FMO-guided design of darunavir analogs as HIV-1 protease inhibitors.

The prevalence of HIV-1 infection continues to pose a significant global public health issue, highlighting the need for antiretroviral drugs that target viral proteins to reduce viral replication. One such target is HIV-1 protease (PR), responsible for cleaving viral polyproteins, leading to the maturation of viral proteins. While darunavir (DRV) is a potent HIV-1 PR inhibitor, drug resistance can arise due to mutations in HIV-1 PR. To address this issue, we developed a novel approach using the fragment molecular orbital (FMO) method and structure-based drug design to create DRV analogs. Using combinatorial programming, we generated novel analogs freely accessible via an on-the-cloud mode implemented in Google Colab, Combined Analog generator Tool (CAT). The designed analogs underwent cascade screening through molecular docking with HIV-1 PR wild-type and major mutations at the active site. Molecular dynamics (MD) simulations confirmed the assess ligand binding and susceptibility of screened designed analogs. Our findings indicate that the three designed analogs guided by FMO, 19-0-14-3, 19-8-10-0, and 19-8-14-3, are superior to DRV and have the potential to serve as efficient PR inhibitors. These findings demonstrate the effectiveness of our approach and its potential to be used in further studies for developing new antiretroviral drugs.

Machine learning-guided design of potent darunavir analogs targeting HIV-1 proteases: A computational approach for antiretroviral drug discovery.

In the pursuit of novel antiretroviral therapies for human immunodeficiency virus type-1 (HIV-1) proteases (PRs), recent improvements in drug discovery have embraced machine learning (ML) techniques to guide the design process. This study employs ensemble learning models to identify crucial substructures as significant features for drug development. Using molecular docking techniques, a collection of 160 darunavir (DRV) analogs was designed based on these key substructures and subsequently screened using molecular docking techniques. Chemical structures with high fitness scores were selected, combined, and one-dimensional (1D) screening based on beyond Lipinski's rule of five (bRo5) and ADME (absorption, distribution, metabolism, and excretion) prediction implemented in the Combined Analog generator Tool (CAT) program. A total of 473 screened analogs were subjected to docking analysis through convolutional neural networks scoring function against both the wild-type (WT) and 12 major mutated PRs. DRV analogs with negative changes in binding free energy ( ΔΔ G bind ) compared to DRV could be categorized into four attractive groups based on their interactions with the majority of vital PRs. The analysis of interaction profiles revealed that potent designed analogs, targeting both WT and mutant PRs, exhibited interactions with common key amino acid residues. This observation further confirms that the ML model-guided approach effectively identified the substructures that play a crucial role in potent analogs. It is expected to function as a powerful computational tool, offering valuable guidance in the identification of chemical substructures for synthesis and subsequent experimental testing.

Synthesis and biological evaluation of 2'-hydroxychalcone derivatives as AMPK activators.

A series of 2'-hydroxychalcone derivatives with various substituents on B-ring were synthesized and evaluated for AMP-activated protein kinase (AMPK) activation activity in podocyte cells. The results displayed that hydroxy, methoxy and methylenedioxy groups on B-ring could enhance the activitiy better than O-saturated alkyl, O-unsaturated alkyl or other alkoxy groups. Compounds 27 and 29 possess the highest fold change of 2.48 and 2.73, respectively, which were higher than those of reference compound (8) (1.28) and metformin (1.88). Compounds 27 and 29 were then subjected to a concentration-response study to obtain the EC50 values of 2.0 and 4.8 µM, respectively and MTT assays also showed that cell viability was not influenced by the exposure of podocytes to compounds 27 and 29 at concentrations up to 50 µM. In addition, compound 27 was proved to activate AMPK via calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß)-dependent pathway without affecting intracellular calcium levels. The computational study showed that the potent compounds exhibited stronger ligand-binding strength to CaMKKß, particularly compounds 27 (-8.4 kcal/mol) and 29 (-8.0 kcal/mol), compared to compound 8 (-7.5 kcal/mol). Fragment molecular orbital (FMO) calculation demonstrated that compound 27 was superior to compound 29 due to the presence of methyl group, which amplified the binding by hydrophobic interactions. Therefore, compound 27 would represent a promising AMPK activator for further investigation of the treatment of diabetes and diabetic nephropathy.

Structural and thermodynamic insights into antibody light chain tetramer formation through 3D domain swapping.

Overexpression of antibody light chains in small plasma cell clones can lead to misfolding and aggregation. On the other hand, the formation of amyloid fibrils from antibody light chains is related to amyloidosis. Although aggregation of antibody light chain is an important issue, atomic-level structural examinations of antibody light chain aggregates are sparse. In this study, we present an antibody light chain that maintains an equilibrium between its monomeric and tetrameric states. According to data from X-ray crystallography, thermodynamic and kinetic measurements, as well as theoretical studies, this antibody light chain engages in 3D domain swapping within its variable region. Here, a pair of domain-swapped dimers creates a tetramer through hydrophobic interactions, facilitating the revelation of the domain-swapped structure. The negative cotton effect linked to the ß-sheet structure, observed around 215 nm in the circular dichroism (CD) spectrum of the tetrameric variable region, is more pronounced than that of the monomer. This suggests that the monomer contains less ß-sheet structures and exhibits greater flexibility than the tetramer in solution. These findings not only clarify the domain-swapped structure of the antibody light chain but also contribute to controlling antibody quality and advancing the development of future molecular recognition agents and drugs.

Structure-yeast α-glucosidase inhibitory activity relationship of 9-O-berberrubine carboxylates.

Thirty-five 9-O-berberrubine carboxylate derivatives were synthesized and evaluated for yeast α-glucosidase inhibitory activity. All compounds demonstrated better inhibitory activities than the parent compounds berberine (BBR) and berberrubine (BBRB), and a positive control, acarbose. The structure-activity correlation study indicated that most of the substituents on the benzoate moiety such as methoxy, hydroxy, methylenedioxy, benzyloxy, halogen, trifluoromethyl, nitro and alkyl can contribute to the activities except multi-methoxy, fluoro and cyano. In addition, replacing benzoate with naphthoate, cinnamate, piperate or diphenylacetate also led to an increase in inhibitory activities except with phenyl acetate. 9, 26, 27, 28 and 33 exhibited the most potent α-glucosidase inhibitory activities with the IC50 values in the range of 1.61-2.67 µM. Kinetic study revealed that 9, 26, 28 and 33 interacted with the enzyme via competitive mode. These four compounds were also proved to be not cytotoxic at their IC50 values. The competitive inhibition mechanism of these four compounds against yeast α-glucosidase was investigated using molecular docking and molecular dynamics simulations. The binding free energy calculations suggest that 26 exhibited the strongest binding affinity, and its binding stability is supported by hydrophobic interactions with D68, F157, F158 and F177. Therefore, 9, 26, 28 and 33 would be promising candidates for further studies of antidiabetic activity.

A sulfonamide chalcone inhibited dengue virus with a potential target at the SAM-binding site of viral methyltransferase.

Dengue infection is a global health problem as climate change facilitates the spread of mosquito vectors. Infected patients could progress to severe plasma leakage and hemorrhagic shock, where current standard treatment remains supportive. Previous reports suggested that several flavonoid derivatives inhibited mosquito-borne flaviviruses. This work aimed to explore sulfonamide chalcone derivatives as dengue inhibitors and to identify molecular targets. We initially screened 27 sulfonamide chalcones using cell-based antiviral and cytotoxic screenings. Two potential compounds, SC22 and SC27, were identified with DENV1-4 EC50s in the range of 0.71-0.94 and 3.15-4.46 µM, and CC50s at 14.63 and 31.02 µM, respectively. The compounds did not show any elevation in ALT or Cr in C57BL/6 mice on the 1st, 3rd, and 7th days after being administered intraperitoneally with 50 mg/kg SC22 or SC27 in a single dose. Moreover, the SAM-binding site of NS5 methyltransferase was a potential target of SC27 identified by computational and enzyme-based assays. The main target of SC22 was in a late stage of viral replication, but the exact target molecule had yet to be identified. In summary, a sulfonamide chalcone, SC27, was a potential DENV inhibitor that targeted viral methyltransferase. Further investigation should be the study of the structure-activity relationship of SC27 derivatives for higher potency and lower toxicity.

Design of electron-donating group substituted 2-PAM analogs as antidotes for organophosphate insecticide poisoning.

The use of organophosphate (OPs) pesticides is widespread in agriculture and horticulture, but these chemicals can be lethal to humans, causing fatalities and deaths each year. The inhibition of acetylcholinesterase (AChE) by OPs leads to the overstimulation of cholinergic receptors, ultimately resulting in respiratory arrest, seizures, and death. Although 2-pralidoxime (2-PAM) is the FDA-approved drug for treating OP poisoning, there is difficulty in blood-brain barrier permeation. To address this issue, we designed and evaluated a series of 2-PAM analogs by substituting electron-donating groups on the para and/or ortho positions of the pyridinium core using in silico techniques. Our PCM-ONIOM2 (MP2/6-31G*:PM7//B3LYP/6-31G*:UFF) binding energy results demonstrated that 13 compounds exhibited higher binding energy than 2-PAM. The analog with phenyl and methyl groups substituted on the para and ortho positions, respectively, showed the most favorable binding characteristics, with aromatic residues in the active site (Y124, W286, F297, W338, and Y341) and the catalytic residue S203 covalently bonding with paraoxon. The results of DS-MD simulation revealed a highly favorable apical conformation of the potent analog, which has the potential to enhance reactivation of AChE. Importantly, newly designed compound demonstrated appropriate drug-likeness properties and blood-brain barrier penetration. These results provide a rational guide for developing new antidotes to treat organophosphate insecticide toxicity.

The 8-bromobaicalein alleviated chikungunya-induced musculoskeletal inflammation and reduced the viral load in healthy adult mice.

Chikungunya virus is a re-emerging arbovirus that has caused epidemic outbreaks in recent decades. Patients in older age groups with high viral load and severe immunologic response during acute infection are likely to develop chronic arthritis and severe joint pain. Currently, no antiviral drug is available. Previous studies suggested that a flavone derivative, 8-bromobaicalein, was a potential dengue and Zika replication inhibitor in a cell-based system targeting flaviviral polymerase. Here we characterized that 8-bromobaicalein inhibited chikungunya virus replication with EC50 of 0.49 ± 0.11 µM in Vero cells. The molecular target predicted at viral nsP1 methyltransferase using molecular binding and fragment molecular orbital calculation. Additionally, oral administration of 250 mg/kg twice daily treatment alleviated chikungunya-induced musculoskeletal inflammation and reduced viral load in healthy adult mice. Pharmacokinetic analysis indicated that the 250 mg/kg administration maintained the compound level above EC99.9 for 12 h. Therefore, 8-bromobaicalein should be a potential candidate for further development as a pan-arboviral drug.

Identification of Promising Sulfonamide Chalcones as Inhibitors of SARS-CoV-2 3CLpro through Structure-Based Virtual Screening and Experimental Approaches.

3CLpro is a viable target for developing antiviral therapies against the coronavirus. With the urgent need to find new possible inhibitors, a structure-based virtual screening approach was developed. This study recognized 75 pharmacologically bioactive compounds from our in-house library of 1052 natural product-based compounds that satisfied drug-likeness criteria and exhibited good bioavailability and membrane permeability. Among these compounds, three promising sulfonamide chalcones were identified by combined theoretical and experimental approaches, with SWC423 being the most suitable representative compound due to its competitive inhibition and low cytotoxicity in Vero E6 cells (EC50 = 0.89 ± 0.32 µM; CC50 = 25.54 ± 1.38 µM; SI = 28.70). The binding and stability of SWC423 in the 3CLpro active site were investigated through all-atom molecular dynamics simulation and fragment molecular orbital calculation, indicating its potential as a 3CLpro inhibitor for further SARS-CoV-2 therapeutic research. These findings suggested that inhibiting 3CLpro with a sulfonamide chalcone such as SWC423 may pave the effective way for developing COVID-19 treatments.

Designing Potent α-Glucosidase Inhibitors: A Synthesis and QSAR Modeling Approach for Biscoumarin Derivatives.

Nineteen biscoumarins were synthesized, well-characterized, and evaluated against α-glucosidases in vitro. Of these, six compounds (10, 12, 16, and 17-19) were newly synthesized and not previously reported in the chemical literature. The majority of the synthesized derivatives demonstrated significant inhibitory activity. A quantitative structure-activity relationship (QSAR) model was developed, revealing a strong correlation between the anti-α-glucosidase activity and selected molecular descriptors. Based on this model, two new compounds (18 and 19) were designed, which exhibited the strongest inhibition with IC50 values of 0.62 and 1.21 µM, respectively, when compared to the positive control (acarbose) with an IC50 value of 93.63 µM. Enzyme kinetic studies of compounds 18 and 19 revealed their competitive inhibition with Ki values of 3.93 and 1.80 µM, respectively. Computational studies demonstrated that compound 18 could be inserted into the original binding site (OBS) of α-glucosidase MAL12 and form multiple hydrophobic interactions with nearby amino acids, with the bromo group playing an essential role in enhancing the binding strength and stability at the OBS of the enzyme based on the quantum mechanical calculations using the fragment molecular orbital method. These findings provide valuable insights into the design of potent α-glucosidase inhibitors, which may have potential therapeutic applications in the treatment of diabetes and related diseases.

Determination of the Association between Mesotrione Sensitivity and Conformational Change of 4-Hydroxyphenylpyruvate Dioxygenase via Free-Energy Analyses.

One widely known herbicide target is 4-hydroxyphenylpyruvate dioxygenase (HPPD). Avena sativa HPPD is less sensitive to mesotrione (herbicide) than Arabidopsis thaliana HPPD. HPPD inhibitor-sensitivity is governed by the dynamic behavior of the C-terminal α-helix of HPPD (H11) in closed and open forms. However, the specific relationship between the plant inhibitor sensitivity and H11 dynamic behavior remains unclear. Herein, we determined the conformational changes in H11 to understand the inhibitor-sensitivity mechanism based on free-energy calculations using molecular dynamics simulations. The calculated free-energy landscapes revealed that Arabidopsis thaliana HPPD preferred the open form of H11 in the apo form and the closed-like form in complex with mesotrione, whereas Avena sativa HPPD exhibited the opposite tendency. We also identified some important residues involved in the dynamic behavior of H11. Therefore, inhibitor sensitivity is governed by indirect interactions due to the protein flexibility caused by the conformational changes of H11.

Sulfonylated Indeno[1,2-c]quinoline Derivatives as Potent EGFR Tyrosine Kinase Inhibitors.

The epidermal growth factor receptor (EGFR) has been considered a potential target for lung cancer therapy due to its essential role in regulating the survival and proliferation of cancer cells. Although erlotinib, a potent EGFR tyrosine kinase (EGFR-TK) inhibitor, has been used as the first-line drug for lung cancer treatment, acquired drug resistance caused by the T790M secondary mutation of EGFR-TK inevitably develops after a median response duration of 9-13 months. Thus, the search for promising compounds to effectively target EGFR-TK has become an imperative necessity. In this study, the kinase inhibitory activities of a series of sulfonylated indeno[1,2-c]quinolines (SIQs) against EGFR-TK were experimentally and theoretically investigated. Among the 23 SIQ derivatives studied, eight compounds showed enhanced EGFR-TK inhibitory activity (IC50 values of ca. 0.6-10.2 nM) compared to the known drug erlotinib (IC50 of ∼20 nM). In a cell-based assay in human cancer cell lines with EGFR overexpression (A549 and A431 cells), the eight selected SIQs all showed more significant cytotoxicity against A431 than A549 cells, consistent with the higher EGFR expression in A431 cells. Molecular docking and FMO-RIMP2/PCM calculations revealed that SIQ17 occupies the ATP-binding site of EGFR-TK, where its sulfonyl group is mainly stabilized by C797, L718, and E762 residues. Triplicate 500 ns molecular dynamics (MD) simulations also confirmed the binding strength of SIQ17 in complex with EGFR. Overall, the potent SIQ compounds obtained in this work could be further optimized for developing novel anticancer drug candidates targeting EGFR-TK.

In silico screening of chalcones and flavonoids as potential inhibitors against yellow head virus 3C-like protease.

Yellow head virus (YHV) is one of the most important pathogens in prawn cultivation. The outbreak of YHV could potentially result in collapses in aquaculture industries. Although a flurry of development has been made in searching for preventive and therapeutic approaches against YHV, there is still no effective therapy available in the market. Previously, computational screening has suggested a few cancer drugs to be used as YHV protease (3CLpro) inhibitors. However, their toxic nature is still of concern. Here, we exploited various computational approaches, such as deep learning-based structural modeling, molecular docking, pharmacological prediction, and molecular dynamics simulation, to search for potential YHV 3CLpro inhibitors. A total of 272 chalcones and flavonoids were in silico screened using molecular docking. The bioavailability, toxicity, and specifically drug-likeness of hits were predicted. Among the hits, molecular dynamics simulation and trajectory analysis were performed to scrutinize the compounds with high binding affinity. Herein, the four selected compounds including chalcones cpd26, cpd31 and cpd50, and a flavonoid DN071_f could be novel potent compounds to prevent YHV and GAV propagation in shrimp. The molecular mechanism at the atomistic level is also enclosed that can be used to further antiviral development.

The 8-bromobaicalein inhibited the replication of dengue, and Zika viruses and targeted the dengue polymerase.

Dengue and Zika viruses are mosquito-borne flaviviruses burdening millions every year with hemorrhagic fever and neurological symptoms. Baicalein was previously reported as a potential anti-flaviviral candidate and halogenation of flavones and flavanones potentiated their antiviral efficacies. Here, we reported that a chemically modified 8-bromobaicalein effectively inhibited all dengue serotypes and Zika viruses at 0.66-0.88 micromolar in cell-based system. The compound bound to dengue serotype 2 conserved pocket and inhibited the dengue RdRp activity with 6.93 fold more than the original baicalein. Moreover, the compound was mildly toxic against infant and adult C57BL/6 mice despite administering continuously for 7 days. Therefore, the 8-bromobaicalein should be investigated further in pharmacokinetics and efficacy in an animal model.

X-ray crystallographic and mutational analysis of the NylC precursor: catalytic mechanism of autocleavage and substrate hydrolysis of nylon hydrolase.

Nylon hydrolase (NylC), a member of the N-terminal nucleophile (Ntn) hydrolase superfamily, is responsible for the degradation of various aliphatic nylons, including nylon-6 and nylon-66. NylC is initially expressed as an inactive precursor (36 kDa), but the precursor is autocatalytically cleaved at Asn266/Thr267 to generate an active enzyme composed of 27 and 9 kDa subunits. We isolated various mutants with amino acid changes at the catalytic centre. X-ray crystallographic analysis revealed that the NylC precursor forms a doughnut-shaped quaternary structure composed of four monomers (molecules A-D) with D2 symmetry. Catalytic residues in the precursor are covered by loop regions at the A/B interface (equivalent to the C/D interface). However, the catalytic residues are exposed to the solvent environment through autocleavage followed by movements of the loop regions. T267A, D306A and D308A mutations resulted in a complete loss of autocleavage. By contrast, in the T267S mutant, autocleavage proceeded slowly at a constant reaction rate (k = 2.8 × 10-5  s-1 ) until complete conversion, but the reaction was inhibited by K189A and N219A mutations. Based on the crystallographic and molecular dynamic simulation analyses, we concluded that the Asp308-Asp306-Thr267 triad, resembling the Glu-Ser-Ser triad conserved in Ntn-hydrolase family enzymes, is responsible for autocleavage and that hydrogen-bonding networks connecting Thr267 with Lys189 and Asn219 are required for increasing the nucleophilicity of Thr267-OH in both the water accessible and water inaccessible systems. Furthermore, we determined that NylC employs the Asp308-Asp306-Thr267 triad as catalytic residues for substrate hydrolysis, but the reaction requires Lys189 and Tyr146 as additional catalytic/substrate-binding residues specific for nylon hydrolysis.

N-Containing α-Mangostin Analogs via Smiles Rearrangement as the Promising Cytotoxic, Antitrypanosomal, and SARS-CoV-2 Main Protease Inhibitory Agents.

New N-containing xanthone analogs of α-mangostin were synthesized via one-pot Smiles rearrangement. Using cesium carbonate in the presence of 2-chloroacetamide and catalytic potassium iodide, α-mangostin (1) was subsequently transformed in three steps to provide ether 2, amide 3, and amine 4 in good yields at an optimum ratio of 1:3:3, respectively. The evaluation of the biological activities of α-mangostin and analogs 2-4 was described. Amine 4 showed promising cytotoxicity against the non-small-cell lung cancer H460 cell line fourfold more potent than that of cisplatin. Both compounds 3 and 4 possessed antitrypanosomal properties against Trypanosoma brucei rhodesiense at a potency threefold stronger than that of α-mangostin. Furthermore, ether 2 gave potent SARS-CoV-2 main protease inhibition by suppressing 3-chymotrypsinlike protease (3CLpro) activity approximately threefold better than that of 1. Fragment molecular orbital method (FMO-RIMP2/PCM) indicated the improved binding interaction of 2 in the 3CLpro active site regarding an additional ether moiety. Thus, the series of N-containing α-mangostin analogs prospectively enhance druglike properties based on isosteric replacement and would be further studied as potential biotically active chemical entries, particularly for anti-lung-cancer, antitrypanosomal, and anti-SARS-CoV-2 main protease applications.

Lichen-Derived Diffractaic Acid Inhibited Dengue Virus Replication in a Cell-Based System.

Dengue is a mosquito-borne flavivirus that causes 21,000 deaths annually. Depsides and depsidones of lichens have previously been reported to be antimicrobials. In this study, our objective was to identify lichen-derived depsides and depsidones as dengue virus inhibitors. The 18 depsides and depsidones of Usnea baileyi, Usnea aciculifera, Parmotrema dilatatum, and Parmotrema tsavoense were tested against dengue virus serotype 2. Two depsides and one depsidone inhibited dengue virus serotype 2 without any apparent cytotoxicity. Diffractaic acid, barbatic acid, and Parmosidone C were three active compounds further characterized for their efficacies (EC50), cytotoxicities (CC50), and selectivity index (SI; CC50/EC50). Their EC50 (SI) values were 2.43 ± 0.19 (20.59), 0.91 ± 0.15 (13.33), and 17.42 ± 3.21 (8.95) µM, respectively. Diffractaic acid showed the highest selectivity index, and similar efficacies were also found in dengue serotypes 1-4, Zika, and chikungunya viruses. Cell-based studies revealed that the target was mainly in the late stage with replication and the formation of infectious particles. This report highlights that a lichen-derived diffractaic acid could become a mosquito-borne antiviral lead as its selectivity indices ranged from 8.07 to 20.59 with a proposed target at viral replication.

Design, synthesis, in vitro, in silico, and SAR studies of flavone analogs towards anti-dengue activity.

Flavone has recently been proved as a promising scaffold for the development of a novel drug against dengue fever, one of the major health threats globally. However, the structure-activity relationship study of flavones on the anti-dengue activity remains mostly limited to the natural-occuring analogs. Herein, 27 flavone analogs were successfully synthesized, of which 5 analogs (5e, 5h, 5o, 5q, and 5r) were novel. In total, 33 analogs bearing a diverse range of substituents were evaluated for their efficacy against DENV2-infected LLC/MK2 cells. The introduction of electron-withdrawing groups on ring B such as Br (5m) or NO2 (5n and 5q) enhanced the activity significantly. In particular, the tri-ester 5d and di-ester 5e exhibited low toxicity against normal cell, and exceptional DENV2 inhibition with the EC50 as low as 70 and 68 nM, respectively, which is over 300-fold more active compared to the original baicalein reference. The viral targets for these potent flavone analogs were predicted to be NS5 MTase and NS5 RdRp, as suggested by the likelihood ratios from the molecular docking study. The great binding interaction energy of 8-bromobaicalein (5f) confirms the anti-dengue activity at atomistic level. The physicochemical property of all the synthetic flavone analogs in this study were predicted to be within the acceptable range. Moreover, the QSAR model showed the strong correlation between the anti-dengue activity and the selected molecular descriptors. This study emphasizes the great potential of flavone as a core structure for further development as a novel anti-dengue agent in the future.

Promising SARS-CoV-2 main protease inhibitor ligand-binding modes evaluated using LB-PaCS-MD/FMO.

Parallel cascade selection molecular dynamics-based ligand binding-path sampling (LB-PaCS-MD) was combined with fragment molecular orbital (FMO) calculations to reveal the ligand path from an aqueous solution to the SARS-CoV-2 main protease (Mpro) active site and to customise a ligand-binding pocket suitable for delivering a potent inhibitor. Rubraxanthone exhibited mixed-inhibition antiviral activity against SARS-CoV-2 Mpro, relatively low cytotoxicity, and high cellular inhibition. However, the atomic inhibition mechanism remains ambiguous. LB-PaCS-MD/FMO is a hybrid ligand-binding evaluation method elucidating how rubraxanthone interacts with SARS-CoV-2 Mpro. In the first step, LB-PaCS-MD, which is regarded as a flexible docking, efficiently samples a set of ligand-binding pathways. After that, a reasonable docking pose of LB-PaCS-MD is evaluated by the FMO calculation to elucidate a set of protein-ligand interactions, enabling one to know the binding affinity of a specified ligand with respect to a target protein. A possible conformation was proposed for rubraxanthone binding to the SARS-CoV-2 Mpro active site, and allosteric inhibition was elucidated by combining blind docking with k-means clustering. The interaction profile, key binding residues, and considerable interaction were elucidated for rubraxanthone binding to both Mpro sites. Integrated LB-PaCS-MD/FMO provided a more reasonable complex structure for ligand binding at the SARS-CoV-2 Mpro active site, which is vital for discovering and designing antiviral drugs.