the biophysical observations more directly to functional enzymatic outcomes and providing more quantitative rigor in some analyses. While the study is overall strong, addressing its weaknesses and limitations would elevate the impact and translational relevance of the current manuscript.
We thank the reviewer for these comments, which have helped to iMprove the quality and impact of our manuscript.
(1) Correlation with Functional Activity:
The most significant gap is the lack of direct enzymatic activity assays under the exact conditions used for MS and HDX. While EC50 values are listed from literature, demonstrating how the observed dimer stabilization (by peptidomimetics) or dimer disruption (by ebselen) directly correlates with inhibition of proteolytic activity in the same experimental setup would solidify the functional relevance of the biophysical observations. For instance, does the fraction of monomer measured by native MS quantitatively predict the loss of activity? Also, the single inhibitor concentration used in each MS experiment needs to be specified in the main text and legends. A discussion on whether the inhibitor concentrations required to observe these dimerization effects (in native MS) or structural dynamics (in HDX-MS) align with EC50 values would be helpful for contextualizing the findings.
We thank the reviewer for these important points. To link the biophysical observations more directly to function, we compared the oligomeric states and proteolytic activities of WT, C300S, and C300F Mpro. C300F was predominantly monomeric and retained only approximately 10% of WT activity, whereas C300S remained predominantly dimeric and retained approximately 70% activity. We further evaluated ebselen inhibition using a matched 1:3 Mpro-to-ebselen molar ratio. Ebselen reduced WT activity to approximately 53% of its untreated control but reduced C300S activity only to approximately 78%, demonstrating that removal of the C300 thiol significantly attenuated the functional effect of ebselen. These data support a relationship between C300-dependent dimer destabilization and reduced proteolytic activity. The Methods (Lines 627–648), and Results (Lines 397–453) have been revised accordingly, with Figures S14–S16 newly added, in the revised manuscript. We did not expect a linear relationship between the monomer fraction measured by native MS and enzymatic activity loss, because ebselen can modify multiple cysteine residues, and individual modification events may have distinct effects on Mpro dimerization and catalytic function. The concentrations and molar ratios used in the native MS, HDX-MS, and activity assays have now been stated in the figure legends. The ebselen concentrations used for native MS and HDX-MS were optimized for biophysical characterization and comparison, and therefore, these concentrations might not be directly related to their IC50 or EC50 values. In these experiments, ebselen was applied at a 3-fold molar excess relative to Mpro, consistent with the enzymatic assay. The observed dimer disruption and conformational changes were consistent with functional inhibition, supporting their mechanistic relevance.
(2) For the two Cys residues found to be targeted by ebselen, what are their respective modification stoichiometry related to the ebselen concentration? Especially for the covalent binding site C300, which is proposed in this study to represent a novel allosteric inhibition mechanism of ebselen, more direct experimental evidence is needed to support this major hypothesis. Does mutation or modification of C300 affect the Mpro dimerization/monomer equilibrium and alter the enzymatic activity? If ebselen acts as a covalent inhibitor linked to multiple Cys, why is its activity only in the μM range?
We thank the reviewer for the insightful comments. Our LC-MS/MS data identified C44 and C300 as ebselen-modified residues, but they do not permit reliable site-resolved occupancy measurements because modified and unmodified peptides can differ in digestion efficiency and MS response. We have therefore clarified that these data provide qualitative site identification rather than absolute modification stoichiometry. To obtain direct functional evidence for C300, we generated C300S and C300F mutants. C300S preserved dimer formation and substantial activity, whereas C300F was mainly monomeric and showed severe activity loss. Importantly, although ebselen-bound C300S species were still detected by native MS, ebselen did not markedly redistribute C300S toward the monomeric state, and its inhibition was reduced from approximately 47% for WT to approximately 22% for C300S. These results indicate that C300 is an important contributor to ebselen-induced dimer disruption, while residual binding and inhibition indicate additional reactive sites. Corresponding revisions have been made to the (Lines 627–648), and Results (Lines 397–453) of the manuscript, together with the newly added figures (Figures S14–S16). The moderate micromolar potency of ebselen is consistent with its heterogeneous, multi-site covalent reactivity: modification occupancy and functional consequence are site-dependent, and not every adduct produces complete inhibition.
(3) For the allosteric inhibitor pelitinib with low-μM activity, no significant differences in deuterium uptake of Mpro were observed. In terms of the binding affinity, what is the difference between pelitinib and ebselen? Some explanations could be provided about the different HDX-MS results between the two non-peptidomimetic inhibitors with similar activities.
We agree with the reviewer that the absence of significant HDX changes for pelitinib requires clarification. Different from ebselen that forms covalent bond with multiple cysteine residues of Mpro, which could lead to sustained conformational changes that are more readily detected by HDX-MS, pelitinib non-covalently binds Mpro and might not induce significant perturbations in backbone dynamics that are detectable at the peptide level by HDX-MS. These points have been integrated into the revised manuscript (Lines 333-337).
(4) Native MS Quantification:
The analysis of monomer-dimer ratios from native MS spectra appears qualitative or semi-quantitative. A more rigorous and quantified analysis of the percentage of dimer/monomer species under each condition, with statistical replicates, would strengthen the equilibrium shift claims. For native MS analysis of each inhibitor, the representative spectrum can be shown in the main figure together with quantified dimer/monomer fractions from replicates to show significance by statistical tests.
We thank the reviewer for the suggestion. We have performed a quantitative analysis of the monomer-dimer equilibrium based on triplicate native MS measurements for each condition. Representative spectra, quantified monomer/dimer ratios, and statistical analyses have been added to Figures 1 and S3. The quantitative results have also been described in the Results section (Lines 158–161, 165-168, 172-174, 177-179, 199-200).
(5) Changes of HDX rates in certain regions seem very subtle. For example, as it states 'residues 296-304 in the C-terminal region of Mpro were more flexible upon ebselen binding (Figure 4c)', the difference is barely observable. The percentage of HDX rate changes between two conditions (with p values) can be specified in the text for each fragment discussed, and any change below 5% or 10% is negligible.
We agree with the reviewer about the need for quantitative rigor in reporting HDX changes. We have calculated the fractional deuterium uptake difference for each peptide fragment discussed in the text between the inhibitor-bound and unbound states. These values, along with their statistical significance (p-values from a two-tailed t-test), have been provided in the revised manuscript (Legends for Figures 3 and 4). Although the HDX change of residues 296–306 is relatively small (<5%), this region showed a reproducible difference with low experimental variability and statistical significance (p < 0.05). Given its location within the C-terminal dimerization interface and its consistency with native MS, we interpret this change as a subtle local conformational perturbation.
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
Major points:
(1) The study lacks validation through inhibitor binding site mutagenesis assays, especially peptidomimetic inhibitor PF-07321332 and ebselen, which would strengthen the mechanistic conclusions.
We appreciate this suggestion. For PF-07321332, the inhibitor forms a covalent interaction with the catalytic residue C145 and inhibits Mpro activity through a distinct mechanism. Previous studies have shown that mutation of C145, such as C145A, completely abolishes Mpro catalytic activity (Bhandari, D, et al. Communications Biology 2025, 8, 1061), making it difficult to directly evaluate the contribution of this residue to inhibitor-induced inhibition using enzymatic assays alone. This limitation and the relevant literature have now been discussed in the revised manuscript (Lines 272–277). Therefore, we focused on C300-dependent regulation of ebselen, which represents a distinct inhibitory mechanism involving modulation of Mpro structural dynamics and dimer stability.
To validate the role of C300 in ebselen-mediated regulation of Mpro, we generated C300S and C300F mutants and performed additional biochemical and structural characterization. The enzymatic assay showed that the C300F mutation significantly affected Mpro activity, and the inhibitory effect of ebselen on C300S Mpro was markedly reduced compared with WT Mpro. Furthermore, native MS analysis demonstrated that ebselen could still bind to C300S Mpro but failed to induce a significant shift in the monomer-dimer equilibrium observed for WT Mpro. These results indicate that C300 is not the only site involved in ebselen binding but is critical for mediating ebselen-induced structural perturbation and dimer destabilization. The manuscript has been revised accordingly for the (Lines 627–648), and Results (Lines 397–453), with new figures (Figures S14–S16) included, further supporting the functional contribution of C300 in ebselen-mediated Mpro regulation.
(2) MR6-31-2 is an ebselen derivative and exhibits a lower EC50 (1.78 μM) compared to ebselen (4.67 μM). It would be helpful to discuss why their activities differ, probably based on the assay conditions or binding behavior.
We agree with the reviewer that the difference in antiviral activity between MR6-31-2 and ebselen requires further clarification. The lower EC50 of MR6-31-2 may result from iMproved cellular properties, including compound stability, permeability, intracellular exposure, and potentially altered interactions with Mpro and/or iMproved cellular properties. Although MR6-31-2 shares the ebselen scaffold, the modified chemical structure may affect its binding behavior and biological activity. However, EC50 values obtained from cellular assays cannot directly reflect the biochemical inhibition potency against purified Mpro. These points have been integrated into the revised Introduction (Lines 98–101).
(3) In Figures 2, S1, S2, S4, S6, and S11, adding the drug name under each panel would make the data much clearer for readers.
The corresponding drug names have been added to panels to iMprove figure clarity.
Minor points:
(1) Line 62-63 refers to the "long linker loop," while Figure 1a labels it as the "long loop linker." Please keep this consistent.
The terminology has been unified as “long loop linker” throughout the manuscript.
(2) Table 1 should be cited at line 80, and PDB code 7BAK should be included in Table 1.
PDB code 7BAK has been included in Table 1, and Table 1 has been cited in the context, as suggested.
(3) Figure 1a should include the corresponding PDB code in the figure legend.
The corresponding PDB code has been added to the Figure 1a legend, as suggested.
(4) It would be helpful to indicate in Figure 1a that the upper structure represents the dimer and the lower structure represents the monomer.
The upper and lower structures in Figure 1a have been indicated as dimeric and monomeric Mpro, respectively, as suggested.
(5) In the Figure S1 legend, it should mention that some inhibitor structures (like ebselen and MR6-31-2) are not fully resolved. Also, the Se atom in ebselen should be shown in Figure S1f (PDB: 7BAK).
The Figure S1 legend has been revised to indicate that some inhibitor structures, including ebselen and MR6-31-2, are partially unresolved, and the selenium atom of ebselen has also been shown in Figure S1f, as suggested.
(6) Pelitinib is an allosteric, non-covalently binding inhibitor. However, in Figure S3, the native MS profile shows dimer species (13+ to 15+) compared with unbound Mpro (14+ to 17+). Please clarify this difference.
We thank the reviewer for raising this good point. Protein charge-state distributions can be influenced by solution-phase conformation, conformational flexibility, solvent properties, and electrospray droplet charging (Susa AC, et al. J Am Soc Mass Spectrom 2017, 28, 332-340). The observed shift in charge state distribution in native MS might suggest that the addition of pelitinib caused changes in the protein conformation, solvent property and electrospray droplet charging. The relevant literature and discussion have been added in the revised manuscript (Lines 200–204).
(7) Line 172: "S1are" should be corrected to "S1 are."
Corrected.
