The small molecule inhibitor of SARS-CoV-2 3CLpro EDP-235 prevents viral replication and transmission in vivo.

The COVID-19 pandemic has led to the deaths of millions of people and severe global economic impacts. Small molecule therapeutics have played an important role in the fight against SARS-CoV-2, the virus responsible for COVID-19, but their efficacy has been limited in scope and availability, with many people unable to access their benefits, and better options are needed. EDP-235 is specifically designed to inhibit the SARS-CoV-2 3CLpro, with potent nanomolar activity against all SARS-CoV-2 variants to date, as well as clinically relevant human and zoonotic coronaviruses. EDP-235 maintains potency against variants bearing mutations associated with nirmatrelvir resistance. Additionally, EDP-235 demonstrates a ≥ 500-fold selectivity index against multiple host proteases. In a male Syrian hamster model of COVID-19, EDP-235 suppresses SARS-CoV-2 replication and viral-induced hamster lung pathology. In a female ferret model, EDP-235 inhibits production of SARS-CoV-2 infectious virus and RNA at multiple anatomical sites. Furthermore, SARS-CoV-2 contact transmission does not occur when naïve ferrets are co-housed with infected, EDP-235-treated ferrets. Collectively, these results demonstrate that EDP-235 is a broad-spectrum coronavirus inhibitor with efficacy in animal models of primary infection and transmission.

Profiling of coronaviral Mpro and enteroviral 3Cpro specificity provides a framework for the development of broad-spectrum antiviral compounds.

The main protease from coronaviruses and the 3C protease from enteroviruses play a crucial role in processing viral polyproteins, making them attractive targets for the development of antiviral agents. In this study, we employed a combinatorial chemistry approach-HyCoSuL-to compare the substrate specificity profiles of the main and 3C proteases from alphacoronaviruses, betacoronaviruses, and enteroviruses. The obtained data demonstrate that coronavirus Mpros exhibit overlapping substrate specificity in all binding pockets, whereas the 3Cpro from enterovirus displays slightly different preferences toward natural and unnatural amino acids at the P4-P2 positions. However, chemical tools such as substrates, inhibitors, and activity-based probes developed for SARS-CoV-2 Mpro can be successfully applied to investigate the activity of the Mpro from other coronaviruses as well as the 3Cpro from enteroviruses. Our study provides a structural framework for the development of broad-spectrum antiviral compounds.

Inhibitors of SARS-CoV-2 Main Protease (Mpro) as Anti-Coronavirus Agents.

The main protease (Mpro) of SARS-CoV-2 is an essential enzyme that plays a critical part in the virus's life cycle, making it a significant target for developing antiviral drugs. The inhibition of SARS-CoV-2 Mpro has emerged as a promising approach for developing therapeutic agents to treat COVID-19. This review explores the structure of the Mpro protein and analyzes the progress made in understanding protein-ligand interactions of Mpro inhibitors. It focuses on binding kinetics, origin, and the chemical structure of these inhibitors. The review provides an in-depth analysis of recent clinical trials involving covalent and non-covalent inhibitors and emerging dual inhibitors targeting SARS-CoV-2 Mpro. By integrating findings from the literature and ongoing clinical trials, this review captures the current state of research into Mpro inhibitors, offering a comprehensive understanding of challenges and directions in their future development as anti-coronavirus agents. This information provides new insights and inspiration for medicinal chemists, paving the way for developing more effective Mpro inhibitors as novel COVID-19 therapies.

Novel Pan-Coronavirus 3CL Protease Inhibitor MK-7845: Biological and Pharmacological Profiling.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) continues to be a global threat due to its ability to evolve and generate new subvariants, leading to new waves of infection. Additionally, other coronaviruses like Middle East respiratory syndrome coronavirus (MERS-CoV, formerly known as hCoV-EMC), which first emerged in 2012, persist and continue to present a threat of severe illness to humans. The continued identification of novel coronaviruses, coupled with the potential for genetic recombination between different strains, raises the possibility of new coronavirus clades of global concern emerging. As a result, there is a pressing need for pan-CoV therapeutic drugs and vaccines. After the extensive optimization of an HCV protease inhibitor screening hit, a novel 3CLPro inhibitor (MK-7845) was discovered and subsequently profiled. MK-7845 exhibited nanomolar in vitro potency with broad spectrum activity against a panel of clinical SARS-CoV-2 subvariants and MERS-CoV. Furthermore, when administered orally, MK-7845 demonstrated a notable reduction in viral burdens by >6 log orders in the lungs of transgenic mice infected with SARS-CoV-2 (K18-hACE2 mice) and MERS-CoV (K18-hDDP4 mice).

Structure-Guided Design of Potent Coronavirus Inhibitors with a 2-Pyrrolidone Scaffold: Biochemical, Crystallographic, and Virological Studies.

Zoonotic coronaviruses are known to produce severe infections in humans and have been the cause of significant morbidity and mortality worldwide. SARS-CoV-2 was the largest and latest contributor of fatal cases, even though MERS-CoV has the highest case-fatality ratio among zoonotic coronaviruses. These infections pose a high risk to public health worldwide warranting efforts for the expeditious discovery of antivirals. Hence, we hereby describe a novel series of inhibitors of coronavirus 3CLpro embodying an N-substituted 2-pyrrolidone scaffold envisaged to exploit favorable interactions with the S3-S4 subsites and connected to an invariant Leu-Gln P2-P1 recognition element. Several inhibitors showed nanomolar antiviral activity in enzyme and cell-based assays, with no significant cytotoxicity. High-resolution crystal structures of inhibitors bound to the 3CLpro were determined to probe and identify the molecular determinants associated with binding, to inform the structure-guided optimization of the inhibitors, and to confirm the mechanism of action of the inhibitors.

Fixing the Achilles Heel of Pfizer's Paxlovid for COVID-19 Treatment.

Nirmatrelvir (PF-07321332), a first-in-class inhibitor of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) main protease (Mpro), was developed by Pfizer under intense pressure during the pandemic to treat COVID-19. A weakness of nirmatrelvir is its limited metabolic stability, which led to the development of a combination therapy (paxlovid), involving coadministration of nirmatrelvir with the cytochrome P450 inhibitor ritonavir. However, limitations in tolerability of the ritonavir component reduce the scope of paxlovid. In response to these limitations, researchers at Pfizer have now developed the second-generation Mpro inhibitor PF-07817883 (ibuzatrelvir). Structurally related to nirmatrelvir, including with the presence of a trifluoromethyl group, albeit located differently, ibuzatrelvir manifests enhanced oral bioavailability, so it does not require coadministration with ritonavir. The development of ibuzatrelvir is an important milestone, because it is expected to enhance the treatment of COVID-19 without the drawbacks associated with ritonavir. Given the success of paxlovid in treating COVID-19, it is likely that ibuzatrelvir will be granted approval as an improved drug for treatment of COVID-19 infections, so complementing vaccination efforts and improving pandemic preparedness. The development of nirmatrelvir and ibuzatrelvir dramatically highlights the power of appropriately resourced modern medicinal chemistry to very rapidly enable the development of breakthrough medicines. Consideration of how analogous approaches can be used to develop similarly breakthrough medicines for infectious diseases such as tuberculosis and malaria is worthwhile.

Competitive Interaction of the SGFRKMAF Peptide with 3CLpro Dimerization Intermediates: A Brownian Dynamics Investigation.

The SGFRKMAF peptide is known to inhibit the dimerization of 3CLpro monomers, which is essential for SARS-CoV-2 replication. The mechanism behind this, however, is largely unknown. In this work, we used Brownian dynamics simulations to compare and contrast 3CLpro monomer-monomer interactions and 3CLpro monomer-SGFRKMAF peptide interactions. We found that formation of the 3CLpro wild-type dimer could potentially involve formation of three intermediates that are primarily stabilized by G11-G124, S1-S301, and T118-G278 interactions. Analysis of 3CLpro monomer interaction with the SGFRKMAF peptide, however, revealed the presence of eight basins of interactions where the peptide assumes the highest local densities at the 3CLPro monomer surface. The second highest-density basin was found to coincide with the interface region of the wild-type 3CLpro dimer, thereby directly blocking the 3CLpro dimer-dimer interactions. The other basins, however, were found to lie far from the interface region. Notably, we found that only 6% of the BD trajectories end up directly into the basin at the interface region and ∼39% of the trajectories end up into those basins lying away from the interface region, indicating a greater role for peptide binding at sites away from the dimer interface region. Importantly, the locations of the basins lying away from the interface were found to coincide with the 3CLpro residues involved in stabilization of the 3CLpro monomer-monomer intermediates. Given that the rate constant of the peptide reaching the monomer surface was found to be almost an order of magnitude higher than the rate constant of monomer-monomer association, the SGFRKMAF peptide has the potential to inhibit dimerization of 3CLpro monomers not only through blocking the interface region but also through blocking the formation of the intermediates involved in the dimerization process. This could potentially open new avenues for 3CLpro dimerization inhibitors that transcend traditional X-ray-based discovery approaches.

3-chymotrypsin-like protease in SARS-CoV-2.

Coronaviruses constitute a significant threat to the human population. Severe acute respiratory syndrome coronavirus-2, SARS-CoV-2, is a highly pathogenic human coronavirus that has caused the coronavirus disease 2019 (COVID-19) pandemic. It has led to a global viral outbreak with an exceptional spread and a high death toll, highlighting the need for effective antiviral strategies. 3-Chymotrypsin-like protease (3CLpro), the main protease in SARS-CoV-2, plays an indispensable role in the SARS-CoV-2 viral life cycle by cleaving the viral polyprotein to produce 11 individual non-structural proteins necessary for viral replication. 3CLpro is one of two proteases that function to produce new viral particles. It is a highly conserved cysteine protease with identical structural folds in all known human coronaviruses. Inhibitors binding with high affinity to 3CLpro will prevent the cleavage of viral polyproteins, thus impeding viral replication. Multiple strategies have been implemented to screen for inhibitors against 3CLpro, including peptide-like and small molecule inhibitors that covalently and non-covalently bind the active site, respectively. In addition, allosteric sites of 3CLpro have been identified to screen for small molecules that could make non-competitive inhibitors of 3CLpro. In essence, this review serves as a comprehensive guide to understanding the structural intricacies and functional dynamics of 3CLpro, emphasizing key findings that elucidate its role as the main protease of SARS-CoV-2. Notably, the review is a critical resource in recognizing the advancements in identifying and developing 3CLpro inhibitors as effective antiviral strategies against COVID-19, some of which are already approved for clinical use in COVID-19 patients.

Mutational profiling of SARS-CoV-2 papain-like protease reveals requirements for function, structure, and drug escape.

Papain-like protease (PLpro) is an attractive drug target for SARS-CoV-2 because it is essential for viral replication, cleaving viral poly-proteins pp1a and pp1ab, and has de-ubiquitylation and de-ISGylation activities, affecting innate immune responses. We employ Deep Mutational Scanning to evaluate the mutational effects on PLpro enzymatic activity and protein stability in mammalian cells. We confirm features of the active site and identify mutations in neighboring residues that alter activity. We characterize residues responsible for substrate binding and demonstrate that although residues in the blocking loop are remarkably tolerant to mutation, blocking loop flexibility is important for function. We additionally find a connected network of mutations affecting activity that extends far from the active site. We leverage our library to identify drug-escape variants to a common PLpro inhibitor scaffold and predict that plasticity in both the S4 pocket and blocking loop sequence should be considered during the drug design process.

In Silico Comparative Analysis of Ivermectin and Nirmatrelvir Inhibitors Interacting with the SARS-CoV-2 Main Protease.

Exploring therapeutic options is crucial in the ongoing COVID-19 pandemic caused by SARS-CoV-2. Nirmatrelvir, which is a potent inhibitor that targets the SARS-CoV-2 Mpro, shows promise as an antiviral treatment. Additionally, Ivermectin, which is a broad-spectrum antiparasitic drug, has demonstrated effectiveness against the virus in laboratory settings. However, its clinical implications are still debated. Using computational methods, such as molecular docking and 100 ns molecular dynamics simulations, we investigated how Nirmatrelvir and Ivermectin interacted with SARS-CoV-2 Mpro(A). Calculations using density functional theory were instrumental in elucidating the behavior of isolated molecules, primarily by analyzing the frontier molecular orbitals. Our analysis revealed distinct binding patterns: Nirmatrelvir formed strong interactions with amino acids, like MET49, MET165, HIS41, HIS163, HIS164, PHE140, CYS145, GLU166, and ASN142, showing stable binding, with a root-mean-square deviation (RMSD) of around 2.0 Å. On the other hand, Ivermectin interacted with THR237, THR239, LEU271, LEU272, and LEU287, displaying an RMSD of 1.87 Å, indicating enduring interactions. Both ligands stabilized Mpro(A), with Ivermectin showing stability and persistent interactions despite forming fewer hydrogen bonds. These findings offer detailed insights into how Nirmatrelvir and Ivermectin bind to the SARS-CoV-2 main protease, providing valuable information for potential therapeutic strategies against COVID-19.

Potential SARS-CoV-2 Mpro steroid inhibitors from Aphanamixis polystachya (Wall.) R. N. Parker.

Herein, six previously undescribed steroids (1-6), were isolated from leaves and twigs of Aphanamixis polystachya (Wall.) R. N. Parker (Meliaceae). Their structures were elucidated by comprehensive spectroscopic analysis, including HRESIMS, 1D and 2D NMR, UV, and IR. Antiviral activity of these compounds were evaluated. Compounds 1-6 showed varying degrees of inhibitory activity against the severe acute respiratory syndrome coronavirus 2 main protease (SARS-CoV-2 Mpro) at 200 µM.

Discovery of Novel Nonpeptidic and Noncovalent Small Molecule 3CLpro Inhibitors as anti-SARS-CoV-2 Drug Candidate.

SARS-CoV-2 has still been threatening global public health with its emerging variants. Our previous work reported lead compound JZD-07 that displayed good 3CLpro inhibitory activity. Here, an in-depth structural optimization for JZD-07 was launched to obtain more desirable drug candidates for the therapy of SARS-CoV-2 infection, in which 54 novel derivatives were designed and synthesized by a structure-based drug design strategy. Among them, 24 compounds show significantly enhanced 3CLpro inhibitory potencies with IC50 values less than 100 nM, and 11 compounds dose-dependently inhibit the replication of the SARS-CoV-2 delta variant. In particular, compound 49 has the most desirable antiviral activity with EC50 of 0.272 ± 0.013 µM against the delta variant, which was more than 20 times stronger than JZD-07. Oral administration of 49 could significantly reduce the lung viral copies of mice, exhibiting a more favorable therapeutic potential. Overall, this investigation presents a promising drug candidate for further development to treat SARS-CoV-2 infection.

Ensemble docking based virtual screening of SARS-CoV-2 main protease inhibitors.

During the first years of COVID-19 pandemic, X-ray structures of the coronavirus drug targets were acquired at an unprecedented rate, giving hundreds of PDB depositions in less than a year. The main protease (Mpro) of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is the primary validated target of direct-acting antivirals. The selection of the optimal ensemble of structures of Mpro for the docking-driven virtual screening campaign was thus non-trivial and required a systematic and automated approach. Here we report a semi-automated active site RMSD based procedure of ensemble selection from the SARS-CoV-2 Mpro crystallographic data and virtual screening of its inhibitors. The procedure was compared with other approaches to ensemble selection and validated with the help of hand-picked and peer-reviewed activity-annotated libraries. Prospective virtual screening of non-covalent Mpro inhibitors resulted in a new chemotype of thienopyrimidinone derivatives with experimentally confirmed enzyme inhibition.

N-acylbenzimidazoles as selective Acylators of the catalytic cystein of the coronavirus 3CL protease.

The 3CL protease (3CLpro, Mpro) plays a key role in the replication of the SARS-CoV-2 and was validated as therapeutic target by the development and approval of specific antiviral drugs (nirmatrelvir, ensitrelvir), inhibitors of this protease. Moreover, its high conservation within the coronavirus family renders it an attractive therapeutic target for the development of anti-coronavirus compounds with broad spectrum activity to control COVID-19 and future coronavirus diseases. Here we report on the design, synthesis and structure-activity relationships of a new series of small covalent reversible inhibitors of the SARS-CoV-2 3CLpro. As elucidated thanks to the X-Ray structure of some inhibitors with the 3CLpro, the mode of inhibition involves acylation of the thiol of the catalytic cysteine. The synthesis of 60 analogs led to the identification of compound 56 that inhibits the SARS-CoV-2 3CLpro with high potency (IC50 = 70 nM) and displays antiviral activity in cells (EC50 = 3.1 µM). Notably, compound 56 inhibits the 3CLpro of three other human coronaviruses and exhibit a good selectivity against two human cysteine proteases. These results demonstrate the potential of this electrophilic N-acylbenzimidazole series as a basis for further optimization.

Exploring the therapeutic potential of Thai medicinal plants: in vitro screening and in silico docking of phytoconstituents for novel anti-SARS-CoV-2 agents.

BACKGROUND: The high virulence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for coronavirus disease 2019 (COVID-19), has triggered global health and economic concerns. The absence of specific antiviral treatments and the side effects of repurposed drugs present persistent challenges. This study explored a promising antiviral herbal extract against SARS-CoV-2 from selected Thai medicinal plants based on in vitro efficacy and evaluated its antiviral lead compounds by molecular docking. METHODS: Twenty-two different ethanolic-aqueous crude extracts (CEs) were rapidly screened for their potential activity against porcine epidemic diarrhea virus (PEDV) as a surrogate using a plaque reduction assay. Extracts achieving ≥ 70% anti-PEDV efficacy proceeded to the anti-SARS-CoV-2 activity test using a 50% tissue culture infectious dose method in Vero E6 cells. Molnupiravir and extract-free media served as positive and negative controls, respectively. Potent CEs underwent water/ethyl acetate fractionation to enhance antiviral efficacy, and the fractions were tested for anti-SARS-CoV-2 performance. The fraction with the highest antiviral potency was identified using liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Molecular docking analyses of these compounds against the main protease (Mpro) of SARS-CoV-2 (6LU7) were performed to identify antiviral lead molecules. The top three hits were further evaluated for their conformational stability in the docked complex using molecular dynamics (MD) simulation. RESULTS: The water fraction of mulberry (Morus alba Linn.) leaf CE (WF-MLCE) exhibited the most potent anti-SARS-CoV-2 efficacy with low cytotoxicity profile (CC50 of ~ 0.7 mg/mL), achieving 99.92% in pre-entry mode and 99.88% in postinfection treatment mode at 0.25 mg/mL. Flavonoids and conjugates were the predominant compounds identified in WF-MLCE. Molecular docking scores of several flavonoids against SARS-CoV-2 Mpro demonstrated their superior antiviral potency compared to molnupiravir. Remarkably, myricetin-3-O-ß-D-galactopyranoside, maragrol B, and quercetin 3-O-robinobioside exhibited binding energies of ~ - 9 kcal/mol. The stability of each ligand-protein complex of these compounds with the Mpro system showed stability during MD simulation. These three molecules were pronounced as antiviral leads of WF-MLCE. Given the low cytotoxicity and high antiviral potency of WF-MLCE, it holds promise as a candidate for future therapeutic development for COVID-19 treatment, especially considering its economic and pharmacological advantages.

In vitro selection and analysis of SARS-CoV-2 nirmatrelvir resistance mutations contributing to clinical virus resistance surveillance.

To facilitate the detection and management of potential clinical antiviral resistance, in vitro selection of drug-resistant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) against the virus Mpro inhibitor nirmatrelvir (Paxlovid active component) was conducted. Six Mpro mutation patterns containing T304I alone or in combination with T21I, L50F, T135I, S144A, or A173V emerged, with A173V+T304I and T21I+S144A+T304I mutations showing >20-fold resistance each. Biochemical analyses indicated inhibition constant shifts aligned to antiviral results, with S144A and A173V each markedly reducing nirmatrelvir inhibition and Mpro activity. SARS-CoV-2 surveillance revealed that in vitro resistance-associated mutations from our studies and those reported in the literature were rarely detected in the Global Initiative on Sharing All Influenza Data database. In the Paxlovid Evaluation of Protease Inhibition for COVID-19 in High-Risk Patients trial, E166V was the only emergent resistance mutation, observed in three Paxlovid-treated patients, none of whom experienced COVID-19-related hospitalization or death.

Boosting immunity: synergistic antiviral effects of luteolin, vitamin C, magnesium and zinc against SARS-CoV-2 3CLpro.

SARS-CoV-2 was first discovered in 2019 and has disseminated throughout the globe to pandemic levels, imposing significant health and economic burdens. Although vaccines against SARS-CoV-2 have been developed, their long-term efficacy and specificity have not been determined, and antiviral drugs remain necessary. Flavonoids, which are commonly found in plants, fruits, and vegetables and are part of the human diet, have attracted considerable attention as potential therapeutic agents due to their antiviral and antimicrobial activities and effects on other biological activities, such as inflammation. The present study uses a combination of biochemical, cellular, molecular dynamics, and molecular docking experiments to provide compelling evidence that the flavonoid luteolin (2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-chromen-4-one) has antiviral activity against SARS-CoV-2 3-chymotrypsin-like protease (3CLpro) that is synergistically enhanced by magnesium, zinc, and vitamin C. The IC50 of luteolin against 2 µM 3CLpro is 78 µM and decreases 10-fold to 7.6 µM in the presence of zinc, magnesium, and vitamin C. Thermodynamic stability analyses revealed that luteolin has minimal effects on the structure of 3CLpro, whereas metal ions and vitamin C significantly alter the thermodynamic stability of the protease. Interactome analysis uncovered potential host-virus interactions and functional clusters associated with luteolin activity, supporting the relevance of this flavone for combating SARS-CoV-2 infection. This comprehensive investigation sheds light on luteolin's therapeutic potential and provides insights into its mechanisms of action against SARS-CoV-2. The novel formulation of luteolin, magnesium, zinc, and vitamin C may be an effective avenue for treating COVID-19 patients.

LC/MS-guided isolation and GNPS procedures to identify flavonoid of HCoV-OC43 inhibitors.

The screening of based target compounds supported by LC/MS, MS/MS and Global Natural Products Social (GNPS) used to identify the compounds 1-10 of Butea monsperma. They were evaluated in human malignant embryonic rhabdomyoma cells (RD cells) infected with Human coronavirus OC43 (HCoV-OC43) and showed significant inhibitory activity. Target inhibition tests showed that compounds 6 and 8 inhibited the proteolytic enzyme 3CLpro, which is widely present in coronavirus and plays an important role in the replication process, with an effective IC50 value. The study confirmed that dioxymethylene of compound 8 may be a key active fragment in inhibiting coronavirus (EC50 7.2 µM, SI > 139.1). The results have led to identifying natural bioactive compounds for possible inhibiting HCoV-OC43 and developing drug for Traditional Chinese Medicine (TCM).

Influence of EGCG oxidation on inhibitory activity against the SARS-CoV-2 main protease.

SARS-CoV-2 main protease (Mpro) is a well-recognized target for COVID-19 therapy. Green tea (-)-epigallocatechin-3-gallate (EGCG) possesses Mpro-inhibitory activity; however, the influence of EGCG oxidation on its inhibition activity remains obscure, given its high oxidation propensity. This study reveals that prolonged EGCG oxidation in the presence of Mpro dramatically increases its inhibitory activity with an IC50 of 0.26 µM. The inhibitory mechanism is that EGCG-quinone preferentially binds the active site Mpro-Cys145-SH, which forms a quinoprotein. Though Mpro is present in the cell lysate, EGCG preferentially depletes its thiols. Non-cytotoxic EGCG effectively generates a quinoprotein in living cells, thus EGCG might selectively inhibit Mpro in SARS-CoV-2 infected cells. Chlorogenic acid facilitates EGCG oxidation. Together, they synergistically deplete multiple Mpro thiols though this is not more beneficial than EGCG alone. By contrast, excessive EGCG oxidation prior to incubation with Mpro largely compromises its inhibitory activity. Overall, the low IC50 and the high selectivity imply that EGCG is a promising dietary Mpro inhibitor. While EGCG oxidation in the presence of Mpro has a pivotal role in inhibition, enhancing EGCG oxidation by chlorogenic acid no longer increases its inhibitory potential. EGCG oxidation in the absence of Mpro should be avoided to maximize its Mpro-inhibitory activity.

Structural basis for the inhibition of the HCoV-NL63 main protease Mpro by X77.

Human coronaviruses are a group of pathogens that primarily cause respiratory and intestinal diseases. Infection can easily cause respiratory symptoms, as well as a variety of serious complications. There are several types of human coronaviruses, such as SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, and SARS-CoV-2. The prevalence of COVID-19 has led to a growing focus on drug research against human coronaviruses. The main protease (Mpro) from human coronaviruses is a relatively conserved that controls viral replication. X77 was discovered to have extremely high inhibitory activity against SARS-CoV-2 Mpro through the use of computer-simulated docking. In this paper, we have resolved the crystal structure of the HCoV-NL63 Mpro complexed with X77 and analyzed their interaction in detail. This data provides essential information for solving their binding modes and their structural determinants. Then, we compared the binding modes of X77 with SARS-CoV-2 Mpro and HCoV-NL63 Mpro in detail. This study illustrates the structural basis of HCoV-NL63 Mpro binding to the inhibitor X77. The structural insights derived from this study will inform the development of new drugs with broad-spectrum resistance to human coronaviruses.