Revealing the molecular mechanisms of proteolysis of SARS-CoV-2 Mpro by QM/MM computational methods

SARS-CoV-2 M^pro is one of the enzymes essential for the replication process of the virus responsible for the COVID-19 pandemic. This work is focused on exploring its proteolysis reaction by means of QM/MM methods. The resulting free energy landscape of the process provides valuable information on the species appearing along the reaction path and suggests that the mechanism of action of this enzyme, taking place in four steps, slightly differs from that of other cysteine proteases. Our predictions, which are in agreement with some recently published experimental data, can be used to guide the design of COVID-19 antiviral compounds with clinical potential.

The molecular mechanism of the proteolysis reaction catalyzed by SARS-CoV-2 M^pro, one of the enzymes essential for the replication process of the virus responsible for the COVID-19 pandemic, is described using computational QM/MM methods.     

Selective covalent targeting of SARS-CoV-2 main protease by enantiopure chlorofluoroacetamide

The coronavirus disease 2019 (COVID-19) pandemic has necessitated the development of antiviral agents against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The main protease (M^pro) is a promising target for COVID-19 treatment. Here, we report an irreversible SARS-CoV-2 M^pro inhibitor possessing chlorofluoroacetamide (CFA) as a warhead for the covalent modification of M^pro. Ugi multicomponent reaction using chlorofluoroacetic acid enabled the rapid synthesis of dipeptidic CFA derivatives that identified 18 as a potent inhibitor of SARS-CoV-2 M^pro. Among the four stereoisomers, (R,R)-18 exhibited a markedly higher inhibitory activity against M^pro than the other isomers. Reaction kinetics and computational docking studies suggest that the R configuration of the CFA warhead is crucial for the rapid covalent inhibition of M^pro. Our findings highlight the prominent influence of the CFA chirality on the covalent modification of proteinous cysteines and provide the basis for improving the potency and selectivity of CFA-based covalent inhibitors.

Chlorofluoroacetamide (CFA) was used as the warhead for covalent targeting of SARS-CoV-2 M^pro. The chirality at CFA showed marked influence on inhibitory activity, suggesting stereospecific activation of CFA for cysteine modification in the protein.     

The S1′–S3′ Pocket of the SARS-CoV-2 Main Protease Is Critical for Substrate Selectivity and Can Be Targeted with Covalent Inhibitors

The main protease (M^pro) of SARS-CoV-2 is a well-characterized target for antiviral drug discovery. To date, most antiviral drug discovery efforts have focused on the S4–S1′ pocket of M^pro; however, it is still unclear whether the S1′–S3′ pocket per se can serve as a new site for drug discovery. In this study, the S1′–S3′ pocket of M^pro was found to differentially recognize viral peptidyl substrates. For instance, S3′ in M^pro strongly favors Phe or Trp, and S1′ favors Ala. The peptidyl inhibitor D-4–77, which possesses an α-bromoacetamide warhead, was discovered to be a promising inhibitor of M^pro, with an IC₅₀ of 0.95 μM and an antiviral EC₅₀ of 0.49 μM. The M^pro/inhibitor co-crystal structure confirmed the binding mode of the inhibitor to the S1′–S3′ pocket and revealed a covalent mechanism. In addition, D-4–77 functions as an immune protectant and suppresses SARS-CoV-2 M^pro-induced antagonism of the host NF-κB innate immune response. These findings indicate that the S1′–S3′ pocket of SARS-CoV-2 M^pro is druggable, and that inhibiting SARS-CoV-2 M^pro can simultaneously protect human innate immunity and inhibit virion assembly.     

Discovery of SARS-CoV-2 Mpro peptide inhibitors from modelling substrate and ligand binding

The main protease (M^pro) of SARS-CoV-2 is central to viral maturation and is a promising drug target, but little is known about structural aspects of how it binds to its 11 natural cleavage sites. We used biophysical and crystallographic data and an array of biomolecular simulation techniques, including automated docking, molecular dynamics (MD) and interactive MD in virtual reality, QM/MM, and linear-scaling DFT, to investigate the molecular features underlying recognition of the natural M^pro substrates. We extensively analysed the subsite interactions of modelled 11-residue cleavage site peptides, crystallographic ligands, and docked COVID Moonshot-designed covalent inhibitors. Our modelling studies reveal remarkable consistency in the hydrogen bonding patterns of the natural M^pro substrates, particularly on the N-terminal side of the scissile bond. They highlight the critical role of interactions beyond the immediate active site in recognition and catalysis, in particular plasticity at the S2 site. Building on our initial M^pro-substrate models, we used predictive saturation variation scanning (PreSaVS) to design peptides with improved affinity. Non-denaturing mass spectrometry and other biophysical analyses confirm these new and effective ‘peptibitors’ inhibit M^pro competitively. Our combined results provide new insights and highlight opportunities for the development of M^pro inhibitors as anti-COVID-19 drugs.

The main protease (M^pro) of SARS-CoV-2 is central to viral maturation and is a promising drug target. In silico methods reveal structural aspects of how it binds to its 11 natural cleavage sites, the design of novel peptide inhibitors, and insights into drug design.     

Mechanism of covalent binding of ibrutinib to Bruton's tyrosine kinase revealed by QM/MM calculations

Ibrutinib is the first covalent inhibitor of Bruton''s tyrosine kinase (BTK) to be used in the treatment of B-cell cancers. Understanding the mechanism of covalent inhibition will aid in the design of safer and more selective covalent inhibitors that target BTK. The mechanism of covalent inhibition in BTK has been uncertain because there is no appropriate residue nearby that can act as a base to deprotonate the cysteine thiol prior to covalent bond formation. We investigate several mechanisms of covalent modification of C481 in BTK by ibrutinib using combined quantum mechanics/molecular mechanics (QM/MM) molecular dynamics reaction simulations. The lowest energy pathway involves direct proton transfer from C481 to the acrylamide warhead in ibrutinib, followed by covalent bond formation to form an enol intermediate. There is a subsequent rate-limiting keto–enol tautomerisation step (ΔG^‡ = 10.5 kcal mol⁻¹) to reach the inactivated BTK/ibrutinib complex. Our results represent the first mechanistic study of BTK inactivation by ibrutinib to consider multiple mechanistic pathways. These findings should aid in the design of covalent drugs that target BTK and other similar targets.

QM/MM simulations show that covalent modification of BTK by ibrutinib proceeds via an intramolecular proton transfer from C481 to the acrylamide warhead of ibrutinib, followed by covalent bond formation and subsequent keto–enol tautomerisation.      

Inhibitor binding influences the protonation states of histidines in SARS-CoV-2 main protease

The main protease (M^pro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an attractive target for antiviral therapeutics. Recently, many high-resolution apo and inhibitor-bound structures of M^pro, a cysteine protease, have been determined, facilitating structure-based drug design. M^pro plays a central role in the viral life cycle by catalyzing the cleavage of SARS-CoV-2 polyproteins. In addition to the catalytic dyad His41–Cys145, M^pro contains multiple histidines including His163, His164, and His172. The protonation states of these histidines and the catalytic nucleophile Cys145 have been debated in previous studies of SARS-CoV M^pro, but have yet to be investigated for SARS-CoV-2. In this work we have used molecular dynamics simulations to determine the structural stability of SARS-CoV-2 M^pro as a function of the protonation assignments for these residues. We simulated both the apo and inhibitor-bound enzyme and found that the conformational stability of the binding site, bound inhibitors, and the hydrogen bond networks of M^pro are highly sensitive to these assignments. Additionally, the two inhibitors studied, the peptidomimetic N3 and an α-ketoamide, display distinct His41/His164 protonation-state-dependent stabilities. While the apo and the N3-bound systems favored N_δ (HD) and N_ϵ (HE) protonation of His41 and His164, respectively, the α-ketoamide was not stably bound in this state. Our results illustrate the importance of using appropriate histidine protonation states to accurately model the structure and dynamics of SARS-CoV-2 M^pro in both the apo and inhibitor-bound states, a necessary prerequisite for drug-design efforts.

The main protease (M^pro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an attractive target for antiviral therapeutics.     

The Discovery of Small Allosteric and Active Site Inhibitors of the SARS-CoV-2 Main Protease via Structure-Based Virtual Screening and Biological Evaluation

The main protease enzyme (M^pro) of SARS-CoV-2 is one of the most promising targets for COVID-19 treatment. Accordingly, in this work, a structure-based virtual screening of 3.8 million ligand libraries was carried out. After rigorous filtering, docking, and post screening assessments, 78 compounds were selected for biological evaluation, 3 of which showed promising inhibition of the M^pro enzyme. The obtained hits (CB03, GR04, and GR20) had reasonable potencies with K_i values in the medium to high micromolar range. Interestingly, while our most potent hit, GR20, was suggested to act via a reversible covalent mechanism, GR04 was confirmed as a noncompetitive inhibitor that seems to be one of a kind when compared to the other allosteric inhibitors discovered so far. Moreover, all three compounds have small sizes (~300 Da) with interesting fittings in their relevant binding sites, and they possess lead-like characteristics that can introduce them as very attractive candidates for the future development of COVID-19 treatments.     

Azapeptide activity-based probes for the SARS-CoV-2 main protease enable visualization of inhibition in infected cells

The COVID-19 pandemic has revealed the vulnerability of the modern, global society. With expected waves of future infections by SARS-CoV-2, treatment options for infected individuals will be crucial in order to decrease mortality and hospitalizations. The SARS-CoV-2 main protease is a validated drug target, for which the first inhibitor has been approved for use in patients. To facilitate future work on this drug target, we designed a solid-phase synthesis route towards azapeptide activity-based probes that are capped with a cysteine-reactive electrophile for covalent modification of the active site of M^pro. This design led to the most potent ABP for M^pro and one of the most potent inhibitors reported thus far. We demonstrate that this ABP can be used to visualize M^pro activity and target engagement by drugs in infected cells.

The COVID-19 pandemic has revealed the vulnerability of the modern, global society.     

Interaction of small molecules with the SARS-CoV-2 main protease in silico and in vitro validation of potential lead compounds using an enzyme-linked immunosorbent assay

Caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the COVID-19 pandemic is ongoing, with no proven safe and effective vaccine to date. Further, effective therapeutic agents for COVID-19 are limited, and as a result, the identification of potential small molecule antiviral drugs is of particular importance. A critical antiviral target is the SARS-CoV-2 main protease (M^pro), and our aim was to identify lead compounds with potential inhibitory effects. We performed an initial molecular docking screen of 300 small molecules, which included phenolic compounds and fatty acids from our OliveNet™ library (224), and an additional group of curated pharmacological and dietary compounds. The prototypical α-ketoamide 13b inhibitor was used as a control to guide selection of the top 30 compounds with respect to binding affinity to the M^pro active site. Further studies and analyses including blind docking were performed to identify hypericin, cyanidin-3-O-glucoside and SRT2104 as potential leads. Molecular dynamics simulations demonstrated that hypericin (ΔG = -18.6 and -19.3 kcal/mol), cyanidin-3-O-glucoside (ΔG = -50.8 and -42.1 kcal/mol), and SRT2104 (ΔG = -8.7 and -20.6 kcal/mol), formed stable interactions with the M^pro active site. An enzyme-linked immunosorbent assay indicated that, albeit, not as potent as the covalent positive control (GC376), our leads inhibited the M^pro with activity in the micromolar range, and an order of effectiveness of hypericin and cyanidin-3-O-glucoside > SRT2104 > SRT1720. Overall, our findings, and those highlighted by others indicate that hypericin and cyanidin-3-O-glucoside are suitable candidates for progress to in vitro and in vivo antiviral studies.     

Inhibition of Cysteine Proteases by 6,6′-Dihydroxythiobinupharidine (DTBN) from Nuphar lutea

The specificity of inhibition by 6,6′-dihydroxythiobinupharidine (DTBN) on cysteine proteases was demonstrated in this work. There were differences in the extent of inhibition, reflecting active site structural-steric and biochemical differences. Cathepsin S (IC₅₀ = 3.2 μM) was most sensitive to inhibition by DTBN compared to Cathepsin B, L and papain (IC₅₀ = 1359.4, 13.2 and 70.4 μM respectively). DTBN is inactive for the inhibition of M^pro of SARS-CoV-2. Docking simulations suggested a mechanism of interaction that was further supported by the biochemical results. In the docking results, it was shown that the cysteine sulphur of Cathepsin S, L and B was in close proximity to the DTBN thiaspirane ring, potentially forming the necessary conditions for a nucleophilic attack to form a disulfide bond. Covalent docking and molecular dynamic simulations were performed to validate disulfide bond formation and to determine the stability of Cathepsins-DTBN complexes, respectively. The lack of reactivity of DTBN against SARS-CoV-2 M^pro was attributed to a mismatch of the binding conformation of DTBN to the catalytic binding site of M^pro. Thus, gradations in reactivity among the tested Cathepsins may be conducive for a mechanism-based search for derivatives of nupharidine against COVID-19. This could be an alternative strategy to the large-scale screening of electrophilic inhibitors.     

Fullerenes against COVID-19: Repurposing C60 and C70 to Clog the Active Site of SARS-CoV-2 Protease

The persistency of COVID-19 in the world and the continuous rise of its variants demand new treatments to complement vaccines. Computational chemistry can assist in the identification of moieties able to lead to new drugs to fight the disease. Fullerenes and carbon nanomaterials can interact with proteins and are considered promising antiviral agents. Here, we propose the possibility to repurpose fullerenes to clog the active site of the SARS-CoV-2 protease, M^pro. Through the use of docking, molecular dynamics, and energy decomposition techniques, it is shown that C₆₀ has a substantial binding energy to the main protease of the SARS-CoV-2 virus, M^pro, higher than masitinib, a known inhibitor of the protein. Furthermore, we suggest the use of C₇₀ as an innovative scaffold for the inhibition of SARS-CoV-2 M^pro. At odds with masitinib, both C₆₀ and C₇₀ interact more strongly with SARS-CoV-2 M^pro when different protonation states of the catalytic dyad are considered. The binding of fullerenes to M^pro is due to shape complementarity, i.e., vdW interactions, and is aspecific. As such, it is not sensitive to mutations that can eliminate or invert the charges of the amino acids composing the binding pocket. Fullerenic cages should therefore be more effective against the SARS-CoV-2 virus than the available inhibitors such as masinitib, where the electrostatic term plays a crucial role in the binding.     

From Repurposing to Redesign: Optimization of Boceprevir to Highly Potent Inhibitors of the SARS-CoV-2 Main Protease

The main protease (M^pro) of the betacoronavirus SARS-CoV-2 is an attractive target for the development of treatments for COVID-19. Structure-based design is a successful approach to discovering new inhibitors of the M^pro. Starting from crystal structures of the M^pro in complexes with the Hepatitis C virus NS3/4A protease inhibitors boceprevir and telaprevir, we optimized the potency of the alpha-ketoamide boceprevir against the M^pro by replacing its P1 cyclobutyl moiety by a γ-lactam as a glutamine surrogate. The resulting compound, MG-78, exhibited an IC₅₀ of 13 nM versus the recombinant M^pro, and similar potency was observed for its P1′ N-methyl derivative MG-131. Crystal structures confirmed the validity of our design concept. In addition to SARS-CoV-2 M^pro inhibition, we also explored the activity of MG-78 against the M^pro of the alphacoronavirus HCoV NL63 and against enterovirus 3C proteases. The activities were good (0.33 µM, HCoV-NL63 M^pro), moderate (1.45 µM, Coxsackievirus 3C^pro), and relatively poor (6.7 µM, enterovirus A71 3C^pro), respectively. The structural basis for the differences in activities was revealed by X-ray crystallo-graphy. We conclude that the modified boceprevir scaffold is suitable for obtaining high-potency inhibitors of the coronavirus M^pros but further optimization would be needed to target enterovirus 3C^pros efficiently.     

In-silico investigation of phenolic compounds from leaves of Phillyrea angustifolia L. as a potential inhibitor against the SARS-CoV-2 main protease (Mpro PDB ID:5R83) using a virtual screening method

There is currently a global COVID-19 pandemic caused by the severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) and its variants. This highly contagious viral disease continues to pose a major health threat global. The discovery of vaccinations is not enough to prevent their spread and dire consequences. To take advantage of the current drugs and isolated compounds, and immediately qualifying approach is required. The aim of our research is evaluation the potency for natural antiviral compounds against the SARS CoV-2 M^pro. Molecular docking of four phenolic compounds from Phillyrea angustifolia leaves with SARS-CoV-2 M^pro has been conducted. Similarly, the stability of selected ligand–protein interactions has been determined using MD simulations. Moreover, the quantitative structure–activity relationship (QSAR), MMGBSA binding energies, pharmacokinetics, and drug-likeness predictions for selected phenolic have been reported. The selected phenolic compounds (Luteolin-7-O-glucoside, Apigenin-7-O-glucoside, Demethyl-oleuropein, and Oleuropein aglycone) revealed strong binding contacts in the two active pockets of a target protein of SARS-CoV-2 M^pro with the docking scores and highest binding energies with a binding energy of ?8.2 kcal/mol; ?7.8 kcal/mol; ?7.2 kcal/mol and ?7.0 kcal/mol respectively. Both Demethyloleoeuropein and Oleuropein aglycone can interact with residues His41 and Cys145 (catalytic dyad) and other amino acids of the binding pocket of M^pro. According to QSAR, studies on pharmacokinetics and drug-like properties suggested that oleuropein aglycone could be the best inhibitor of SARS-CoV-2 for new drug design and development. Further in vivo, in vitro, and clinical studies are highly needed to examine the potential of these phenolic compounds in the fight against COVID-19.     

Design of Novel Enantiopure Dispirooxindolopyrrolidine-Piperidones as Promising Candidates toward COVID-19: Asymmetric Synthesis,Crystal Structure and In Silico Studies

Despite the effectiveness of COVID-19 vaccines, there is still an urgent need for discovering new anti-viral drugs to address the awful spread and transmission of the rapidly modifiable virus. In this study, the ability of a small library of enantiomerically pure spirooxindolopyrrolidine-grafted piperidones to inhibit the main protease of SARS-CoV-2 (M^pro) is evaluated. These spiroheterocycles were synthesized by 1,3-dipolar cycloaddition of various stabilized azomethine ylides with chiral dipolarophiles derived from N-[(S)-(-)-methylbenzyl]-4-piperidone. The absolute configuration of contiguous carbons was confirmed by a single crystal X-ray diffraction analysis. The binding of these compounds to SARS-CoV-2 M^pro was investigated using molecular docking and molecular dynamics simulation. Three compounds 4a, 4b and 4e exhibited stable binding modes interacting with the key subsites of the substrate-binding pocket of SARS-CoV-2 M^pro. The synthesized compounds represent potential leads for the development of novel inhibitors of SARS-CoV-2 main protease protein for COVID-19 treatment.     

Design,Microwave-Assisted Synthesis and In Silico Prediction Study of Novel Isoxazole Linked Pyranopyrimidinone Conjugates as New Targets for Searching Potential Anti-SARS-CoV-2 Agents

A series of novel naphthopyrano[2,3-d]pyrimidin-11(12H)-one containing isoxazole nucleus 4 was synthesized under microwave irradiation and classical conditions in moderate to excellent yields upon 1,3-dipolar cycloaddition reaction using various arylnitrile oxides under copper(I) catalyst. A one-pot, three-component reaction, N-propargylation and Dimroth rearrangement were used as the key steps for the preparation of the dipolarophiles3. The structures of the synthesized compounds were established by ¹H NMR, ¹³C NMR and HRMS-ES means. The present study aims to also predict the theoretical assembly of the COVID-19 protease (SARS-CoV-2 M^pro) and to discover in advance whether this protein can be targeted by the compounds 4a–1 and thus be synthesized. The docking scores of these compounds were compared to those of the co-crystallized native ligand inhibitor (N3) which was used as a reference standard. The results showed that all the synthesized compounds (4a–l) gave interesting binding scores compared to those of N3 inhibitor. It was found that compounds 4a, 4e and 4i achieved greatly similar binding scores and modes of interaction than N3, indicating promising affinity towards SARS-CoV-2 M^pro. On the other hand, the derivatives 4k, 4h and 4j showed binding energy scores (−8.9, −8.5 and −8.4 kcal/mol, respectively) higher than the M^pro N3 inhibitor (−7.0 kcal/mol), revealing, in their turn, a strong interaction with the target protease, although their interactions were not entirely comparable to that of the reference N3.     

Interaction of the prototypical α-ketoamide inhibitor with the SARS-CoV-2 main protease active site in silico: Molecular dynamic simulations highlight the stability of the ligand-protein complex

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes an illness known as COVID-19, which has been declared a global pandemic with over 2 million confirmed cases and 137,000 deaths in 185 countries and regions at the time of writing (16 April 2020), over a quarter of these cases being in the United States. In the absence of a vaccine, or an approved effective therapeutic, there is an intense interest in repositioning available drugs or designing small molecule antivirals. In this context, in silico modelling has proven to be an invaluable tool. An important target is the SARS-CoV-2 main protease (M^pro), involved in processing translated viral proteins. Peptidomimetic α-ketoamides represent prototypical inhibitors of M^pro. A recent attempt at designing a compound with enhanced pharmacokinetic properties has resulted in the synthesis and evaluation of the α-ketoamide 13b analogue. Here, we performed molecular docking and molecular dynamics simulations to further characterize the interaction of α-ketoamide 13b with the active site of the SARS-CoV-2 M^pro. We included the widely used antibiotic, amoxicillin, for comparison. Our findings indicate that α-ketoamide 13b binds more tightly (predicted GlideScore = -8.7 and -9.2 kcal/mol for protomers A and B, respectively), to the protease active site compared to amoxicillin (-5.0 and -4.8 kcal/mol). Further, molecular dynamics simulations highlight the stability of the interaction of the α-ketoamide 13b ligand with the SARS-CoV-2 M^pro (ΔG = -25.2 and -22.3 kcal/mol for protomers A and B). In contrast, amoxicillin interacts unfavourably with the protease (ΔG = +32.8 kcal/mol for protomer A), with unbinding events observed in several independent simulations. Overall, our findings are consistent with those previously observed, and highlight the need to further explore the α-ketoamides as potential antivirals for this ongoing COVID-19 pandemic.     

Synergistic inhibition of SARS-CoV-2 cell entry by otamixaban and covalent protease inhibitors: pre-clinical assessment of pharmacological and molecular properties

SARS-CoV-2, the cause of the COVID-19 pandemic, exploits host cell proteins for viral entry into human lung cells. One of them, the protease TMPRSS2, is required to activate the viral spike protein (S). Even though two inhibitors, camostat and nafamostat, are known to inhibit TMPRSS2 and block cell entry of SARS-CoV-2, finding further potent therapeutic options is still an important task. In this study, we report that a late-stage drug candidate, otamixaban, inhibits SARS-CoV-2 cell entry. We show that otamixaban suppresses TMPRSS2 activity and SARS-CoV-2 infection of a human lung cell line, although with lower potency than camostat or nafamostat. In contrast, otamixaban inhibits SARS-CoV-2 infection of precision cut lung slices with the same potency as camostat. Furthermore, we report that otamixaban''s potency can be significantly enhanced by (sub-) nanomolar nafamostat or camostat supplementation. Dominant molecular TMPRSS2-otamixaban interactions are assessed by extensive 109 μs of atomistic molecular dynamics simulations. Our findings suggest that combinations of otamixaban with supplemental camostat or nafamostat are a promising option for the treatment of COVID-19.

SARS-CoV-2, the cause of the COVID-19 pandemic, exploits host proteins for viral entry into human lung cells and is blocked by otamixaban in combination with a covalent protease inhibitor.     

Elucidation of Binding Features and Dissociation Pathways of Inhibitors and Modulators in SARS-CoV-2 Main Protease by Multiple Molecular Dynamics Simulations

COVID-19 can cause different neurological symptoms in some people, including smell, inability to taste, dizziness, confusion, delirium, seizures, stroke, etc. Owing to the issue of vaccine effectiveness, update and coverage, we still need one or more diversified strategies as the backstop to manage illness. Characterizing the structural basis of ligand recognition in the main protease (M^pro) of SARS-CoV-2 will facilitate its rational design and development of potential drug candidates with high affinity and selectivity against COVID-19. Up to date, covalent-, non-covalent inhibitors and allosteric modulators have been reported to bind to different active sites of M^pro. In the present work, we applied the molecular dynamics (MD) simulations to systematically characterize the potential binding features of catalytic active site and allosteric binding sites in M^pro using a dataset of 163 3D structures of M^pro-inhibitor complexes, in which our results are consistent with the current studies. In addition, umbrella sampling (US) simulations were used to explore the dissociation processes of substrate pathway and allosteric pathway. All the information provided new insights into the protein features of M^pro and will facilitate its rational drug design for COVID-19.