TL;DR
Structurally optimized triazine-based nucleoside analogues are identified as potential SARS-CoV-2 Mpro inhibitors and the utility of an integrated computational strategy for the rational design and prioritization of antiviral candidates for further experimental validation is demonstrated.
. The main protease of SARS-CoV-2 (Mpro, 3CLpro) is a crucial viral enzyme required for the cleavage of viral polyproteins during coronavirus replication. Its catalytic activity relies on the conserved His41–Cys145 dyad, making the main protease (Mpro) an important molecular target for the discovery of antiviral drugs. In the present study, four artemisinin-derived compounds namely, artesunate, artemether, artemisinin and dihydroartemisinin were evaluated as potential Mpro binders using molecular docking and post docking interaction analysis . Docking simulations were performed using the crystal structure of SARS-CoV-2 Mpro and AutoDock Vina. The docking grid was centered on the catalytic pocket defined by His41 and Cys145. The generated poses were assessed based on predicted affinity, proximity to catalytic residues, hydrogen bond-like O/N contacts, hydrophobic C–C contacts, and multivariate interaction profiles. Among the tested ligands, artesunate showed the most favorable predicted binding affinity with docking score
Although the acute phase of the SARS-CoV-2 pandemic has subsided, the continued emergence of viral variants underscores the need for structurally diverse antiviral inhibitors. In this study, molecular docking followed by molecular dynamics (300 ns) simulations and binding free energy calculations using the Molecular Mechanics Poisson–Boltzmann Surface Area (MM-PBSA) method were employed to evaluate substituted flavonoids derived from Taraxacum officinale and Urtica dioica as potential inhibitors of the SARS-CoV-2 main protease (Mpro/3CLpro). Docking analysis identified several derivatives with favorable binding scores; however, dynamic refinement revealed differential stability among the ligand–protein complexes. Among the evaluated compounds, the luteolin derivative LND-17 showed the most consistent performance, exhibiting binding free energy estimates approaching those obtained for the reference inhibitors nirmatrelvir and ensitrelvir, sustained catalytic pocket occupancy, and energetic contributions involving the catalytic dyad (His41 and Cys145). Additional derivatives, including LNG-04, QND-07, and QNG-20, showed moderate stabilization but lower overall consistency. These findings highlight glycosylated flavonoids as promising scaffolds for future structure-based optimization and provide structural insights to guide experimental validation.
SARS-CoV-2 main protease (MPro) is a proven target for drug discovery of small-molecule antiviral agents due to its crucial role in viral polyprotein processing, high structural conservation across numerous divergent variants, and the lack of similar human enzymes. Unlike covalent compounds, noncovalent inhibitors of MPro do not modify the enzyme's active site, and may offer improved safety profiles, greater chemical tractability, and better oral bioavailability without the need for pharmacokinetic enhancement. In this study, we designed, synthesized and characterized thirteen noncovalent nonpeptidic SARS-CoV-2 MPro inhibitors clustered into two series (KK and KB) of compounds. The inhibitors were designed based on our recently discovered Mcule-5948770040 and its analogue HL-3-68 designed through the structure–activity relationship study. To obtain atomic details of the inhibitors' binding we solved room-temperature X-ray structures of the MPro/inhibitor complexes, and to quantify their binding and antiviral properties we performed in vitro DSF and ITC measurements and TCID50 antiviral assays. In addition, a room-temperature neutron structure of the MPro/KB-5 complex allowed direct determination of hydrogen positions, mapping intermolecular interactions and directly visualizing the protonation states and hydrogen bonding. Improved binding affinities of KK-7 and KB-3 through KB-6 could be attributed to the observed nonconventional S–H⋯F hydrogen bond and an additional conventional hydrogen bond between the carboxamide moieties and Q189. Our study provides binding details for the designed compounds and demonstrates the feasibility of our joint X-ray/neutron structure-assisted drug design approach to generate more potent noncovalent nonpeptidic MPro inhibitors.
The COVID-19 pandemic severely threatened global public health, and the SARS-CoV-2 main protease (3CLpro) is a vital target for anti-COVID-19 drug development. Nevertheless, most reported 3CLpro orthosteric inhibitors readily trigger viral drug resistance, limiting their long-term clinical application. Herein, we identified a novel 3CLpro allosteric site AS1 via AlloSite and AlloReverse tools. Through virtual screening and structural optimization, a series of potent 3CLpro allosteric derivatives were designed and synthesized. FRET assays confirmed that derivatives E42 (IC50 = 59.85 ± 4.10 μM) and E60 (IC50 = 7.64 ± 0.25 μM) possessed superior inhibitory potency to the lead compound E0. Structure-activity relationship and molecular docking analyses revealed that the p-trifluoromethylphenethylamine fragment, trimethylene linker, amide bond, methyl group and 4-nitropyrazole ring are key active structural motifs, enabling E42 to specifically bind the AS1 allosteric site. The antiviral activity and cytotoxicity of E42 and E60 were evaluated using a SARS-CoV-2 trans-complementation cell culture system (containing mNG/Fluc) and the CCK-8 assay, respectively. The luciferase reporter assay revealed a consistent trend in antiviral activity among nirmatrelvir, E42, and E60, with E42 exhibiting moderate potency (EC50 = 13.79 ± 1.28 μM). Molecular dynamics simulations indicated that E42 inhibits 3CLpro activity by stabilizing its inactive conformation and disrupting protein allosteric equilibrium. Compared with conventional orthosteric inhibitors prone to drug resistance and poorly active, unsafe reported allosteric inhibitors, the optimized compounds in this study combine favorable potency and biosafety. This work provides novel lead structures and mechanistic insights for developing anti-COVID-19 3CLpro allosteric inhibitors.
The main protease (MPro) of coronaviruses (CoVs) is an essential enzyme involved in viral replication and represents an attractive target for antiviral drug discovery. Based on the similar binding pocket residues within the MPro of different CoVs, this study aimed to identify potential inhibitors of SARS-CoV-2 MPro from PDB ID 6M2N using integrated computational approaches. Interaction-based pharmacophore modeling, virtual screening, molecular docking, MM-GBSA binding energy calculation, and molecular dynamics simulation (MDS) were performed using BIOVIA Discovery Studio. The validated pharmacophore model was utilized to screen the ZINC database, followed by docking and 100 ns MDS analyses of the top-ranked compounds. The pharmacophore model 01 demonstrated favorable predictive performance (AUC = 0.781). Virtual screening identified 483 compounds, from which 15 compounds were selected for docking studies. Among them, ZINC95473654 (Lig-1), ZINC95473725 (Lig-2), and ZINC08792368 (Lig-3) exhibited strong binding affinity toward MPro. Lig-1 demonstrated the best docking score and binding free energy, along with stable interactions with key catalytic residues HIS41, CYS145, and GLU166. MDS analyses further confirmed that Lig-1, Lig-2 and Lig-3 maintained stable conformations. The hydrogen bond distance monitoring and post MDS-MM-GBSA results suggest Lig-1 followed by Lig-3 as an inhibitor for MPro and persistent intermolecular interactions throughout the 100 ns simulation period. The findings suggest that Lig-1, followed by Lig-3, may serve as promising computational lead compounds targeting SARS-CoV-2 MPro, representing promising candidates for further experimental validation.
Abstract The ongoing prevalence of SARS-CoV-2 variations highlights the urgent need for novel antiviral agents targeting key viral proteins. The papain-like protease (PLpro) is pivotal in viral replication and immune evasion, rendering it a compelling therapeutic target. In the present study, a series of novel thiadiazole- and oxadiazole-based derivatives (12-g & 13a-g) were rationally designed based on the structural features of the known PLpro inhibitor GRL0617. Convergent synthetic strategy was employed to synthesize the target compounds involving the construction of 1,3,4-thiadiazole/oxadiazole intermediates followed by amide coupling with a naphthyl-containing scaffold. All compounds were characterized by 1H NHMR, 13C NMR and mass spectrometry and evaluated for their in vitro SARS-CoV-2 PLpro inhibitory activity. Several compounds exhibited potent inhibition, surpassing the reference inhibitor GRL0617 (IC50 = 2.4 ± 1.1 µM). Among them, oxadiazole derivative (13c) featuring 3-cyanophenyl substitution emerged as the most potent inhibitor with IC50 value of 0.06 ± 0.8 µM, followed by compounds (12b, IC50 = 0.3 ± 0.3 µM), (12e, IC50 = 0.4 ± 1.6 µM) and (13e, IC50 = 0.6 ± 0.8 µM) respectively. Structure activity relationship (SAR) analysis revealed that electron-withdrawing substituents, particularly cyano group at meta position, significantly enhanced PLpro inhibition whereas methoxy/methyl groups resulted in reduced inhibition. Furthermore, molecular docking studies demonstrated favorable binding interactions of the compounds within the PLpro catalytic pocket through H-bonding, hydrophobic and π–π interactions. Moreover, molecular dynamic simulations confirmed the stability of the ligand-protein complexes throughout the simulation period supporting the experimental findings. The integrated rational design-based synthesis, biological evaluation, and computational investigations collectively identified thiadiazole/oxadiazole scaffolds as promising candidates for the development of SARS-CoV-2 inhibitors, offering valuable insights for next-generation antiviral agents targeting coronavirus proteases.