Natural flavonoid apigenin and xanthoangelol E are potent antivirals against human metapneumovirus: an in-silico study

Natural flavonoid apigenin and xanthoangelol E are potent antivirals against human metapneumovirus: an in-silico study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Natural flavonoid apigenin and xanthoangelol E are potent antivirals against human metapneumovirus: an in-silico study Hasan Huzayfa Rahaman, Nadim Sharif, Wasifuddin Ahmed, Nazmul Sharif, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6892264/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Human metapneumovirus (hMPV) is one of the potential pandemic pathogens, and it is a concern for elderly subjects and immunocompromised patients. There is no vaccine or specific antiviral available for hMPV. We conducted an in-silico study to develop antivirals against human metapneumovirus. Our methodology included protein modeling, stability assessment, molecular docking, molecular simulation, analysis of non-covalent interactions, bioavailability, carcinogenicity, and pharmacokinetic profiling. We pinpointed four plant-derived bio-compounds as antiviral candidates. Among the compounds, apigenin showed the highest binding affinity, with values of -8.0 kcal/mol for the hMPV-F protein and -7.6 kcal/mol for the hMPV-N protein. Molecular dynamic simulations and further analyses confirmed that the protein-ligand docked complexes exhibited significantly acceptable stability compared to two standard antiviral drugs. Additionally, these four compounds yielded satisfactory outcomes in bioavailability, drug-likeness, and ADME-Tox (absorption, distribution, metabolism, excretion, and toxicity) and STopTox analyses. This study highlights the highest potential of apigenin and xanthoangelol E as effective antivirals, underscoring the necessity for preclinical and clinical trials and wet-lab evaluation to consider them as treatments for human metapneumovirus infection. Biological sciences/Drug discovery Biological sciences/Microbiology/Antimicrobials/Antiviral agents Human metapneumovirus Antivirals Drug discovery In-silico Molecular docking Dynamic simulation Pharmacokinetics ADME-Tox Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction Human metapneumovirus (hMPV) is one of the common viral etiological agents of acute respiratory tract infection (ARI) contributing to a significant percentage of morbidity and mortality, among the immunocompromised and elderly people, especially in Southeast Asia [ 1 , 2 ]. Although the prevalence of hMPV varies globally from 5–15% among children, the altered genotypes can cause higher incidence and larger outbreaks [ 1 – 4 ]. The virus was first discovered by genetic analysis of nasopharyngeal aspirate samples taken from 28 hospitalized children in the Netherlands. These samples, taken from children with ARIs during the past 20 years, had symptoms ranging from mild respiratory problems to severe cough, bronchiolitis, and pneumonia. Some of them were hospitalized and needed mechanical ventilation [ 2 ]. Respiratory viruses pose a persistent and significant threat to global public health, with human metapneumovirus (hMPV) ranking among the most concerning pathogens. The hMPV, a leading cause of respiratory tract infections, disproportionately affects vulnerable populations, including young children, the elderly, and immunocompromised individuals [ 3 – 7 ]. The virus is transmitted by direct or close contact with the respiratory secretions (through sneezing and coughing) of people infected with the virus or by contact with objects and surfaces that have the virus on them [ 6 , 7 ]. Children and adults with other medical conditions like asthma, chronic lung disease, congenital heart disease, neuromuscular disorders, cancer or chronic obstructive pulmonary disease (COPD) also tend to acquire hMPV infection that may require hospitalization [ 7 ]. HMPV possesses a 13.3 kb negative-sense RNA genome containing eight genes: 3’-N-P-M-F-M2-SH-G-L-5’. The F, SH, and G proteins constitute surface glycoproteins [ 8 , 9 ]. The M and M2 genes encode the matrix protein M, and the M2-1 and M2-2 proteins via overlapping ORFs [ 10 , 11 ]. The negative-sense, single-stranded RNA genome encodes eight genes, namely N, nucleocapsid; P, phosphoprotein; M, matrix; F, fusion; M2; SH, small hydrophobic; G, glycoprotein; and L, polymerase [ 12 , 13 ]. The fusion (F) protein of hMPV is highly conserved among hMPV subgroups, and it shares similar structural topology and approximately 30% amino acid sequence homology with the respiratory syncytial virus (RSV) F protein. The prefusion conformation of hMPV F is meta-stable and undergoes conformational rearrangement to the post-fusion state during the process of membrane fusion [ 14 , 15 ]. As the only target of neutralizing antibodies, hMPV F has been stabilized in both prefusion and post-fusion conformations to facilitate recombinant expression and vaccine development [ 16 , 17 ]. The viral genome is encapsidated in a sheath of oligomerized copies of the nucleoprotein N, forming a ribonucleoprotein complex termed nucleocapsid [ 18 ]. The nucleocapsid is the template for transcription and replication by L, which is also dependent on the obligate polymerase cofactor, the phosphoprotein P, together forming the active L/ P holoenzyme [ 19 ]. Recently, the increased incidence of hMPV across many countries in Asia has been an alarming health issue. However, like many other viruses, specific antivirals and vaccines are not available against hMPV. Focusing on the existing gaps in the development of hMPV antivirals, we performed this study to find the most suitable antiviral compounds and conduct in silico analysis to evaluate their binding and therapeutic potentials against all the major proteins of the virus. Methods Preparation, Identification and modeling of the proteins Both protein models were identified based on their resolution [ 20 – 22 ]. These proteins were retrieved from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (PDB). The PDB IDs of the selected proteins were 7SEJ (2.51 Å resolution) and 8PDO (3.10 Å resolution). The binding sites of these retrieved proteins were poorly characterized in the database. As a result, we conducted the docking analysis to predict the best pocket and binding sites. For this purpose, all redundant heteroatoms, ions, and water molecules were removed, and RNA was also removed from the nucleoprotein (hMPV-N), and the missing hydrogen atoms and Gasteiger charges were provided to the protein structures [ 23 – 29 ]. We also optimized the hMPV protein's 3-D structures by removing the ions, water molecules, and heteroatoms. We also provided the missing hydrogen atoms and charges to the structures. We use the BIOVA Discovery Studio 2024 Client 24.1 and Swiss-PDB-Viewer for the preparation of the 3-D structures [ 22 – 25 ]. The predicted models of the target proteins were validated by using the Ramachandran plot analysis in the ProCheck [ 22 ]. Further, we estimated the overall quality by using the ERRAT2 program [ 23 ]. The ProSA webserver ( https://prosa.services.came.sbg.ac.at/prosa.php ), was used to create an energy plot for the model validation and calculate the Z-score of the selected proteins [ 24 ]. We created the PDB file format of the selected proteins and their receptor targets for molecular docking. Ligand preparation The ligand was built by using the derivatives of plant lead agents against common respiratory diseases. The ligands that are docked with the human metapneumovirus (hMPV) proteins. The 3-D crystal structures of these four compounds, apigenin, 4-terpineol, cinnamaldehyde and xanthoangelol E in SDF format and simplified molecular input line entry system strings were taken from PubChem database ( https://pubchem.ncbi.nlm.nih.gov/ ) for use in the molecular docking analysis and pharmacokinetics studies. The ligands were converted to the energetically most stable structure using energy minimization for the following docking [ 25 – 31 ]. We used two currently available antiviral ribavirin (RBVN) and favipiravir (FPVR) ( https://pubchem.ncbi.nlm.nih.gov/compound/Ribavirin ) as the control for the molecular docking analysis and pharmacokinetics study. Molecular docking and protein-ligand interaction analysis Molecular docking, which is an essential tool for in silico drug discovery, predicts the favored pose of a ligand within the target (receptor) protein by forming a stable (protein-ligand) complex through intermolecular interactions [ 23 ]. All of the molecular and protein-ligand docking experiments were performed by using the PyRx software (Virtual Screening software, available at https://pyrx.sourceforge.io/ ) because it offers more accuracy in predicting ligand-protein interactions and is very suitable for multiple ligand docking. We performed docking for all the selected hMPV proteins. We performed blind docking of the selected proteins, and protein grid boxes were prepared accordingly, with respective centers (x, y, z) 230.66, 162.88, 213.49.11 Å (for hMPV-N protein), 10.69, 3.80.93, 53.19 Å (for hMPV-F protein) [ 26 – 28 ]. The final visualization of the docked structure was performed using the BIOVA Discovery Studio Visualizer 2024 Client 24.1 ( https://discover.3ds.com/discovery-studio-visualizer-download ). Molecular dynamics simulations We used the representative both protein model for the molecular dynamic simulations (MDS) by CABS-flex 2.0 webserver ( http://biocomp.chem.uw.edu.pl/CABSflex2 ) [ 29 ]. In this analysis, we determined the root mean square fluctuation (RMSF) values by simulating the flexibility of protein and proper amino acid interactions and complexes. The system was completed with the simulation time of 10 ns, applying default parameters in respect to the MD trajectory, or the NMR ensemble, for recording the RMSF values (as the measure of protein flexibility) of all individual amino acids residues of hMPV-F, and hMPV-F_apigenin, and hMPV-N, and hMPV-N_apigenin docked complexes [ 20 , 29 , 32 ]. Thus, all these analyses find out the conformational stability of protein and protein-ligand complex. Protein contacts atlas The Protein Contacts Atlas (PCA) is a platform to visualize and analyze the structural insights and the non-covalent contacts within a single protein, protein complex, and between protein and ligands. We have used the Protein Contacts Atlas (at: https://pca.mbgroup.bio/ ), to analyze the human metapneumovirus (hMPV-F) fusion protein. We took several outcomes from the analysis, including (a) Chord plots revealing the non-covalent contacts at the atomic level of proteins' secondary structures, (b) Asteroid plot showing the amino acid residues, and (c) Scatter plot matrix providing per-residue statistics [ 20 , 33 – 35 ]. Non-covalent interaction analysis We found apigenin and xanthoangelol E as the most potent drug candidate by molecular docking studies against hMPV proteins. Additionally, we selected the hMPV-F_apigenin, hMPV-N_apigenin, HMPV-F_xanthoangelol E and hMPV-N_ xanthoangelol E docked complexes for the non-covalent interaction (NCI) study. The reference RBVN was used for comparison. Atom-atom NCIs analysis in the molecules (hMPV-F_apigenin, hMPV-N_apigenin, hMPV-F_xanthoangelol E and hMPV-N_ xanthoangelol E complexes) was conducted depending on the reduced density gradient to determine the non-covalent interactions using the Bader’s Quantum Theory of Atoms in Molecules (QTAIM). The electron density (ED) was defined by following the previous studies [ 33 – 37 ]. We evaluated the NCIs using the pro-molecular density of atoms following the equation of previous studies. We used the Multiwfn ( http://sobereva.com/multiwfn ) package for calculating and visualizing the NCIs [ 38 ]. Drug likeness, pharmacokinetics, and ADME-Tox analysis We perform the ADMET (absorption, distribution, metabolism, excretion, toxicity) analysis for the ligands and the control compound using the SwissADME ( http://www.swissadme.ch/ ), and ADMETlab 2.0 ( https://admetmesh.scbdd.com/ ) [ 39 , 40 ]. We conducted this analysis to evaluate the (i) ADME-Tox (absorption, distribution, metabolism, excretion, and toxicity), ii) drug-likeness of the ligands by adopting Pfizer Rule, GSK Rule, Golden Triangle criteria, along with the Lipinski's RO5, iii) compounds’ bioavailability score by radar plot analysis, (iv) medicinal chemistry properties Further, we determined the quantitative structure-activity relationship (QSAR) by using the STopTox server ( https://stoptox.mml.unc.edu/ ). For this analysis, six organs were selected as toxicity points, including acute oral toxicity, acute inhalation toxicity, acute dermal toxicity, skin sensitization, eye irritation and corrosion, and skin irritation and corrosion. Additionally, for toxicity analysis, the compounds were subjected to carcinogenicity prediction via the CarcinoPred-EL web server ( http://112.126.70.33/toxicity/CarcinoPred-EL/index.html ) [ 41 , 42 ]. Results Model quality assessment and validation of target protein structures Structural models of the N-RNA complex and fusion proteins of human metapneumovirus (hMPV) were evaluated and validated using ProSA web-based servers. To verify the accuracy of these protein models, the SAVES v6.0 webserver (https://saves.mbi.ucla.edu/) was employed, whereas the ProSA web tool (https://prosa.services.came.sbg.ac.at/prosa.php) was used to assess the quality of the 3-D protein structures. The ProSA results, including the Z-scores and energy plots, are presented in the supplementary file. The ERRAT2 web-based program calculated the overall quality factors for the N-RNA complex and hMPV-F proteins to be 92.97 and 97.79, respectively (Supplementary Figure 1). The Z-scores for the N-RNA complex and hMPV-F proteins were -9.09 and -7.86, respectively, with energy plots showing sequence positions in favorable regions (Supplementary Figure 1). Overall, the examined models demonstrated high quality and stability, making these proteins suitable targets for antiviral development. The quality of these protein model structures was further confirmed through Ramachandran Plot analysis using the ProCheck web tool from the EMBL-EBI web server (https://www.ebi.ac.uk/thornton-srv/software/PROCHECK/). According to the ProCheck-mediated Ramachandran plots, approximately 92.6% and 90.4% of the residues were located in the most favored regions of the N-RNA complex and hMPV-F proteins, respectively (Supplementary Figure 2). Visualization of Docking The experimental findings demonstrated potent interactions between the four prospective drug candidates of plant-derived natural products as lead agents against common respiratory diseases, along with two standard drugs (FPVR, RBVN) (Table 1), against two human metapneumovirus proteins: nucleoprotein (hMPV-N) (we docked all of the compounds only in the nucleoprotein) and hMPV-F protein. Following the successful docking of these compounds to the proteins, various drug-protein interaction modes were generated, each associated with a specific docking score (binding energy). For both the compounds and docking, the binding mode with the lowest binding energy was considered optimal. The investigation also documented the specific amino acids involved in the drug-protein interactions. Visualization of all docked structures was conducted utilizing BIOVIA Discovery Studio 2024 Client 24.1( https://discover.3ds.com/discovery-studio-visualizer-download ). The plant-derived four natural compounds interacting with the proteins with involved amino acid residues and bond types, are explained for all the protein-ligand complexes (Figure 1). In this study, apigenin demonstrated the highest affinity for the target proteins hMPV-N and hMPV-F, with binding energies of -7.6 and -8.0 kcal/mol, respectively (Figure. 2 part A and Figure 3 part A). This compound formed four hydrophobic interactions with the hMPV-F protein (Figure. 4 part C) and established one conventional hydrogen bond with the LYS283 residue, along with three hydrophobic interactions with the hMPV-N protein (Figure. 5 part C). The hMPV-N protein also interacted with 4-Terpineol, showing a binding affinity of -5.7 kcal/mol (Figure. 3 part D). The binding of this compound in the specific pocket of the hMPV-N protein was confirmed by two hydrogen bonds between the oxygen of a hydroxyl group on the A chain and the residues PRO215 and GLY255, along with some hydrophobic interactions (Figure. 6 part A). Cinnamaldehyde displayed a strong binding affinity with the hMPV-N protein, with an interaction energy of -5.3 kcal/mol (Figure. 3 part B). Noteworthy conventional hydrogen bonding was observed between the carbonyl group of the ligand and the ARG293 residue of the A chain (Figure. 5 part B). Xanthoangelol E also formed one conventional hydrogen bond with the LYS283 residue of the A chain in the hMPV-N protein complex, with a binding energy of -5.1 kcal/mol (Figure. 3 part C and Figure. 4 part D). Docking studies of the hMPV-F protein showed excellent interactions with these ligands (Figure 1). 4-terpineol formed one conventional hydrogen bond with the GLY255 residue of the A chain and two hydrophobic interactions, with a binding energy of -5.7 kcal/mol (Figure 2 part D and Figure 3 part A). Cinnamaldehyde and xanthoangelol E interacted with the hMPV-F protein, showing binding affinities of -5.3 and -7.6 kcal/mol, respectively (Figure 2 part B-C). Cinnamaldehyde formed one conventional hydrogen bond with the TYR310 residue of the A chain, and both cinnamaldehyde and xanthoangelol E also engaged in some hydrophobic interactions (Figure 3 part B and D). In this study, we also predicted the binding potential of two standard antivirals targeting both the hMPV-N and hMPV-F proteins. FPVR exhibited binding energies of -6.5 and -6.7 kcal/mol, while RBVN showed values of -5.1 and -6.3 kcal/mol (Figure 3 part E-F and Figure 2 part E-F). When interacting with the hMPV target proteins, FPVR and RBVN displayed hydrogen bonds and hydrophobic interactions. RBVN and FPVR formed four and three conventional hydrogen bonds with the hMPV-N protein, respectively. The hMPV-F protein also interacted with RBVN and FPVR, forming four and seven conventional hydrogen bonds, respectively (Figure 5 part E-F and Figure part 4 E-F). Molecular simulations We conducted molecular dynamic simulation using CABS-flex 2.0 to compute the RMSF values hMPV-F, and hMPV-F_apigenin, hMPV-N, and hMPV-N_apigenin docked complexes (Figure 5 part A-D). We found low fluctuation and higher stability in the receptor protein conformation after molecular docking. This study demonstrated that both proteins have multiple amino acids in different regions to form a significantly stable complex with the ligand. In our study, hMPV-F protein displayed amino acid residues with RMSF values within 0.00 and 3.00 Å, while a total of 847, 680, and 688 amino acid residues of the docked complexes hMPV-F_apigenin, hMPV-N, and hMPV-N_apigenin, respectively, displayed the RMSFs within the mentioned range. On the other hand, hMPV-F, hMPV-F_apigenin, hMPV-N and hMPV-N_apigenin complexes had average RMSFs of 1.276, 1.309, 0.837 and 0.776 Å, respectively. The hMPV-F, hMPV-F_apigenin, hMPV-N and hMPV-N_apigenin complexes displayed highly flexible residues with RMSFs: 3.047 to 7.243 Å (for 20 residues), 3.014 to 5.061 Å (for 16 residues), 3.177 to 10.142 Å (for 22 residues) and 3.06 to 8.96 Å (for 13 residues) (Table 2). Protein contacts atlas profiles We performed a PCA analysis on the hMPV-F protein of human metapneumovirus (Figure 7). The Chord plots illustrated the essential secondary structure components contributing to the protein's tertiary structure and functionality. These plots indicated a greater number of loops with significant residue contact within the proteins. The Asteroid plots revealed the central residues with the strongest ligand interactions via hydrogen bonds (Figure 7 part B), with the inner circle (first shell) containing NAG601 residues in the hMPV-F protein's asteroid plots. The Scatter plot (Figure 7 part C) provided quantitative data per residue, including solvated area, degree, betweenness, and closeness, with all scatter plots predicting higher acceptable values for the study protein. Non-covalent interaction analysis Non-covalent interaction analysis was conducted to examine intramolecular and intermolecular bonding structures using the RDG indicator. In this study, hMPV-F_apigenin, hMPV-N_apigenin, hMPV-F_xanthoangelol E, and hMPV-N_xanthoangelol E showed a higher number of spikes in steric interaction regions, indicating sign (λ2)ρ (Figure 8 part A-D), evident in the π-interactions (Figure 4 and Figure 5). The hMPV-N_apigenin complex exhibited strong spikes in steric interaction regions (sign(λ2)ρ) (Figure 8 part A) with three π-interactions (Figure 5 part C). Similarly, the hMPV-F_apigenin complex showed higher spikes in steric regions (sign(λ2)ρ) with three π-interactions (Figure 4, Figure 5 and Figure 8D). The hMPV-F_xanthoangelol E and hMPV-N_xanthoangelol E also displayed higher spikes in steric regions (sign(λ2)ρ) with three and four π-interactions, respectively. The six protein-ligand complexes also showed peak points with sign(λ2)ρ by H-bonds, indicating stronger interaction. The non-covalent interaction regions, with sign(λ2)ρ ≈ 0 represented by green color, indicated the VDW bonds (Figure 8). Bioavailability analysis The bioavailability of the plant-derived ligands was provided by using the radar plot (Figure 9). We also provided the bioavailability score of the two reference ligands, including favipiravir and ribavirin. We found that the ligands, 4-terpineol, xanthoangelol E, apigenin, and cinnamaldehyde had acceptable bioavailability scores in the majority of the parameters compared to the reference antivirals (Figure 9). They showed improved drug-likeness compared to the existing antiviral compounds. The bioavailability score was 0.68 for all the ligands. Evaluation of acute toxicity and carcinogenicity of the ligands Evaluation of acute toxicity and carcinogenicity of the ligands was conducted using STopTox (Figure 10 and Figure 11). We predicted the end-point toxicity for apigenin and xanthoangelol E. The most active ligands with antiviral properties against hMPV-F and hMPV-N proteins showed highly acceptable values in the fragment contributions (atoms and structural fragments) models. We found a negative prediction for xanthoangelol E ligand in acute dermal toxicity, skin sensitization, eye irritation and corrosion, and skin irritation and corrosion tests with confidence scores of 58%, 58%, 65%, 50%, and 70%, respectively (Figure 10), while acute inhalation toxicity showed a positive confidence score of 55% (Figure 10). Additionally, apigenin provided acceptable values in three parameters in the STopTox analysis. In three out of six acute toxicity tests, apigenin showed negative predictions with confidence scores ranging from 66% to 73% (Figure 11). Additionally, for assessing the carcinogenic potential of all ligands, we utilized the CarcinoPred-EL webserver (http://ccsipb.lnu.edu.cn/toxicity/CarcinoPred-EL/b), which employs three machine learning techniques (Ensemble SVM, Ensemble RF, and Ensemble XGBoost) to estimate carcinogenicity by calculating average probabilities (Table 3). All compounds in our research exhibited probabilities below 0.5, with apigenin showing the lowest values: 0.27, 0.26, and 0.44 in the SVM, RF, and XGBoost models, respectively. Evaluation of ADMET profiles of the ligands Assessment of the ADMET profiles of the ligands revealed through SwissADME analysis that 4-terpineol complies with Lipinski’s rule of five for drug-likeness, having a molecular weight of 154.25 g/mol (less than 500 g/mol), one H-bond donor (not exceeding 5), one H-bond acceptor (not exceeding 10), one rotatable bond (not exceeding 10), a Topological Polar Surface Area (TPSA) of 20.23 Ų (< 140 Ų), 11 heavy atoms (not exceeding 36), and an AlogP value not exceeding 5 (Table 4). Similarly, all three ligands adhered to Lipinski’s rule of five with no violations, as shown in Table 4. The low logP value and the number of H-bond donors and acceptors of the four ligands suggested good absorption and permeation with balanced hydrophobicity and hydrophilicity. Terpineol and xanthoangelol were found to be BBB positive, and all ligands exhibited high gastrointestinal (GI) absorption. The analysis indicated that 4-terpineol and cinnamaldehyde did not respond to CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4 inhibitors. Apigenin responded to CYP1A2, CYP2D6, and CYP3A4 inhibitors, while xanthoangelol E responded to CYP2C9 and CYP3A4 inhibitors (Table 4). CYP3A4 was identified as a non-substrate/non-inhibitor, suggesting potential liver metabolism. SwissADME analysis showed that apigenin and xanthoangelol E met all drug-like filters, including Ghose (with 4-terpineol having 1 violation and cinnamaldehyde having 2 violations), Veber, Egan, and Muegge, which define drug-likeness constraints through various parameters. A bioavailability score of 0.55 for all four ligands indicated a 55% probability of rat bioavailability. Alerts were observed for PAINS and Brenk, except for apigenin, indicating the specificity of the compounds. All ligands performed well in the ADMET analysis, with some exceptions, and exhibited favorable pharmacokinetic properties (Table 4). Discussion Identifying compounds that exhibit a broad antiviral spectrum by targeting highly conserved structures has proven to be effective against a wide array of viruses [ 20 , 21 , 43 ]. First, to the best of our knowledge, this study represents the most comprehensive investigation of human metapneumovirus (hMPV) drug discovery using plant derived flavonoids. Our predicted flavonoids demonstrated the strongest binding affinity and the lowest binding energy, ranging from − 5.1 to -8.00 kcal/mol, when tested against the anticipated stable structures of the hMPV-N protein and the human metapneumovirus (hMPV-F) fusion protein. Any values below − 5.0 kcal/mol during molecular docking between the ligand and target protein are considered significantly stronger bindings [ 21 – 25 ]. In the majority of the protein-ligand interactions, hydrogen bonds, VWB, and non-covalent bonds were detected. In this context, apigenin and xanthoangelol E have the strongest binding potentials to the predicted protein models. Compared to previous studies, we predicted the lowest binding energy against these proteins [ 43 – 46 ]. Second, we explored a large library of compounds to predict antivirals against metapneumovirus (hMPV), making it a therapeutic molecule that could interact in a variety of ways. When interacting with various proteins, all of the anticipated ligands offer flexibility. Further important information about these compounds' antiviral activity can be added by wet lab study. Third, using the most recent and appropriate techniques and technologies for stable interaction, we have assessed our target protein models. In the Ramachandran plot assay, SAVES v6.0 webserver analysis, and ProSA web-based servers analysis, we discovered that the chosen proteins of the N-RNA complex and human metapneumovirus (hMPV-F) fusion protein were extremely stable. Previous studies have also used these servers and tools to predict the stability of the protein models [ 20 , 21 ]. In comparison to earlier research, the predicted protein models' Z-score and ERRAT overall quality factor were significantly higher than the allowable limit. Based on the available genotypes, our study produced a number of universal therapeutic targets by utilizing reference protein models and evaluating them according to various criteria. The selection of hMPV proteins was based on their significance in virulence and infection. The human metapneumovirus (hMPV-F) fusion protein is essential for viral entry and is a key target of neutralizing antibodies and vaccine development 46 . The hMPV polymerase (L) binds an obligate cofactor, the phosphoprotein (P). During replication and transcription, the L/P complex traverses the viral RNA genome, which is encapsidated within nucleoproteins (N) [ 44 – 47 ]. The viral genome is encapsidated in a sheath of oligomerized copies of the nucleoprotein N, forming a ribonucleoprotein complex termed nucleocapsid [45–47]. After choosing proteins, suitable ligands were created using prior studies as a guide. Blind docking was used to find the right protein pockets. The two proteins under investigation showed different binding scores, and the docking results' lowest binding energy was used to determine the pockets. Additionally, the docking was done to clarify patterns of ligand-protein binding. The study found that apigenin had the best docking score of all the docked proteins, according to the visualization of the docking results, while xanthoangelol E also exhibited a good binding score. For the model proteins, we contrasted our results with those of remdesivir and favipiravir. However, we found that our selected ligands were more compatible and significantly suitable with the lowest binding energy for the target proteins. Fourth, in the molecular simulation, we also found that both proteins and their docked compound with apigenin had multiple residues with suitable flexibility and deformability scores to provide a successful binding through the CABS.flex 2.0 webserver. It has been reported that the residue RMSFs of 1.00–3.00 Å maintained protein conformational stability [20,21,48,49]. While some other studies have shown RMSF values below 3 Å, we discovered average RMSF values of 1.27 Å for the amino acid residues of hMPV-F and 0.837 Å for the hMPV-N protein, which is a highly acceptable result. Additionally, the average RMSFs for the hMPV-F_apigenin and hMPV-N_apigenin complexes were 0.837 and 0.776 Å, respectively. This explains the low fluctuation in the receptor protein conformation after molecular docking with apigenin, signifying the protein-ligand complex stability as has been demonstrated earlier [48,49]. Additionally, both the protein contact atlas and NCI analysis also showed significantly acceptable scores for the predicted docked complexes. Fifth, in the pharmacokinetics, toxicity, bioavailability, and carcinogenicity analysis, these compounds have provided acceptable values, which can be modified further to reduce the existing toxicity. Compared to earlier research, all of the ligands displayed a good bioavailability score of 0.68. The toxicity test battery comprises six acute toxicity parameters to predict for small-molecule drugs in their early development process, and these include acute inhalation toxicity, acute oral toxicity, acute dermal toxicity, eye irritation and corrosion, skin sensitization and skin irritation and corrosion. In the acute toxicity analysis based on the STopTox (one of the most accepted toxicity evaluation tools of ligands), xanthoangelol E showed positive results in five parameters, except acute inhalation toxicity and apigenin showed three negative results and three positive results in the six parameters, respectively. Considering carcinogenicity as a highly toxic endpoint for bioactive molecules for drug development purposes, all the plant-based molecules were subjected to carcinogenicity testing in order to know whether the compounds are carcinogens or non-carcinogens. CarcinoPred-EL result showed, all of the compounds were carcinogenicity negative. Additionally, the ligands' pharmacokinetic characteristics were roughly comparable according to the ADMET (absorption, distribution, metabolism, excretion, toxicity) study. Every compound complied with Lipinski's rule of five. Similar to this, all compounds, aside from apigenin, have high GI adsorption and BBB permeability. Given that 4-terpineol and cinnamon aldehyde are CYP3A4 non-substrates/non-inhibitors, the medication may undergo hepatic metabolism. Based on a comparison of the compounds' ADMET analysis and docking data, it can be concluded that all of the ligands favored the hMPV virulence proteins for binding throughout the study and might be employed as a medication for antiviral therapy of the human metapneumovirus. Flavonoids have been studied as potent antivirals against other viruses in previous studies. Our study is also providing sufficient evidence on the use of flavonoids as antivirals in accordance with previous studies [48–50]. We could not perform any wet lab analysis, despite the fact that we reported plant-derived ligands with notably acceptable results during interaction with human metapneumovirus (hMPV) proteins in silico. In order to develop the ligands as antivirals, they should be manufactured in a medication form and tested in a lab. Conclusion Higher binding affinities for two of the human metapneumovirus virulence proteins were demonstrated by the predicted flavonoid compounds. Multiple binding sites for the chosen ligands were present in the stable protein models. These ligands exhibited considerably acceptable pharmacokinetic characteristics, decreased toxicity, and greater acceptable bioavailability profiles to be suitable antiviral compounds. By providing the in silico insights, this study will contribute to the development of direct treatment options against the human metapneumovirus, and help lower the health burden. Statements and Declarations Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Competing interests The authors have no relevant financial or non-financial interests to disclose. Authors’ contribution All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Hasan Huzayfa Rahaman, Nadim Sharif and Wasifuddin Ahmed. The first draft of the manuscript was written by Hasan Huzayfa Rahaman, Nadim Sharif Wasifuddin Ahmed, Nazmul Sharif, Rista Majumder, Silvia Aparicio Obregon, Rubén Calderón Iglesias, Isabel De la Torre Díez, and Shuvra Kanti Dey and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Availability of Data and Materials All data supporting the findings of this study are available within the paper and its Supplementary Information. Clinical trial number: not applicable Ethics approval and consent to participate Not Applicable. References Williams BG, Gouws E, Boschi-Pinto C, et al (2022) Estimates of world-wide distribution of child deaths from acute respiratory infections. 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(2022) STopTox: An insilico alternative to animal testing for acute systemic and topical toxicity. Environ. Health Perspect. 130:027012. Zhang L, Ai H, Chen W, et al (2017) CarcinoPred-EL: Novel models for predicting the carcinogenicity of chemicals using molecular fingerprints and ensemble learning methods. Sci Rep 7:2118. Hsieh CL, Rush SA, Palomo C, et al (2022) Structure-based design of prefusion-stabilized human metapneumovirus fusion proteins. Nat Commun 13:1299. Whitehead JD, Decool H, Leyrat C, et al (2023) Structure of the N-RNA/P interface indicates mode of L/P recruitment to the nucleocapsid of human metapneumovirus. Nat Commun 14:7627. Tawar RG, Duquerroy S, Vonrhein C, et al (2009) Crystal structure of a nucleocapsid-like nucleoprotein-RNA complex of respiratory syncytial virus. Science 326:1279–1283. Gonnin L, Desfosses A, Bacia-Verloop M, et al (2023) Structural landscape of the respiratory syncytial virus nucleocapsids. Nat Commun 14:5732 Van Den Bergh A, Bailly B, Guillon P, von Itzstein M, Dirr L (2022) Antiviral strategies against human metapneumovirus: Targeting the fusion protein. Antivir Res 207:105405. De A, Bhattacharya S, Debroy B, Bhattacharya A, Pal K (2023) Exploring the pharmacological aspects of natural phytochemicals against SARS-CoV-2 Nsp14 through an in silico approach, In Silico Pharmacol 11:12. Wang L, Song J, Liu A, et al (2020). Research progress of the antiviral bioactivities of natural flavonoids. Nat Prod Bioprospect 10:271-83. Lani R, Hassandarvish P, Shu MH, et al (2016). Antiviral activity of selected flavonoids against Chikungunya virus. Antivir Res 133:50-61. Tables Tables 1 to 4 are available in the Supplementary Files section Additional Declarations No competing interests reported. Supplementary Files Supplimentaryinformation.docx Tables.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6892264","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":471955905,"identity":"44cfd0ae-0c77-4bad-b6cc-9c5bf23b6ecd","order_by":0,"name":"Hasan Huzayfa Rahaman","email":"","orcid":"","institution":"Jahangirnagar University","correspondingAuthor":false,"prefix":"","firstName":"Hasan","middleName":"Huzayfa","lastName":"Rahaman","suffix":""},{"id":471955906,"identity":"5c6a8f12-a089-47b8-a930-4d987e7302bd","order_by":1,"name":"Nadim Sharif","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArklEQVRIiWNgGAWjYHACgwMMFWwMDMwgNhvRWs6QqoWBsQ3GJkaLOfvhjYduzuNL3M7OY8DwoewwYS2WPWkFh3O3sSXubOYxYJxxjggtBgdyDMBaNhxmS2DmbSNGy/k3QC1zoFr+EqXlBsiWBpAW5gPMjMRpeVZwOOcYmzFIy8Gec+nEOCx58+ecmmOyG84fbHzwo8yasBYoOAYmDxCtHghqSFE8CkbBKBgFIw0AALhUPyPNy3knAAAAAElFTkSuQmCC","orcid":"","institution":"Jahangirnagar University","correspondingAuthor":true,"prefix":"","firstName":"Nadim","middleName":"","lastName":"Sharif","suffix":""},{"id":471955907,"identity":"c68d2a47-a9c3-4d23-a9a9-0db7e18f22c3","order_by":2,"name":"Wasifuddin Ahmed","email":"","orcid":"","institution":"Jahangirnagar University","correspondingAuthor":false,"prefix":"","firstName":"Wasifuddin","middleName":"","lastName":"Ahmed","suffix":""},{"id":471955908,"identity":"fef60cf7-d6f4-45e6-b41d-171ddc50eb99","order_by":3,"name":"Nazmul Sharif","email":"","orcid":"","institution":"Rajshahi University of Engineering \u0026 Technology","correspondingAuthor":false,"prefix":"","firstName":"Nazmul","middleName":"","lastName":"Sharif","suffix":""},{"id":471955909,"identity":"bfcb71e4-519e-4500-a82c-044c35901b13","order_by":4,"name":"Rista Majumder","email":"","orcid":"","institution":"University of Rajshahi","correspondingAuthor":false,"prefix":"","firstName":"Rista","middleName":"","lastName":"Majumder","suffix":""},{"id":471955910,"identity":"8c23564a-c5ef-4cd4-8a71-eea8a1dd61c3","order_by":5,"name":"Silvia Aparicio Obregon","email":"","orcid":"","institution":"Universidad Europea del Atlántico. Isabel Torres 21","correspondingAuthor":false,"prefix":"","firstName":"Silvia","middleName":"Aparicio","lastName":"Obregon","suffix":""},{"id":471955911,"identity":"1096f809-28a1-4b87-8ea4-113c2592fd02","order_by":6,"name":"Rubén Calderón Iglesias","email":"","orcid":"","institution":"Universidad Europea del Atlántico. Isabel Torres 21","correspondingAuthor":false,"prefix":"","firstName":"Rubén","middleName":"Calderón","lastName":"Iglesias","suffix":""},{"id":471955912,"identity":"8f9e974e-47cb-4971-9a0c-542eeb91cb1e","order_by":7,"name":"Isabel Torre Díez","email":"","orcid":"","institution":"University of Valladolid","correspondingAuthor":false,"prefix":"","firstName":"Isabel","middleName":"Torre","lastName":"Díez","suffix":""},{"id":471955913,"identity":"83f8efae-772e-47a2-863c-a410786a56c7","order_by":8,"name":"Shuvra Kanti Dey","email":"","orcid":"","institution":"Jahangirnagar University","correspondingAuthor":false,"prefix":"","firstName":"Shuvra","middleName":"Kanti","lastName":"Dey","suffix":""}],"badges":[],"createdAt":"2025-06-14 06:38:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6892264/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6892264/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84919884,"identity":"2b873926-fd55-40fa-b53b-5f3ee3960488","added_by":"auto","created_at":"2025-06-18 19:35:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":8435960,"visible":true,"origin":"","legend":"\u003cp\u003eHuman metapneumovirus a. major genes encoding necessary proteins and other macromolecules, and b. virion with the target proteins and ligands for antivirals.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6892264/v1/91b836fa18172aa1ccb733cc.png"},{"id":84919886,"identity":"9469fd89-3231-49b0-96f2-1e303ffbb0da","added_by":"auto","created_at":"2025-06-18 19:35:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":18421105,"visible":true,"origin":"","legend":"\u003cp\u003eThe 3-D elucidation of molecular docking between hMPV-F protein and four plant-derived respective ligands against common respiratory disease and two standard (FPVR and RBVN): A. hMPV-F_apigenin, B. hMPV-F_cinnamaldehyde, C. hMPV-F_xanthoangelol E, D. hMPV-F_4-terpineol, E. hMPV-F_RBVN and F. hMPV-F_FPVR. hMPV-F: Human metapneumovirus fusion protein, FPVR: favipiravir, RBVN: ribavirin.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6892264/v1/2217fcaca93c2fd5186b1215.png"},{"id":84919915,"identity":"5cf03491-1102-49f3-9cb7-92b5d0465bd2","added_by":"auto","created_at":"2025-06-18 19:35:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":17007659,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe \u003c/strong\u003e3-D elucidation of molecular docking between hMPV-N protein and four plant-derived respective ligands against common respiratory disease and two standard (FPVR and RBVN): A. hMPV-N_apigenin, B. hMPV-N_cinnamaldehyde, C. hMPV-N_xanthoangelol E, D. hMPV-N_4-terpineol, E. hMPV-N_RBVN and F. hMPV-N_FPVR. hMPV-N: Human metapneumovirus nucleoprotein, FPVR: favipiravir, RBVN: ribavirin.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6892264/v1/5a2e7c433d6d869ab5b4ac03.png"},{"id":84919890,"identity":"ddfe0582-fa69-4fe0-bfb7-506b25819db8","added_by":"auto","created_at":"2025-06-18 19:35:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6341179,"visible":true,"origin":"","legend":"\u003cp\u003eTwo-dimensional (2-D) elucidation of interaction between hMPV-F (human metapneumovirus fusion protein) and ligands: A. 4-terpineol, B. cinnamaldehyde, C. apigenin, D. xanthoangelol E, E. ribavirin, F. favipiravir. The number of conventional hydrogen bonds is one in hMPV-F_4-terpineol and hMPV-F_cinnamaldehyde, and four and seven in hMPV-F_ribavirin and hMPV-F_favipiravir complexes. Apigenin and xanthoangelol E displayed no conventional hydrogen bond against hMPV-F.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6892264/v1/9faf4e85c4615600042b0e94.png"},{"id":84919896,"identity":"e1286b90-05ba-4cd7-acfd-17d3de67c158","added_by":"auto","created_at":"2025-06-18 19:35:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":8621600,"visible":true,"origin":"","legend":"\u003cp\u003eTwo-dimensional (2-D) elucidation of interaction between hMPV-N (human metapneumovirus nucleoprotein) and ligands: A. 4-terpineol, B. cinnamaldehyde, C. apigenin, D. xanthoangelol E, E. ribavirin, F. favipiravir. The number of conventional hydrogen bond is one in hMPV-N_apigenin, hMPV-N_cinnamaldehyde and hMPV-N_xanthoangelol E, four in hMPV-N_ribavirin and hMPV-N_favipiravir complexes. Compound 4-terpineol displayed two conventional hydrogen bonds against hMPV-N.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6892264/v1/9ba23c09ec5d87b0ed2e6deb.png"},{"id":84919885,"identity":"0a6ee9df-1ab6-49d2-872b-6e999571d9d7","added_by":"auto","created_at":"2025-06-18 19:35:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5141059,"visible":true,"origin":"","legend":"\u003cp\u003eThe CABS-flex-based MDS analysis showed RMSF (root mean square fluctuation) plot to authenticate the protein structural stability for unbound A. hMPV-F, B. hMPV-N and C. hMPV-F_apigenin, D. hMPV-N_apigenin docked complexes. hMPV-F: Human metapneumovirus fusion protein, hMPV-N: Human metapneumovirus nucleoprotein.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6892264/v1/c66f0b0611359d7868946d0c.png"},{"id":84919907,"identity":"19f2863c-d30d-4d93-9e77-4f895c7b8aa5","added_by":"auto","created_at":"2025-06-18 19:35:33","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":15221550,"visible":true,"origin":"","legend":"\u003cp\u003eProtein contact atlas of selected proteins of hMPV—F protein. A. a Chord plot showing the secondary structure elements of the protein necessary in shaping the tertiary structure, B. an Asteroid plot with the most active residue for ligand-protein interaction through conventional hydrogen bonds (the number of node sizes depicts the number of contacts), and C. provides a Scatter plot of the residues. hMPV-F: Human metapneumovirus fusion protein.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6892264/v1/7b67d4f6a9517ba8288fcad8.png"},{"id":84919935,"identity":"909f9e61-e5d5-4020-a65e-4b7a5e915af7","added_by":"auto","created_at":"2025-06-18 19:35:34","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":29445275,"visible":true,"origin":"","legend":"\u003cp\u003eNon-covalent interaction (NCI) analysis for A. hMPV-N_apigenin, B. hMPV-N_xanthoangelol E, C. hMPV-N_RBVN, D. hMPV-F_apigenin, E. hMPV-F_xanthoangelol E, \u0026nbsp;and F. hMPV-F_RBVN docked complexes based on the reduced density gradient (RDG) using Multiwfn. In the scatter plots, red color indicates the steric effect (repulsion) in regions with sign(λ 2 ) ρ \u0026gt;0, green color represents VDW (van der Waals) interactions in regions with sign (λ 2 with sign(λ 2 ) ρ ) ρ = 0, blue color in the regions \u0026lt; 0 displays hydrogen bonding.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-6892264/v1/7653a93c52a1faa329a67c2d.png"},{"id":84919909,"identity":"f0f33ff2-d3c1-40a2-81a8-7200d767c909","added_by":"auto","created_at":"2025-06-18 19:35:33","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":3751912,"visible":true,"origin":"","legend":"\u003cp\u003eThe bioavailability radar of the ligands, evaluated through SwissADME web tool. The plant-derived compounds used were A. 4-terpineol, B. cinnamaldehyde, C. apigenin, D. xanthoangelol, references, E. favipiravir, and F. ribavirin. The colored zone specifies the relevant physicochemical space for oval bioavailability.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-6892264/v1/c27a40cd186827dd4477702f.png"},{"id":84919928,"identity":"6c792b98-f7bc-4056-bb8d-7d85d57f60a1","added_by":"auto","created_at":"2025-06-18 19:35:34","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":10494810,"visible":true,"origin":"","legend":"\u003cp\u003eSTopTox-generated six-pack end-points toxicity for xanthoangelol E. A. Acute Inhalation toxicity, B. Acute Oral toxicity, C. Acute Dermal toxicity, D. Eye Irritation and Corrosion, E. Skin Sensitization, F. Skin Irritation and Corrosion. In each model, atoms and structural fragments determining the toxicity are indicated with brown continuous lines, and the regions reducing the toxicity are indicated with green dashed lines.\u003c/p\u003e","description":"","filename":"Figure10.png","url":"https://assets-eu.researchsquare.com/files/rs-6892264/v1/0de6d3b782dc15db9410d39a.png"},{"id":84921099,"identity":"6a250ffe-3963-4cc6-8f89-f60fc94ea9ca","added_by":"auto","created_at":"2025-06-18 19:43:33","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":28634839,"visible":true,"origin":"","legend":"\u003cp\u003eSTopTox-generated six-pack end-points toxicity for apigenin. A. Acute Inhalation toxicity, B. Acute Oral toxicity, C. Acute Dermal toxicity, D. Eye Irritation and Corrosion, E. Skin Sensitization, F. Skin Irritation and Corrosion. In each model, atoms and structural fragments determining the toxicity are indicated with brown continuous lines, and the regions reducing the toxicity are indicated with green dashed lines.\u003c/p\u003e","description":"","filename":"Figure11.png","url":"https://assets-eu.researchsquare.com/files/rs-6892264/v1/7c3e5206886887f5a72592d2.png"},{"id":84921096,"identity":"ed8265b4-fb5a-442b-880f-c72ba5e5316c","added_by":"auto","created_at":"2025-06-18 19:43:33","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1088159,"visible":true,"origin":"","legend":"","description":"","filename":"Supplimentaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6892264/v1/cdfe7d3e355108851c7de3d6.docx"},{"id":84919895,"identity":"baa6cf31-21e9-4c33-b6f7-2d9f5ef1973b","added_by":"auto","created_at":"2025-06-18 19:35:33","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":84589,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6892264/v1/414befab3c2383ae9c477f9d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Natural flavonoid apigenin and xanthoangelol E are potent antivirals against human metapneumovirus: an in-silico study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHuman metapneumovirus (hMPV) is one of the common viral etiological agents of acute respiratory tract infection (ARI) contributing to a significant percentage of morbidity and mortality, among the immunocompromised and elderly people, especially in Southeast Asia [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Although the prevalence of hMPV varies globally from 5\u0026ndash;15% among children, the altered genotypes can cause higher incidence and larger outbreaks [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The virus was first discovered by genetic analysis of nasopharyngeal aspirate samples taken from 28 hospitalized children in the Netherlands. These samples, taken from children with ARIs during the past 20 years, had symptoms ranging from mild respiratory problems to severe cough, bronchiolitis, and pneumonia. Some of them were hospitalized and needed mechanical ventilation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRespiratory viruses pose a persistent and significant threat to global public health, with human metapneumovirus (hMPV) ranking among the most concerning pathogens. The hMPV, a leading cause of respiratory tract infections, disproportionately affects vulnerable populations, including young children, the elderly, and immunocompromised individuals [\u003cspan additionalcitationids=\"CR4 CR5 CR6\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The virus is transmitted by direct or close contact with the respiratory secretions (through sneezing and coughing) of people infected with the virus or by contact with objects and surfaces that have the virus on them [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Children and adults with other medical conditions like asthma, chronic lung disease, congenital heart disease, neuromuscular disorders, cancer or chronic obstructive pulmonary disease (COPD) also tend to acquire hMPV infection that may require hospitalization [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHMPV possesses a 13.3 kb negative-sense RNA genome containing eight genes: 3\u0026rsquo;-N-P-M-F-M2-SH-G-L-5\u0026rsquo;. The F, SH, and G proteins constitute surface glycoproteins [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The M and M2 genes encode the matrix protein M, and the M2-1 and M2-2 proteins via overlapping ORFs [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The negative-sense, single-stranded RNA genome encodes eight genes, namely N, nucleocapsid; P, phosphoprotein; M, matrix; F, fusion; M2; SH, small hydrophobic; G, glycoprotein; and L, polymerase [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe fusion (F) protein of hMPV is highly conserved among hMPV subgroups, and it shares similar structural topology and approximately 30% amino acid sequence homology with the respiratory syncytial virus (RSV) F protein. The prefusion conformation of hMPV F is meta-stable and undergoes conformational rearrangement to the post-fusion state during the process of membrane fusion [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. As the only target of neutralizing antibodies, hMPV F has been stabilized in both prefusion and post-fusion conformations to facilitate recombinant expression and vaccine development [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The viral genome is encapsidated in a sheath of oligomerized copies of the nucleoprotein N, forming a ribonucleoprotein complex termed nucleocapsid [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The nucleocapsid is the template for transcription and replication by L, which is also dependent on the obligate polymerase cofactor, the phosphoprotein P, together forming the active L/ P holoenzyme [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Recently, the increased incidence of hMPV across many countries in Asia has been an alarming health issue. However, like many other viruses, specific antivirals and vaccines are not available against hMPV. Focusing on the existing gaps in the development of hMPV antivirals, we performed this study to find the most suitable antiviral compounds and conduct in silico analysis to evaluate their binding and therapeutic potentials against all the major proteins of the virus.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePreparation, Identification and modeling of the proteins\u003c/h2\u003e \u003cp\u003eBoth protein models were identified based on their resolution [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These proteins were retrieved from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (PDB). The PDB IDs of the selected proteins were 7SEJ (2.51 \u0026Aring; resolution) and 8PDO (3.10 \u0026Aring; resolution). The binding sites of these retrieved proteins were poorly characterized in the database. As a result, we conducted the docking analysis to predict the best pocket and binding sites. For this purpose, all redundant heteroatoms, ions, and water molecules were removed, and RNA was also removed from the nucleoprotein (hMPV-N), and the missing hydrogen atoms and Gasteiger charges were provided to the protein structures [\u003cspan additionalcitationids=\"CR24 CR25 CR26 CR27 CR28\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. We also optimized the hMPV protein's 3-D structures by removing the ions, water molecules, and heteroatoms. We also provided the missing hydrogen atoms and charges to the structures. We use the BIOVA Discovery Studio 2024 Client 24.1 and Swiss-PDB-Viewer for the preparation of the 3-D structures [\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The predicted models of the target proteins were validated by using the Ramachandran plot analysis in the ProCheck [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Further, we estimated the overall quality by using the ERRAT2 program [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The ProSA webserver (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://prosa.services.came.sbg.ac.at/prosa.php\u003c/span\u003e\u003cspan address=\"https://prosa.services.came.sbg.ac.at/prosa.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ), was used to create an energy plot for the model validation and calculate the Z-score of the selected proteins [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. We created the PDB file format of the selected proteins and their receptor targets for molecular docking.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLigand preparation\u003c/h3\u003e\n\u003cp\u003eThe ligand was built by using the derivatives of plant lead agents against common respiratory diseases. The ligands that are docked with the human metapneumovirus (hMPV) proteins. The 3-D crystal structures of these four compounds, apigenin, 4-terpineol, cinnamaldehyde and xanthoangelol E in SDF format and simplified molecular input line entry system strings were taken from PubChem database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ) for use in the molecular docking analysis and pharmacokinetics studies. The ligands were converted to the energetically most stable structure using energy minimization for the following docking [\u003cspan additionalcitationids=\"CR26 CR27 CR28 CR29 CR30\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. We used two currently available antiviral ribavirin (RBVN) and favipiravir (FPVR) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/compound/Ribavirin\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov/compound/Ribavirin\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) as the control for the molecular docking analysis and pharmacokinetics study.\u003c/p\u003e\n\u003ch3\u003eMolecular docking and protein-ligand interaction analysis\u003c/h3\u003e\n\u003cp\u003eMolecular docking, which is an essential tool for in silico drug discovery, predicts the favored pose of a ligand within the target (receptor) protein by forming a stable (protein-ligand) complex through intermolecular interactions [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. All of the molecular and protein-ligand docking experiments were performed by using the PyRx software (Virtual Screening software, available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pyrx.sourceforge.io/\u003c/span\u003e\u003cspan address=\"https://pyrx.sourceforge.io/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ) because it offers more accuracy in predicting ligand-protein interactions and is very suitable for multiple ligand docking. We performed docking for all the selected hMPV proteins. We performed blind docking of the selected proteins, and protein grid boxes were prepared accordingly, with respective centers (x, y, z) 230.66, 162.88, 213.49.11 \u0026Aring; (for hMPV-N protein), 10.69, 3.80.93, 53.19 \u0026Aring; (for hMPV-F protein) [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The final visualization of the docked structure was performed using the BIOVA Discovery Studio Visualizer 2024 Client 24.1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://discover.3ds.com/discovery-studio-visualizer-download\u003c/span\u003e\u003cspan address=\"https://discover.3ds.com/discovery-studio-visualizer-download\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ).\u003c/p\u003e\n\u003ch3\u003eMolecular dynamics simulations\u003c/h3\u003e\n\u003cp\u003eWe used the representative both protein model for the molecular dynamic simulations (MDS) by CABS-flex 2.0 webserver (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://biocomp.chem.uw.edu.pl/CABSflex2\u003c/span\u003e\u003cspan address=\"http://biocomp.chem.uw.edu.pl/CABSflex2\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In this analysis, we determined the root mean square fluctuation (RMSF) values by simulating the flexibility of protein and proper amino acid interactions and complexes. The system was completed with the simulation time of 10 ns, applying default parameters in respect to the MD trajectory, or the NMR ensemble, for recording the RMSF values (as the measure of protein flexibility) of all individual amino acids residues of hMPV-F, and hMPV-F_apigenin, and hMPV-N, and hMPV-N_apigenin docked complexes [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Thus, all these analyses find out the conformational stability of protein and protein-ligand complex.\u003c/p\u003e\n\u003ch3\u003eProtein contacts atlas\u003c/h3\u003e\n\u003cp\u003eThe Protein Contacts Atlas (PCA) is a platform to visualize and analyze the structural insights and the non-covalent contacts within a single protein, protein complex, and between protein and ligands. We have used the Protein Contacts Atlas (at: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pca.mbgroup.bio/\u003c/span\u003e\u003cspan address=\"https://pca.mbgroup.bio/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), to analyze the human metapneumovirus (hMPV-F) fusion protein. We took several outcomes from the analysis, including (a) Chord plots revealing the non-covalent contacts at the atomic level of proteins' secondary structures, (b) Asteroid plot showing the amino acid residues, and (c) Scatter plot matrix providing per-residue statistics [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eNon-covalent interaction analysis\u003c/h2\u003e \u003cp\u003eWe found apigenin and xanthoangelol E as the most potent drug candidate by molecular docking studies against hMPV proteins. Additionally, we selected the hMPV-F_apigenin, hMPV-N_apigenin, HMPV-F_xanthoangelol E and hMPV-N_ xanthoangelol E docked complexes for the non-covalent interaction (NCI) study. The reference RBVN was used for comparison. Atom-atom NCIs analysis in the molecules (hMPV-F_apigenin, hMPV-N_apigenin, hMPV-F_xanthoangelol E and hMPV-N_ xanthoangelol E complexes) was conducted depending on the reduced density gradient to determine the non-covalent interactions using the Bader\u0026rsquo;s Quantum Theory of Atoms in Molecules (QTAIM). The electron density (ED) was defined by following the previous studies [\u003cspan additionalcitationids=\"CR34 CR35 CR36\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. We evaluated the NCIs using the pro-molecular density of atoms following the equation of previous studies. We used the Multiwfn (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://sobereva.com/multiwfn\u003c/span\u003e\u003cspan address=\"http://sobereva.com/multiwfn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) package for calculating and visualizing the NCIs [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDrug likeness, pharmacokinetics, and ADME-Tox analysis\u003c/h3\u003e\n\u003cp\u003eWe perform the ADMET (absorption, distribution, metabolism, excretion, toxicity) analysis for the ligands and the control compound using the SwissADME (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.swissadme.ch/\u003c/span\u003e\u003cspan address=\"http://www.swissadme.ch/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and ADMETlab 2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://admetmesh.scbdd.com/\u003c/span\u003e\u003cspan address=\"https://admetmesh.scbdd.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. We conducted this analysis to evaluate the (i) ADME-Tox (absorption, distribution, metabolism, excretion, and toxicity), ii) drug-likeness of the ligands by adopting Pfizer Rule, GSK Rule, Golden Triangle criteria, along with the Lipinski's RO5, iii) compounds\u0026rsquo; bioavailability score by radar plot analysis, (iv) medicinal chemistry properties\u003c/p\u003e \u003cp\u003eFurther, we determined the quantitative structure-activity relationship (QSAR) by using the STopTox server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://stoptox.mml.unc.edu/\u003c/span\u003e\u003cspan address=\"https://stoptox.mml.unc.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). For this analysis, six organs were selected as toxicity points, including acute oral toxicity, acute inhalation toxicity, acute dermal toxicity, skin sensitization, eye irritation and corrosion, and skin irritation and corrosion. Additionally, for toxicity analysis, the compounds were subjected to carcinogenicity prediction via the CarcinoPred-EL web server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://112.126.70.33/toxicity/CarcinoPred-EL/index.html\u003c/span\u003e\u003cspan address=\"http://112.126.70.33/toxicity/CarcinoPred-EL/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eModel quality assessment and validation of target protein structures \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStructural models of the N-RNA complex and fusion proteins of human metapneumovirus (hMPV) were evaluated and validated using ProSA web-based servers. To verify the accuracy of these protein models, the SAVES v6.0 webserver (https://saves.mbi.ucla.edu/) was employed, whereas the ProSA web tool (https://prosa.services.came.sbg.ac.at/prosa.php) was used to assess the quality of the 3-D protein structures. The ProSA results, including the Z-scores and energy plots, are presented in the supplementary file. The ERRAT2 web-based program calculated the overall quality factors for the N-RNA complex and hMPV-F proteins to be 92.97 and 97.79, respectively (Supplementary Figure 1). The Z-scores for the N-RNA complex and hMPV-F proteins were -9.09 and -7.86, respectively, with energy plots showing sequence positions in favorable regions (Supplementary Figure 1). Overall, the examined models demonstrated high quality and stability, making these proteins suitable targets for antiviral development. The quality of these protein model structures was further confirmed through Ramachandran Plot analysis using the ProCheck web tool from the EMBL-EBI web server (https://www.ebi.ac.uk/thornton-srv/software/PROCHECK/). According to the ProCheck-mediated Ramachandran plots, approximately 92.6% and 90.4% of the residues were located in the most favored regions of the N-RNA complex and hMPV-F proteins, respectively (Supplementary Figure 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVisualization of Docking \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental findings demonstrated potent interactions between the four prospective drug candidates of plant-derived natural products as lead agents against common respiratory diseases, along with two standard drugs (FPVR, RBVN) (Table 1), against two human metapneumovirus proteins: nucleoprotein (hMPV-N) (we docked all of the compounds only in the nucleoprotein) and hMPV-F protein. Following the successful docking of these compounds to the proteins, various drug-protein interaction modes were generated, each associated with a specific docking score (binding energy). For both the compounds and docking, the binding mode with the lowest binding energy was considered optimal. The investigation also documented the specific amino acids involved in the drug-protein interactions. Visualization of all docked structures was conducted utilizing BIOVIA Discovery Studio 2024 Client 24.1( https://discover.3ds.com/discovery-studio-visualizer-download ). The plant-derived four natural compounds interacting with the proteins with involved amino acid residues and bond types, are explained for all the protein-ligand complexes (Figure 1). \u003c/p\u003e\n\u003cp\u003eIn this study, apigenin demonstrated the highest affinity for the target proteins hMPV-N and hMPV-F, with binding energies of -7.6 and -8.0 kcal/mol, respectively (Figure. 2 part A and Figure 3 part A). This compound formed four hydrophobic interactions with the hMPV-F protein (Figure. 4 part C) and established one conventional hydrogen bond with the LYS283 residue, along with three hydrophobic interactions with the hMPV-N protein (Figure. 5 part C). The hMPV-N protein also interacted with 4-Terpineol, showing a binding affinity of -5.7 kcal/mol (Figure. 3 part D). The binding of this compound in the specific pocket of the hMPV-N protein was confirmed by two hydrogen bonds between the oxygen of a hydroxyl group on the A chain and the residues PRO215 and GLY255, along with some hydrophobic interactions (Figure. 6 part A). Cinnamaldehyde displayed a strong binding affinity with the hMPV-N protein, with an interaction energy of -5.3 kcal/mol (Figure. 3 part B). Noteworthy conventional hydrogen bonding was observed between the carbonyl group of the ligand and the ARG293 residue of the A chain (Figure. 5 part B). Xanthoangelol E also formed one conventional hydrogen bond with the LYS283 residue of the A chain in the hMPV-N protein complex, with a binding energy of -5.1 kcal/mol (Figure. 3 part C and Figure. 4 part D).\u003c/p\u003e\n\u003cp\u003eDocking studies of the hMPV-F protein showed excellent interactions with these ligands (Figure 1). 4-terpineol formed one conventional hydrogen bond with the GLY255 residue of the A chain and two hydrophobic interactions, with a binding energy of -5.7 kcal/mol (Figure 2 part D and Figure 3 part A). Cinnamaldehyde and xanthoangelol E interacted with the hMPV-F protein, showing binding affinities of -5.3 and -7.6 kcal/mol, respectively (Figure 2 part B-C). Cinnamaldehyde formed one conventional hydrogen bond with the TYR310 residue of the A chain, and both cinnamaldehyde and xanthoangelol E also engaged in some hydrophobic interactions (Figure 3 part B and D).\u003c/p\u003e\n\u003cp\u003eIn this study, we also predicted the binding potential of two standard antivirals targeting both the hMPV-N and hMPV-F proteins. FPVR exhibited binding energies of -6.5 and -6.7 kcal/mol, while RBVN showed values of -5.1 and -6.3 kcal/mol (Figure 3 part E-F and Figure 2 part E-F). When interacting with the hMPV target proteins, FPVR and RBVN displayed hydrogen bonds and hydrophobic interactions. RBVN and FPVR formed four and three conventional hydrogen bonds with the hMPV-N protein, respectively. The hMPV-F protein also interacted with RBVN and FPVR, forming four and seven conventional hydrogen bonds, respectively (Figure 5 part E-F and Figure part 4 E-F).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular simulations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe conducted molecular dynamic simulation using CABS-flex 2.0 to compute the RMSF values hMPV-F, and hMPV-F_apigenin, hMPV-N, and hMPV-N_apigenin docked complexes (Figure 5 part A-D). We found low fluctuation and higher stability in the receptor protein conformation after molecular docking. This study demonstrated that both proteins have multiple amino acids in different regions to form a significantly stable complex with the ligand. In our study, hMPV-F protein displayed amino acid residues with RMSF values within 0.00 and 3.00 \u0026Aring;, while a total of 847, 680, and 688 amino acid residues of the docked complexes hMPV-F_apigenin, hMPV-N, and hMPV-N_apigenin, respectively, displayed the RMSFs within the mentioned range. On the other hand, hMPV-F, hMPV-F_apigenin, hMPV-N and hMPV-N_apigenin complexes had average RMSFs of 1.276, 1.309, 0.837 and 0.776 \u0026Aring;, respectively. The hMPV-F, hMPV-F_apigenin, hMPV-N and hMPV-N_apigenin complexes displayed highly flexible residues with RMSFs: 3.047 to 7.243 \u0026Aring; (for 20 residues), 3.014 to 5.061 \u0026Aring; (for 16 residues), 3.177 to 10.142 \u0026Aring; (for 22 residues) and 3.06 to 8.96 \u0026Aring; (for 13 residues) (Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein contacts atlas profiles \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe performed a PCA analysis on the hMPV-F protein of human metapneumovirus (Figure 7). The Chord plots illustrated the essential secondary structure components contributing to the protein\u0026apos;s tertiary structure and functionality. These plots indicated a greater number of loops with significant residue contact within the proteins. The Asteroid plots revealed the central residues with the strongest ligand interactions via hydrogen bonds (Figure 7 part B), with the inner circle (first shell) containing NAG601 residues in the hMPV-F protein\u0026apos;s asteroid plots. The Scatter plot (Figure 7 part C) provided quantitative data per residue, including solvated area, degree, betweenness, and closeness, with all scatter plots predicting higher acceptable values for the study protein.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNon-covalent interaction analysis \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNon-covalent interaction analysis was conducted to examine intramolecular and intermolecular bonding structures using the RDG indicator. In this study, hMPV-F_apigenin, hMPV-N_apigenin, hMPV-F_xanthoangelol E, and hMPV-N_xanthoangelol E showed a higher number of spikes in steric interaction regions, indicating sign (\u0026lambda;2)\u0026rho; (Figure 8 part A-D), evident in the \u0026pi;-interactions (Figure 4 and Figure 5). The hMPV-N_apigenin complex exhibited strong spikes in steric interaction regions (sign(\u0026lambda;2)\u0026rho;) (Figure 8 part A) with three \u0026pi;-interactions (Figure 5 part C). Similarly, the hMPV-F_apigenin complex showed higher spikes in steric regions (sign(\u0026lambda;2)\u0026rho;) with three \u0026pi;-interactions (Figure 4, Figure 5 and Figure 8D). The hMPV-F_xanthoangelol E and hMPV-N_xanthoangelol E also displayed higher spikes in steric regions (sign(\u0026lambda;2)\u0026rho;) with three and four \u0026pi;-interactions, respectively. The six protein-ligand complexes also showed peak points with sign(\u0026lambda;2)\u0026rho; by H-bonds, indicating stronger interaction. The non-covalent interaction regions, with sign(\u0026lambda;2)\u0026rho; \u0026asymp; 0 represented by green color, indicated the VDW bonds (Figure 8). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBioavailability analysis \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe bioavailability of the plant-derived ligands was provided by using the radar plot (Figure 9). We also provided the bioavailability score of the two reference ligands, including favipiravir and ribavirin. We found that the ligands, 4-terpineol, xanthoangelol E, apigenin, and cinnamaldehyde had acceptable bioavailability scores in the majority of the parameters compared to the reference antivirals (Figure 9). They showed improved drug-likeness compared to the existing antiviral compounds. The bioavailability score was 0.68 for all the ligands. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of acute toxicity and carcinogenicity of the ligands \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEvaluation of acute toxicity and carcinogenicity of the ligands was conducted using STopTox (Figure 10 and Figure 11). We predicted the end-point toxicity for apigenin and xanthoangelol E. The most active ligands with antiviral properties against hMPV-F and hMPV-N proteins showed highly acceptable values in the fragment contributions (atoms and structural fragments) models. We found a negative prediction for xanthoangelol E ligand in acute dermal toxicity, skin sensitization, eye irritation and corrosion, and skin irritation and corrosion tests with confidence scores of 58%, 58%, 65%, 50%, and 70%, respectively (Figure 10), while acute inhalation toxicity showed a positive confidence score of 55% (Figure 10). Additionally, apigenin provided acceptable values in three parameters in the STopTox analysis. In three out of six acute toxicity tests, apigenin showed negative predictions with confidence scores ranging from 66% to 73% (Figure 11). Additionally, for assessing the carcinogenic potential of all ligands, we utilized the CarcinoPred-EL webserver (http://ccsipb.lnu.edu.cn/toxicity/CarcinoPred-EL/b), which employs three machine learning techniques (Ensemble SVM, Ensemble RF, and Ensemble XGBoost) to estimate carcinogenicity by calculating average probabilities (Table 3). All compounds in our research exhibited probabilities below 0.5, with apigenin showing the lowest values: 0.27, 0.26, and 0.44 in the SVM, RF, and XGBoost models, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of ADMET profiles of the ligands\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAssessment of the ADMET profiles of the ligands revealed through SwissADME analysis that 4-terpineol complies with Lipinski\u0026rsquo;s rule of five for drug-likeness, having a molecular weight of 154.25 g/mol (less than 500 g/mol), one H-bond donor (not exceeding 5), one H-bond acceptor (not exceeding 10), one rotatable bond (not exceeding 10), a Topological Polar Surface Area (TPSA) of 20.23 \u0026Aring;\u0026sup2; (\u0026amp;lt; 140 \u0026Aring;\u0026sup2;), 11 heavy atoms (not exceeding 36), and an AlogP value not exceeding 5 (Table 4). Similarly, all three ligands adhered to Lipinski\u0026rsquo;s rule of five with no violations, as shown in Table 4. The low logP value and the number of H-bond donors and acceptors of the four ligands suggested good absorption and permeation with balanced hydrophobicity and hydrophilicity. Terpineol and xanthoangelol were found to be BBB positive, and all ligands exhibited high gastrointestinal (GI) absorption. The analysis indicated that 4-terpineol and cinnamaldehyde did not respond to CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4 inhibitors. Apigenin responded to CYP1A2, CYP2D6, and CYP3A4 inhibitors, while xanthoangelol E responded to CYP2C9 and CYP3A4 inhibitors (Table 4). CYP3A4 was identified as a non-substrate/non-inhibitor, suggesting potential liver metabolism. SwissADME analysis showed that apigenin and xanthoangelol E met all drug-like filters, including Ghose (with 4-terpineol having 1 violation and cinnamaldehyde having 2 violations), Veber, Egan, and Muegge, which define drug-likeness constraints through various parameters. A bioavailability score of 0.55 for all four ligands indicated a 55% probability of rat bioavailability. Alerts were observed for PAINS and Brenk, except for apigenin, indicating the specificity of the compounds. All ligands performed well in the ADMET analysis, with some exceptions, and exhibited favorable pharmacokinetic properties (Table 4).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIdentifying compounds that exhibit a broad antiviral spectrum by targeting highly conserved structures has proven to be effective against a wide array of viruses [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. First, to the best of our knowledge, this study represents the most comprehensive investigation of human metapneumovirus (hMPV) drug discovery using plant derived flavonoids. Our predicted flavonoids demonstrated the strongest binding affinity and the lowest binding energy, ranging from \u0026minus;\u0026thinsp;5.1 to -8.00 kcal/mol, when tested against the anticipated stable structures of the hMPV-N protein and the human metapneumovirus (hMPV-F) fusion protein. Any values below \u0026minus;\u0026thinsp;5.0 kcal/mol during molecular docking between the ligand and target protein are considered significantly stronger bindings [\u003cspan additionalcitationids=\"CR22 CR23 CR24\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In the majority of the protein-ligand interactions, hydrogen bonds, VWB, and non-covalent bonds were detected. In this context, apigenin and xanthoangelol E have the strongest binding potentials to the predicted protein models. Compared to previous studies, we predicted the lowest binding energy against these proteins [\u003cspan additionalcitationids=\"CR44 CR45\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Second, we explored a large library of compounds to predict antivirals against metapneumovirus (hMPV), making it a therapeutic molecule that could interact in a variety of ways. When interacting with various proteins, all of the anticipated ligands offer flexibility. Further important information about these compounds' antiviral activity can be added by wet lab study. Third, using the most recent and appropriate techniques and technologies for stable interaction, we have assessed our target protein models. In the Ramachandran plot assay, SAVES v6.0 webserver analysis, and ProSA web-based servers analysis, we discovered that the chosen proteins of the N-RNA complex and human metapneumovirus (hMPV-F) fusion protein were extremely stable. Previous studies have also used these servers and tools to predict the stability of the protein models [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In comparison to earlier research, the predicted protein models' Z-score and ERRAT overall quality factor were significantly higher than the allowable limit. Based on the available genotypes, our study produced a number of universal therapeutic targets by utilizing reference protein models and evaluating them according to various criteria.\u003c/p\u003e \u003cp\u003eThe selection of hMPV proteins was based on their significance in virulence and infection. The human metapneumovirus (hMPV-F) fusion protein is essential for viral entry and is a key target of neutralizing antibodies and vaccine development\u003csup\u003e46\u003c/sup\u003e. The hMPV polymerase (L) binds an obligate cofactor, the phosphoprotein (P). During replication and transcription, the L/P complex traverses the viral RNA genome, which is encapsidated within nucleoproteins (N) [\u003cspan additionalcitationids=\"CR45 CR46\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The viral genome is encapsidated in a sheath of oligomerized copies of the nucleoprotein N, forming a ribonucleoprotein complex termed nucleocapsid [45\u0026ndash;47]. After choosing proteins, suitable ligands were created using prior studies as a guide. Blind docking was used to find the right protein pockets. The two proteins under investigation showed different binding scores, and the docking results' lowest binding energy was used to determine the pockets. Additionally, the docking was done to clarify patterns of ligand-protein binding. The study found that apigenin had the best docking score of all the docked proteins, according to the visualization of the docking results, while xanthoangelol E also exhibited a good binding score. For the model proteins, we contrasted our results with those of remdesivir and favipiravir. However, we found that our selected ligands were more compatible and significantly suitable with the lowest binding energy for the target proteins.\u003c/p\u003e \u003cp\u003eFourth, in the molecular simulation, we also found that both proteins and their docked compound with apigenin had multiple residues with suitable flexibility and deformability scores to provide a successful binding through the CABS.flex 2.0 webserver. It has been reported that the residue RMSFs of 1.00\u0026ndash;3.00 \u0026Aring; maintained protein conformational stability [20,21,48,49]. While some other studies have shown RMSF values below 3 \u0026Aring;, we discovered average RMSF values of 1.27 \u0026Aring; for the amino acid residues of hMPV-F and 0.837 \u0026Aring; for the hMPV-N protein, which is a highly acceptable result. Additionally, the average RMSFs for the hMPV-F_apigenin and hMPV-N_apigenin complexes were 0.837 and 0.776 \u0026Aring;, respectively. This explains the low fluctuation in the receptor protein conformation after molecular docking with apigenin, signifying the protein-ligand complex stability as has been demonstrated earlier [48,49]. Additionally, both the protein contact atlas and NCI analysis also showed significantly acceptable scores for the predicted docked complexes.\u003c/p\u003e \u003cp\u003eFifth, in the pharmacokinetics, toxicity, bioavailability, and carcinogenicity analysis, these compounds have provided acceptable values, which can be modified further to reduce the existing toxicity. Compared to earlier research, all of the ligands displayed a good bioavailability score of 0.68. The toxicity test battery comprises six acute toxicity parameters to predict for small-molecule drugs in their early development process, and these include acute inhalation toxicity, acute oral toxicity, acute dermal toxicity, eye irritation and corrosion, skin sensitization and skin irritation and corrosion. In the acute toxicity analysis based on the STopTox (one of the most accepted toxicity evaluation tools of ligands), xanthoangelol E showed positive results in five parameters, except acute inhalation toxicity and apigenin showed three negative results and three positive results in the six parameters, respectively. Considering carcinogenicity as a highly toxic endpoint for bioactive molecules for drug development purposes, all the plant-based molecules were subjected to carcinogenicity testing in order to know whether the compounds are carcinogens or non-carcinogens. CarcinoPred-EL result showed, all of the compounds were carcinogenicity negative. Additionally, the ligands' pharmacokinetic characteristics were roughly comparable according to the ADMET (absorption, distribution, metabolism, excretion, toxicity) study. Every compound complied with Lipinski's rule of five. Similar to this, all compounds, aside from apigenin, have high GI adsorption and BBB permeability. Given that 4-terpineol and cinnamon aldehyde are CYP3A4 non-substrates/non-inhibitors, the medication may undergo hepatic metabolism. Based on a comparison of the compounds' ADMET analysis and docking data, it can be concluded that all of the ligands favored the hMPV virulence proteins for binding throughout the study and might be employed as a medication for antiviral therapy of the human metapneumovirus. Flavonoids have been studied as potent antivirals against other viruses in previous studies. Our study is also providing sufficient evidence on the use of flavonoids as antivirals in accordance with previous studies [48\u0026ndash;50].\u003c/p\u003e \u003cp\u003eWe could not perform any wet lab analysis, despite the fact that we reported plant-derived ligands with notably acceptable results during interaction with human metapneumovirus (hMPV) proteins in silico. In order to develop the ligands as antivirals, they should be manufactured in a medication form and tested in a lab.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eHigher binding affinities for two of the human metapneumovirus virulence proteins were demonstrated by the predicted flavonoid compounds. Multiple binding sites for the chosen ligands were present in the stable protein models. These ligands exhibited considerably acceptable pharmacokinetic characteristics, decreased toxicity, and greater acceptable bioavailability profiles to be suitable antiviral compounds. By providing the in silico insights, this study will contribute to the development of direct treatment options against the human metapneumovirus, and help lower the health burden.\u003c/p\u003e"},{"header":"Statements and Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Hasan Huzayfa Rahaman, Nadim Sharif and Wasifuddin Ahmed. The first draft of the manuscript was written by Hasan Huzayfa Rahaman, Nadim Sharif Wasifuddin Ahmed, Nazmul Sharif, Rista Majumder, Silvia Aparicio Obregon, Rubén Calderón Iglesias, Isabel De la Torre Díez, and Shuvra Kanti Dey and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are available within the paper and its Supplementary Information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number:\u0026nbsp;\u003c/strong\u003enot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eWilliams BG, Gouws E, Boschi-Pinto C, et al (2022) Estimates of world-wide distribution of child deaths from acute respiratory infections. Lancet Infect Dis 2:25-32.\u003c/li\u003e\n \u003cli\u003evan den Hoogen BG, de Jong JC, Groen J, et al (2001) A newly discovered human pneumovirus isolated from young children with respiratory tract disease. 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Antivir Res 133:50-61.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 4 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":false,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Human metapneumovirus, Antivirals, Drug discovery, In-silico, Molecular docking, Dynamic simulation, Pharmacokinetics, ADME-Tox ","lastPublishedDoi":"10.21203/rs.3.rs-6892264/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6892264/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHuman metapneumovirus (hMPV) is one of the potential pandemic pathogens, and it is a concern for elderly subjects and immunocompromised patients. There is no vaccine or specific antiviral available for hMPV. We conducted an in-silico study to develop antivirals against human metapneumovirus. Our methodology included protein modeling, stability assessment, molecular docking, molecular simulation, analysis of non-covalent interactions, bioavailability, carcinogenicity, and pharmacokinetic profiling. We pinpointed four plant-derived bio-compounds as antiviral candidates. Among the compounds, apigenin showed the highest binding affinity, with values of -8.0 kcal/mol for the hMPV-F protein and -7.6 kcal/mol for the hMPV-N protein. Molecular dynamic simulations and further analyses confirmed that the protein-ligand docked complexes exhibited significantly acceptable stability compared to two standard antiviral drugs. Additionally, these four compounds yielded satisfactory outcomes in bioavailability, drug-likeness, and ADME-Tox (absorption, distribution, metabolism, excretion, and toxicity) and STopTox analyses. This study highlights the highest potential of apigenin and xanthoangelol E\u003cstrong\u003e \u003c/strong\u003eas effective antivirals, underscoring the necessity for preclinical and clinical trials and wet-lab evaluation to consider them as treatments for human metapneumovirus infection.\u003c/p\u003e","manuscriptTitle":"Natural flavonoid apigenin and xanthoangelol E are potent antivirals against human metapneumovirus: an in-silico study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-18 19:35:27","doi":"10.21203/rs.3.rs-6892264/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d799a338-f8ee-4d9a-9138-c3d3ecf65dd6","owner":[],"postedDate":"June 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":50117991,"name":"Biological sciences/Drug discovery"},{"id":50117992,"name":"Biological sciences/Microbiology/Antimicrobials/Antiviral agents"}],"tags":[],"updatedAt":"2025-06-30T07:39:08+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-18 19:35:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6892264","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6892264","identity":"rs-6892264","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}