The dynamic States of Hepatitis B virus Capsid Monomers under the Impact of Different Class of Capsid-Assembly Modulators

The dynamic States of Hepatitis B virus Capsid Monomers under the Impact of Different Class of Capsid-Assembly Modulators | 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 The dynamic States of Hepatitis B virus Capsid Monomers under the Impact of Different Class of Capsid-Assembly Modulators Fatemeh Sana Askari, Alireza Mohebbi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6244861/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Sep, 2025 Read the published version in Scientific Reports → Version 1 posted 4 You are reading this latest preprint version Abstract Hepatitis B virus (HBV) remains a global health challenge. Capsid assembly modulators (CAMs) represent a promising class of antiviral agents that disrupt HBV core protein (HBc) function. Understanding the structural and dynamic impact of CAMs on HBc is crucial for the development of next-generation antiviral therapies. This study employed molecular dynamics (MD) simulations to evaluate the conformational behavior of HBc monomers in unbound and ligand-bound states. Different classes of CAMs—Heteroaryldihydropyrimidine (HAP), Sulfamoylbenzamide (SBA), and Ciclopirox—were analyzed to assess their impact on HBc stability, flexibility, and interaction energy. RMSD analysis revealed that HAP binding stabilized HBc, reducing backbone fluctuations, whereas SBA and PPA increased HBc flexibility. RMSF calculations demonstrated that CAM interactions influenced loop and terminal region dynamics. PCA suggested ligand-specific alterations in HBc’s essential motions, with Sulfamoylbenzamide inducing the highest variance. Salt bridge analysis indicated that Ciclopirox formed the strongest electrostatic interactions, stabilizing its binding. DSSP secondary structure analysis showed that CAMs disrupted α-helical content, with Sulfamoylbenzamide and Ciclopirox exhibiting the most pronounced structural rearrangements. This study provides novel insights into CAM-induced conformational changes in HBc. While HAP stabilizes the core protein, SBA and Ciclopirox increase flexibility, potentially leading to misassembled or destabilized capsids. These findings contribute to the rational design of CAM-based antiviral therapies and highlight key structural determinants for future drug optimization. Biological sciences/Computational biology and bioinformatics Biological sciences/Microbiology/Virology/Hepatitis b virus Hepatitis B virus Capsid assembly modulators Molecular dynamics simulation Ciclopirox Heteroaryldihydropyrimidine Sulfamoylbenzamide Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Introduction Hepatitis B virus (HBV) remains a global health concern, affecting over 296 million individuals chronically and leading to approximately 820,000 deaths annually due to liver cirrhosis and hepatocellular carcinoma 1 , 2 . Despite the availability of vaccines and nucleos(t)ide analog therapies, these treatments do not eliminate covalently closed circular DNA (cccDNA) and are challenged by resistance issues 3 . Therefore, innovative therapeutic strategies are imperative for managing chronic HBV infection. HBV capsid assembly modulators (CAMs) have emerged as a promising class of antiviral agents, targeting the HBV core protein (HBc) essential for viral replication and cccDNA formation. CAMs, such as Heteroaryldihydropyrimidines (HAPs), Isoquinolinone derivatives, and compounds like AB-836, function by inducing defective or empty capsids, thereby hindering the encapsidation of pregenomic RNA (pgRNA) and viral polymerase, critical for viral replication and persistence 4 . The HBc, an icosahedral structure formed by 120 homodimers of Cp, relies on precise interactions at the dimer-dimer interface for its assembly. Residues such as Val1240, Trp1250, Arg1270, Thr1280, and Pro1340 play critical roles in maintaining capsid stability through hydrophobic and hydrogen bonding interactions. CAMs exploit these interactions by either misdirecting assembly into non-functional capsids or stabilizing incomplete intermediates 4 . HAPs, for example, bind to a hydrophobic pocket at the dimer-dimer interface, inducing allosteric changes that accelerate improper assembly or destabilize preformed capsids. HAP-1, in particular, destabilizes mature capsids while promoting the formation of aberrant structures 5 . Sulfamoylbenzamides (SBAs) and phenylpropenamides (PPAs) offer distinct mechanisms of action. SBAs, such as AB-836, inhibit pgRNA-containing nucleocapsid formation, thereby reducing cccDNA production and viral replication 6 . Molecular dynamics (MD) simulations and structure-activity relationship (SAR) studies have further elucidated the binding dynamics of these CAMs. Hydrophobic interactions and van der Waals forces are essential for stabilizing inhibitor-protein complexes, while specific substitutions on CAM scaffolds enhance efficacy 7 . CAMs antiviral effects are multifaceted and it is not completely understood. It has been reprted that CAMs impact capsid stability by inducing structural rearrangements in the spike and base regions of Cp. CAM-A compounds increase dynamics within the spike region, while CAM-E compounds stabilize inter-subunit interactions, promoting tighter capsid assembly 8 . These effects are critical for disrupting the HBV lifecycle, as they prevent encapsidation of pgRNA and inhibit reverse transcription. HAP18 disrupts the hydrogen bonding networks within HBV dimers and stabilizes capsid structures, demonstrating allosteric communication that modulates capsid dynamics 4 . Isoquinolinone-based CAMs have been shown to suppress HBV replication by binding at the dimer:dimer interface of the HBV core protein, locking the preferred conformation and enhancing drug efficacy 8 . Moreover, AB-836, a potent CAM, inhibits viral replication by preventing the formation of cccDNA, thereby suppressing HBV antigen production and transcription during de novo infection 5 . Therefore, the therapeutic potential of CAMs extends beyond direct antiviral activity. By targeting HBV capsid assembly, CAMs effectively suppress antigen production, reduce immune evasion, and limit cccDNA replenishment. Their specificity for the HBV core protein minimizes off-target effects, making them suitable for combination therapies with existing antivirals. In the last study of our group 9 , it was observed that HAP dynamically stabilize HBc and compound with structurally similar features, like Lovastatin and Simvastatin, also induce identical activities. To further uncover the dynamic of HBc under unbounded and bounded conditions, this study aims to explore the behaviour of the HBV capsid monomers alone or in complexes with HAP, SBA, and PPA (Ciclopirox). Leveraging computational and experimental approaches, we seek to uncover structural determinants of CAM efficacy and contribute to the rational design of next-generation antivirals. Results Molecular dynamic simulation An MD simulation study was performed, considering it an efficacious approach for validating the stability of the HBcAg complexes with Ciclopirox and Sulfamoylbenzamide. MD analysis would provide valuable information regarding the dynamic behavior of the Ciclopirox and Sulfamoylbenzamide complexes with HBcAg and ligands' movements within their corresponding binding sites within the receptor. Also, we could see how HBc monomers are conformationally affected by the binding of two classes of HBV CAMs. Therefore, the trajectories were analyzed within 100 ns all-atom MD simulations. Trajectory analysis of HBcAg apo and holo systems The stability of HBcAg-Heteroaryldihydroprymidine, HBcAg-Ciclopirox, and HBcAg-Sulfamoylbenzamide systems was monitored using the GROMACS rms module to estimate their respective RMS values throughout the 100 ns simulation runs. Furthermore, RMSD fluctuations were measured to estimate the convergent time. Accordingly, RMSD fluctuations were measured at the backbone of HBcAg apo or holo systems (Fig. 1 ). The backbone RMSD fluctuations of HBcAg and its complexes with Ciclopirox, Sulfamoylbenzamide, and Heteroaryldihydroprymidine were analyzed over a 100 ns molecular dynamics (MD) simulation (Fig. 1 a). The assumed convergence time was determined at approximately 15 ns ( see the Black dashed line in Fig. 1 ), beyond which all systems exhibited stable fluctuations. The HBcAg apo system maintained an average RMSD of 0.35 ± 0.04 nm. The HBc_Ciclopirox complex exhibited slightly higher structural deviations (0.41 ± 0.04 nm) than the apo system, implying a stable yet more flexible binding state. The HBc_Sulfamoylbenzamide complex showed the highest RMSD (0.47 ± 0.05 nm), indicating notable structural perturbations upon ligand binding. In contrast, the HBc_Heteroaryldihydroprymidine complex showed the lowest HBcAg flexibility ( RMSD of 0.29 ± 0.07 nm). Ligand stability within the binding pocket was assessed by evaluating the RMSD of individual ligands (Fig. 1 b). The Heteroaryldihydroprymidine ligand exhibited the lowest RMSD (0.07 ± 0.02 nm), indicating a highly stable binding mode with minimal fluctuation. Conversely, Sulfamoylbenzamide displayed a higher RMSD (0.195 ± 0.039 nm), suggesting moderate movement within the pocket. Ciclopirox exhibited RMSD fluctuations around 0.084 ± 0.036 nm, indicating a relatively stable yet dynamic interaction. The radial distribution function (RDF) analysis revealed distinct patterns of ligand distribution around HBcAg. Heteroaryldihydropyrimidine exhibits the smallest average radial distance from the protein (1.84 nm) and an average g(r) of 2.70, achieving the highest cumulative number of ligand molecules (24.66) within that radius. By contrast, Ciclopirox showed the largest average g(r) of 4.80 at an distance of 2.06 nm, indicating strong local enrichment at specific separations; however, its cumulative occupancy (14.92) is the lowest among the three ligands, implying that it does not remain as persistently in close proximity to the protein. Sulfamoylbenzamide occupies an intermediate position, with an average distance of 2.21 nm, an average g(r) of 3.73, and a cumulative number of 22.39. This result indicates that Heteroaryldihydropyrimidine remains most consistently near HBcAg, whereas Ciclopirox, despite occasionally forming strong local interactions, spends less total time in close contact, and Sulfamoylbenzamide falls between these two extremes in both distance and occupancy. It was also observed that Sulfamoylbenzamide disassociates transiently and slide on the surface of HBc protein. Backbone RMSF analysis To see if the lower HBcAg’s backbone RMSD was due to the size of the ligands, per-residue (Fig. 2 a) and per-atom (Fig. 3 ) fluctuations were evaluated using the gromacs rmsf module after system convergence time. Accordingly, The HBcAg apo system (blue line) exhibited the lowest fluctuations, indicating the unbound protein rigidity. The HBcAg complex with the Ciclopirox complex (yellow line) displayed slightly increased fluctuations, particularly in loop regions. The HBcAg-Sulfamoylbenzamide complex (green line) exhibited higher fluctuations, particularly in regions near the ligand binding site. Moreover, The HBcAg-Heteroaryldihydroprymidine system (red line) showed the highest backbone fluctuations, particularly in terminal and loop regions, indicating localized structural flexibility upon ligand binding. It was also observed that all systems exhibited the highest fluctuations at the N-terminal region (M1-I13) and C-terminal residues (A131-S141), which are typically flexible regions of the HBcAg protein. To assess ligand stability within the binding pocket, RMSF per atom of each ligand was calculated (Fig. 3 ). Heteroaryldihydroprymidine exhibited the lowest average RMSF (0.07 nm), indicating highly stable binding. The highest fluctuations were observed at C30 (0.0788 nm), O31 (0.1442 nm), and O32 (0.1504 nm), suggesting minor flexibility in functional groups. Sulfamoylbenzamide displayed higher fluctuations (0.195 nm on average), suggesting moderate movement within the binding pocket. Significant fluctuations were observed in C02 (0.2395 nm), F18 (0.2166 nm), and H2 (0.3352 nm), and some degree of instability was observed in these regions. Also, Ciclopirox had relatively stable interactions, with an average RMSF of 0.084 nm. Higher fluctuations were detected at H3 (0.1141 nm), H5 (0.1133 nm), and H8 (0.0946 nm), suggesting that hydrogen atoms in its structure may contribute to flexible interactions. Interaction energy analysis Molecular mechanics calculations were performed to investigate the binding interaction energies between HBcAg and three ligands: Ciclopirox, Heteroaryldihydroprymidine, and Sulfamoylbenzamide. The short-range Coulombic (Coul-SR) and Lennard-Jones (LJ-SR) interaction energies, along with the 1–4 electrostatic (Coul-14) and 1–4 van der Waals (LJ-14) interactions, were analyzed over the convergence time (15–100 ns). The HBcAg-Ciclopirox complex exhibited a moderate Coulombic short-range interaction energy (-24.76 kJ/mol) and a strong van der Waals stabilization (-79.46 kJ/mol), indicating that hydrophobic interactions contribute significantly to the ligand binding. Interestingly, the Coul-14 interaction energy was highly negative (-102.19 kJ/mol). This could be due to a strong electrostatic attraction, likely facilitated by salt bridge formation between Ciclopirox's charged functional groups and basic HBcAg residues. The LJ-14 component was slightly positive (49.67 kJ/mol), indicating minor steric repulsions within the binding site. The Heteroaryldihydroprymidine complex exhibited the most favorable interaction energy profile among the three ligands, with a highly stabilizing Coulombic short-range energy (-52.08 kJ/mol) and strong van der Waals interactions (-128.32 kJ/mol). These findings suggest that the ligand fits well within the binding pocket, forming extensive non-covalent interactions. However, the Coul-14 interaction was highly positive (665.89 kJ/mol), implying the presence of electrostatic repulsion between the ligand and nearby protein residues. Despite this, the negative LJ-SR (-128.32 kJ/mol) and favorable LJ-14 (86.97 kJ/mol) interactions suggest that van der Waals forces help stabilize the complex. Moreover, the Sulfamoylbenzamide complex showed moderate Coulombic stabilization (-39.19 kJ/mol) but a robust Heteroaryldihydroprymidine -comparable van der Waals interactions (-127.38 kJ/mol), indicating significant hydrophobic contributions to ligand binding. The Coul-14 energy (-1221.26 kJ/mol) was the most negative among all complexes, suggesting extensive charge-charge interactions, possibly involving multiple salt bridges with charged residues in the binding pocket ( see Fig. 4 , Table panel). Salt bridge analysis Owing to the meaningful Coulombic 1–4 interaction energies between ligands and HBcAg, index files containing positively (Lys, Arg, and His) and negatively (Glu and Asp) charged HBcAg residues were generated. The files were used to evaluate the number of salt bridge formations and possible hydrogen bondings between ligands and the receptor through Gromacs mindist and hbond modules. The salt bridge formation was analyzed at a maximum distance of 3.5 Å and 30° angle. As expected, Ciclopirox and Sulfamoylbenzamide form high amounts of salt bridges with positively and negatively charged amino acids of HBcAg ( see Fig. 5 ). The HBcAg-Ciclopirox complex exhibited the highest salt bridge formation among the three ligands. The average number of salt bridges with negatively charged residues was 13.99 ± 7.40, while positive residues formed an even higher number of salt bridges (25.91 ± 11.53). This suggests that Ciclopirox forms strong electrostatic interactions with both acidic and basic residues in the binding pocket, likely contributing to its highly negative Coul-14 energy (-102.19 kJ/mol) observed in the interaction energy analysis. The significant fluctuations in the number of contacts over time indicate dynamic interactions, possibly due to ligand flexibility or conformational shifts observed in the ligand RMSD. The Sulfamoylbenzamide complex displayed moderate salt bridge formation, but its interactions were more selective. The average number of salt bridges with negatively charged residues was only 0.38 ± 1.48, while interactions with positively charged residues were higher (7.80 ± 8.84). However, the significantly lower interaction with negatively charged residues indicates a lack of strong anionic interactions, which could explain why its binding was not as stable as Ciclopirox. Interestingly, Heteroaryldihydroprymidine formed no salt bridges with positively charged residues (0 contacts at > 3.5 Å), while it displayed weak interactions with negatively charged residues (0.41 ± 1.01). This suggests that electrostatic forces are not the primary contributors to the stability of this complex. Instead, van der Waals interactions (as shown by LJ-SR: -128.32 kJ/mol) likely play a dominant role in ligand stabilization. The lack of salt bridge formation may explain why Heteroaryldihydroprymidine exhibited positive Coul-14 interactions (665.89 kJ/mol), indicating repulsion rather than attraction. I have looked into the residue names to make more sense of the interaction at close distances. As depicted in Fig. 6 , Ciclopirox makes high numbers of bind with positively and negatively charged HBc residues. However, it only establishes salt bridges with one negatively charged amino acid residue, Glu42 (0.25 ± 0.09 nm), three possible positively charged residues, Arg38 (0.28 ± 0.13 nm), Arg55 (0.30 ± 0.11 nm), and weakly with Arg27 (0.51 ± 21) (Fig. 7 ). For Sulfamoylbenzamide (Fig. 8 ), only one positively charged residue, Arg149 (0.46 ± 0.31 nm), was observed in the range of 3.5 Å. In addition, a weakly possible salt bridge was observed between Heteroaryldihydroprymidine and a negatively charged residue, Asp28 (0.44 ± 0.09 nm) ( see Fig. 9 ). I have further investigated the hydrogen bond formation between the ligand and the proposed residues in the range of salt bridge formation. Accordingly, an additional index file was generated for each system. For the HBcAg-Ciclopirox complex, the index file was composed of Glu42, Arg38, Arg55, and Arg27. For Sulfamoylbenzamide and Heteroaryldihydroprymidine, the index file comprised Arg149 and Asp28, respectively. Cluster analysis Cluster analysis was performed to evaluate the RMS distributions across unbounded state of HBcAg and its complexes with Ciclopirox, Heteroaryldihydropyrimidine, and Sulfamoylbenzamide. The mean RMS values for the primary clusters (likely representing intra-cluster stability) were relatively low across all groups, ranging from 0.173 ± 0.101 nm (Ciclopirox) to 0.227 ± 0.132 nm (Sulfamoylbenzamide), with HBcAg (0.178 ± 104 nm) and Heteroaryldihydropyrimidine (0.212 ± 0.124 nm) falling within this spectrum (Fig. 10 . Also, the mean number of clusters was similar (0.356 nm) for all groups. However, variability in these clusters was markedly greater, with standard deviations ranging from 0.623 nm (Ciclopirox and Sulfamoylbenzamide) to 0.683 nm (HBcAg). SASA analysis As shown in Fig. 11 , analysis of Solvent Accessible Surface Area (SASA) showed differences in solvent exposure profiles. The average SASA values ranged from 90.64 ± 1.67 nm² (HBcAg) to 101.03 ± 2.39 nm² (Sulfamoylbenzamide-complex), with intermediate values observed for HBc-complexed Heteroaryldihydroprymidine (93.87 ± 1.75 nm²) and HBc-complexed Ciclopirox (99.55 ± (2.20 nm²). HBc protein exhibited the highest mean solvent accessibility in complex with Sulfamoylbenzamide. The result showed a relatively compact or shielded surface of HBcAg when it is in complex with Heteroaryldihydroprymidine. Secondary structure calculation Analysis of the HBcAg simulation from 15 to 100 ns via DSSP indicates that the protein maintains a predominantly α-helical conformation throughout this convergent timeframe. The data reveal a high mean count of α-helix assignments (5143 out of 8502 possible states, corresponding to over half of the residues being helical on average), with minimal to no β-strand content detected. Small proportions of 3 10 ​-helix, π-helix, and turns are present, mostly localized to loop or boundary regions. This stable α-helical propensity is also reflected in individual residue frequencies, where many central residues remain in an α-helix for over 90% of the simulation. Only a few segments exhibit noticeable transitions into coil or turn states, suggesting that the overall fold remains stable and does not significantly deviate from its helical core during the 15–100 ns interval. Compared to the unbound HBcAg, which retained a predominantly α-helical fold, the HBcAg–Ciclopirox complex exhibits a marked reduction in regular secondary structure. DSSP analysis reveals a complete loss of α-helix content (mean of 0) and a pronounced increase in bends (“S”) and coil (“~”) states. Indeed, bends dominate the conformational ensemble with an average of 7077 out of 8501 possible states, whereas polyproline (“P”) conformations also appear intermittently. These findings suggest that Ciclopirox disrupts the native α-helical architecture observed in HBcAg alone, instead favoring more less ordered secondary structures as observed by the RMSA comparision, which may reflect alterations in the protein’s overall stability or function. Compared to unbound HBcAg, which retains a predominantly α-helical fold, complexation with Sulfamoylbenzamide leads to a nearly complete loss of α-helix and an increase in more disordered or bend-like (“S”) states. The DSSP data show that most residues occupy bend (mean of over 7000 out of 8501 possible states) and coil (“~”) conformations, with occasional polyproline (“P”) content. This shift away from the stable helical architecture suggests that Sulfamoylbenzamide substantially perturbs the protein’s native fold, favoring a more flexible or partially disordered conformation similar to the behavior induced upon Ciclopirox binding, and what was observed in RMSD results. Similarly, Heteroaryldihydroprymidine again leads to the near-complete loss of α-helical content. DSSP analysis reveals a pronounced shift toward bend-like (“S”) and coil (“~”) conformations, with an average of over 7000 out of 8501 possible states classified as bends and a smaller fraction of polyproline (“P”) structures. This disruption of the native α-helix is consistent with the previously observed behavior for the other CAM-bound complexes (Ciclopirox and Sulfamoylbenzamide), suggesting that Heteroaryldihydroprymidine likewise induces an even more flexible or partially disordered secondary structure in HBcAg relative to its unbound form. PCA analysis PCA of the HBcAg systems revealed differences in overall atomic fluctuations and the distribution of essential motions upon ligand binding. The sum of eigenvalues, representing the total variance in atomic displacements, was 13.82 nm² for unbound HBcAg, 10.70 nm² for the Ciclopirox complex, 23.90 nm² for the Sulfamoylbenzamide complex, and 14.89 nm² for the Heteroaryldihydroprymidine complex. Focusing on the first ten principal components—which captured over 80% of the variance in the unbound state—the cumulative variance accounted for was 81.77% for HBcAg alone, 79.05% for the Ciclopirox complex, 87.54% for the Sulfamoylbenzamide complex, and 85.99% for the Heteroaryldihydroprymidine complex. These results indicate that while the essential dynamics of HBcAg are largely confined to the top ten PCs, ligand binding alters the overall fluctuation landscape in a ligand-dependent manner, potentially influencing the protein’s functional motions and stability. The evolution time of HBcAg’s projection onto the first ten eigenvectors showed all PCs are actively contributing in atomic projections. Except one major projection at 50 nm time-point in PC1, the system was stable (Fig. 13 A). Similar behaviours were also observed among the HBcAg complexes in the PC5 to PC10. But in the first five PCs the projections were diverse, indicating the involvement of the earlier PCs in reflecting system projections. However, for the sake of riching 80% or higher trajectory data capture by PC, ten components were selected. Furthermore, the mode of motion for different component of HBcAg and its complex with the ligands is depicted in Fig. 13 B. As a finding, except of PC1, PC2, and PC4, mostly C-terminal of the protein has some peak of total (Black lines) atomic motions mostly in horizontal (x and y) axis. Also, most of the central domain atomic movments were also observed almost in all PCs. For Ciclopirox-complexed HBc protein, the atomic motions were more pronounced in both C-terminal (atoms 370 to 420) and cental domains (atoms 120 to 150 and 200 to 250). For Sulfamoylbenzamide complex, first 20 atoms at the N-terminal domain were significantly mobile mostly in 2D horizontal axis. Here, two other domain of atomic motion were observed excel the ones of N-terminal. In this regard, similar motion like that observed in Ciclopirox complex at central domain was observed but in half of PCs. However, the C-terminal domain was significantly motile at the ending atomes compare to HBc unbounded state and Ciclopirox complex ( see Fig. 13 B). In the contrary to these, the complex of HBcAg to Heteroaryldihydroprymidine was very stable at the C-terminal domain. This could also explain the induced flexibility of HBcAg in complex by Heteroaryldihydroprymidine as observed in the RMSD analysis. Neverthless, like other complex the HBcAg was mobile at the N-terminal atomes and central domain similarly. The RMSF of individual atoms was also evaluated for the given PCs. In accordance to the eigenvalues analysis for atomic motion, the RMSF findings revealed significant atomic peaks at the already mentioned domain. This finding also supporded RMSF of amino acid residues mentioned earlier (Fig. 14 ). Further conformational substates of HBcAg were assessed (Fig. 15 ). According to the 2D projection analysis over the first PC with highest motions and the last PC with more stable state, HBcAg spend most of the time of simulation in a stable state at two places with alsmost similar motions as observed in both PCs. The less condensed bridge between these two clusters (black snapshots between two condensed clouds; Fig. 15 ) is also indicates a possible conformational transformation between these two states. This finding was used as a base conformational structure for making the possibility of comparing the unbounded state with conditions that HBcAg is complex with CAMs. Accordingly (Fig. 14 A), upon bounding with Ciclopirox, the bridge mentioned between two states is almost disappeared. This possibly confers HBcAg to has a rapid conformational transform state, and structurally rearrange in the second place. Even more interestingly, HBc had a more structurally flexible transitions when it was in complex with Sulfamoylbenzamide, since it was observed that the bridge was extended for almost 2 nm, and this made a new cluster in space. Moreover, the first structure was more condenced in a new space almost 1 nm away from the one observed in unbounded state of HBc. These new conformational states or substates are possibly involve in the structural flexibili induced by Sulfamoylbenzamide. HBcAg was more stable and the trajectory snapshots were more condenced in the first structure in its complex with Heteroaryldihydroprymidine (Fig. 15 C). However, the second place was also nesteded within the second conformation but less dence. Moreover, a new palce in space was also observe that HBc met in this complex. This finding suggested that HBc preserved and stable when bounded to Sulfamoylbenzamide, and also has a new substate (3rd ), which was similar to the one induced by Sulfamoylbenzamide. This supports how Sulfamoylbenzamide reduces the flexibility of HBcAg as observed in RMSD analysis (and also in a movie made for the complexes [data are not provided]). Discussion HBV remains a significant global health concern, with chronic infections leading to severe liver diseases, including cirrhosis and hepatocellular carcinoma 2 , 10 – 12 . A pivotal component in the HBV lifecycle is the capsid, a protein shell that encases the viral genome and plays essential roles in replication and assembly 9 , 13 . Targeting the capsid assembly process has emerged as a promising therapeutic strategy, leading to the development of CAMs 14 . In this study it was aimed to dynamically evaluate the HBV capsod monomer in unbounded or complexed states.​ The MD simulations and trajectory analyses in the study provided detailed insights into the structural perturbations induced by distinct classes of CAMs, Heteroaryldihydropyrimidine, Ciclopirox, and Sulfamoylbenzamide. These findings contribute to the growing body of evidence that highlights the efficacy and mechanistic of CAMs in disrupting HBV replication and capsid stability. HBV capsid assembly is a critical step in the viral lifecycle, with the capsid protein forming an icosahedral structure composed of 120 homodimers 4 , 14 . The stability and structural integrity of these capsids are essential for the encapsidation of pregenomic RNA (pgRNA) and subsequent reverse transcription. Capsid assembly inhibitors function through two primary mechanisms: (1) misdirecting assembly into non-functional structures and (2) stabilizing immature capsids to prevent RNA encapsidation 5 , 8 , 15 . Our molecular dynamics simulations reveal distinct effects of CAMs on HBcAg conformation and stability, aligning with previous studies that highlight the potential of CAMs as therapeutic agents 16 . Accordingly, RMSD analysis indicated differential impacts of CAMs on the HBcAg backbone. Heteroaryldihydropyrimidine exhibited the lowest fluctuations, suggesting a strong stabilizing effect. Conversely, Sulfamoylbenzamide and Ciclopirox induced a relatively higher RMSD, indicating significant structural perturbations. These findings align with previous reports that classify CAMs into different mechanistic categories. Heteroaryldihydropyrimidines, such as BAY 41-4109, are known to stabilize capsids while promoting aberrant structures at high concentrations 17 . Similarly, the misdirecting effects observed in Sulfamoylbenzamide interactions are consistent with reports on bis-ANS, a small molecule known to misdirect capsid assembly 18 . The binding affinity and stability of CAMs within the HBcAg binding pocket were assessed through RMSD and radial distribution function (RDF) analysis. Heteroaryldihydropyrimidine remained the most tightly bound ligand, exhibiting the smallest average radial distance and the highest cumulative ligand occupancy. These findings suggest strong and persistent interactions, reinforcing its role as a capsid stabilizer 19 . Sulfamoylbenzamide, despite moderate binding affinity, exhibited transient dissociation events, consistent with its proposed sliding and surface-exploring behavior. This aligns with studies showing that sulfamoylbenzamides, including NVR 3-778, effectively inhibit pgRNA-containing nucleocapsid formation 19 . The backbone RMSF analysis demonstrated that ligand binding induced structural flexibility in the HBcAg protein, particularly in the loop and terminal regions. Among the ligands, Heteroaryldihydropyrimidine induced the highest backbone fluctuations, particularly in loop regions. These findings align with previous reports indicating that HBV capsid inhibitors can alter protein flexibility, affecting capsid stability and viral replication 17 , 20 , 21 . Further analysis of per-atom ligand RMSF revealed that Heteroaryldihydropyrimidine exhibited the lowest average RMSF, indicating a stable binding within the HBcAg binding pocket. Conversely, Sulfamoylbenzamide displayed higher fluctuations, particularly at functional groups involved in hydrophobic interactions. Ciclopirox maintained relatively stable interactions but showed some flexibility at specific atomic positions. These variations in RMSF suggest that the ligands differ in their ability to anchor within the binding site, a factor that has been previously reported as critical for HBV inhibitor efficacy. Binding interaction energies provided insight into the stabilizing forces governing CAM-protein interactions. Heteroaryldihydropyrimidine showed the most favorable interaction energy profile, with a strong short-range Coulombic interaction and significant van der Waals forces. However, its highly positive Coul-14 energy suggests electrostatic repulsion, which may affect its overall stability within the binding pocket. In contrast, Sulfamoylbenzamide exhibited moderate Coulombic stabilization but robust van der Waals interactions, comparable to Heteroaryldihydropyrimidine. The moderate interaction energies observed for Ciclopirox suggest that its binding is driven by hydrophobic interactions rather than electrostatic forces. Previous studies indicate that electrostatic interactions play a pivotal role in CAM binding efficacy. BAY 41-4109, for instance, forms extensive electrostatic contacts with positively charged HBcAg residues, mirroring our findings for Sulfamoylbenzamide 22 . The presence of highly negative Coul-14 interaction energy suggests the formation of electrostatic interactions, potentially via salt bridges. Similar findings have been observed in previous studies where hydrophobic interactions were found to be essential in stabilizing HBV core protein inhibitors 23 , 24 . The salt bridge analysis further elucidated the nature of ligand-protein interactions. Ciclopirox exhibited the highest number of salt bridges, particularly with both positively and negatively charged residues. This finding implies strong electrostatic interactions that enhance its binding stability. The moderate salt bridge formation observed in the Sulfamoylbenzamide complex, particularly with positively charged residues, indicates selective electrostatic interactions. Interestingly, Heteroaryldihydropyrimidine formed no salt bridges with positively charged residues, relying predominantly on van der Waals interactions for stability. These findings are in agreement with previous studies highlighting the role of electrostatic interactions in HBV capsid inhibition. Also, cluster analysis confirmed that all ligand-bound HBcAg systems exhibited similar RMSD, suggesting that the overall structural integrity of the protein remained relatively stable. However, the HBcAg-Heteroaryldihydropyrimidine complex exhibited slightly higher deviations, consistent with the high fluctuations observed in RMSF analysis. SASA analysis further confirmed differences in ligand-induced conformational changes. The HBcAg-Sulfamoylbenzamide complex exhibited the highest solvent accessibility, suggesting a less compact structure, while Heteroaryldihydropyrimidine binding resulted in a more shielded protein surface. Secondary structure calculations via DSSP analysis revealed that HBcAg maintains a predominantly α-helical conformation throughout the simulation. However, upon ligand binding, Ciclopirox exhibited a marked reduction in regular secondary structure, favoring more flexible conformations. This disruption of α-helical architecture suggests that Ciclopirox binding induces significant structural changes, potentially altering HBcAg functionality. Such ligand-induced structural alterations have been previously reported as a mechanism by which HBV inhibitors misdirect capsid assembly, ultimately disrupting viral replication 17 , 18 , 25 , 26 . DSSP analysis demonstrated that all ligands destabilized HBeAg’s native α-helical fold, favoring bends and coils. Ciclopirox and Sulfamoylbenzamide caused near-complete helix loss, while Heteroaryldihydroprymidine retained marginal order, likely due to its compact binding (SASA: 93.87 ± 1.75 nm²). This structural destabilization implies that ligand binding induces conformational shifts. Also, the increased solvent exposure in the Sulfamoylbenzamide complex (SASA: 101.03 ± 2.39 nm²) suggests partial unfolding or loosened packing, which may enhance proteolytic susceptibility or alter antigenicity—a phenomenon documented in HBV core protein mutants 27 . Further conformational state of HBC was further evaluated by PCA. PCA along with cluster analyses revealed ligand-dependent alterations in HBeAg’s essential motions. Sulfamoylbenzamide induced the highest variance, reflecting pronounced flexibility, while Ciclopirox restricted motions, likely due to its stabilizing salt bridges. The emergence of distinct conformational substates in ligand-bound systems—particularly the extended “bridge” in the Sulfamoylbenzamide complex—suggests that ligand chemistry dictates accessible conformational ensembles. These findings align with theories positing that ligands act as “allosteric modulators” by reshaping energy landscapes 28 . Moreover, it was observed that Heteroaryldihydropyrimidine stabilize HBc in space where the protein was already met, but in a more density manner. It was also observed that the protein meet a place where the protein has not met before that was also bridge by transient transformation between other two structures. This finding suggests that Heteroaryldihydropyrimidine stabilize HBc with more rapid reangments between three stable location in space. The destabilization of HBeAg’s α-helical structure by all ligands raises questions about functional consequences. While disordered states may impair viral assembly or secretion 29 , excessive flexibility (as seen with Sulfamoylbenzamide) could reduce target engagement longevity. Ciclopirox’s balanced interaction profile—combining electrostatic and hydrophobic forces—positions it as a promising scaffold for optimization. Future work should explore covalent stabilization of salt bridges or incorporation of hydrophobic moieties to enhance Heteroaryldihydroprymidine’s efficacy. The findings also provide mechanistic insights into the interactions of HBV core protein inhibitors and their potential antiviral effects. The variations in RMSF, interaction energy, salt bridge formation, and secondary structure disruption suggest that each ligand influences HBcAg differently. The strong electrostatic interactions and stability observed with Ciclopirox suggest its potential as a potent inhibitor. Heteroaryldihydropyrimidine, despite favorable interaction energy, exhibits electrostatic repulsion, which may affect its binding efficiency. Sulfamoylbenzamide, while stable, interacts selectively with HBcAg, which could influence its effectiveness as a capsid assembly inhibitor. These findings contribute to the growing body of research on HBV capsid-targeting antivirals and underscore the importance of molecular dynamics simulations in drug development. CAMs represent a promising strategy for disrupting HBV replication by interfering with capsid assembly and stability. The results of our study reinforce the therapeutic potential of targeting capsid assembly through distinct molecular mechanisms. Heteroaryldihydropyrimidines appear particularly effective in stabilizing incomplete capsids, making them attractive candidates for antiviral development 30 . Meanwhile, Sulfamoylbenzamides exhibit misdirecting properties, which could be leveraged to prevent the formation of functional viral particles. But the interaction of Sulfamoylbenzamides was not that stable and visually it was observed that it transiently disassociates HBcAg. The findings of this study have several implications, one of which is combination therapy potential. Given their unique mechanisms, CAMs could be used in combination with nucleos(t)ide analogs to enhance antiviral efficacy while minimizing resistance. Also, personalized medicine approaches. The differential sensitivity of HBV RNA versus DNA to CAMs suggests potential applications in personalized treatment strategies, as proposed in multiscale modeling studies 16 . Furthermore, future structural modifications. The binding dynamics observed suggest that further modifications to the ligand scaffolds could enhance binding affinity and specificity, an approach successfully employed in aminothiazole-based HBV capsid inhibitors 23 . Conclusion The finding of the study demonstrated that different class of anti-HBV CAMs assert their impact either by making stable bounded state through which capsid monomer rapidly rearrange and reduce capsid flexibility. This was observed in Heteroaryldihydroprymidine-complexed HBc. Also, this was supported by secondary structure analysi showing class A CAM, Heteroaryldihydroprymidine, retain α-helical structure that lost by interaction of class B CAMs like Sulfamoylbenzamides. Furthermore, type of interaction energy was differ in two class. Future studies can be done by focusing on repurposing potent CAMs thorough utilizing ligand-based drug discovery approaches. Also, stalization and destabilization of different HBc domain by CAMs could be the focus of future study to discover the important residues affecting stabilization of compounds on HBcAg. This also would help to predict the future potent amino acid substitutions that potentially lead to the rise of CAM-resistant mutants. Methods Data gathering of HBcAg and Ciclopirox The crystallographic structure of HBcAg of HBV genotype D subtype adw was obtained from a protein data bank (PDB) with a PDB ID of 6j10 and 2.30 Å resolution 31 . The structure of HBcAg was cleaned from water molecules and other non-standard fragments, as previously shown 32 , 33 . Furthermore, the chemical structures of Ciclopirox and Sulfamoylbenzamide in complex with the HBcAg were also extracted from the same .pdb file. The complex .pdb files of HBcAg, Ciclopirox, and Sulfamoylbenzamide were used in molecular dynamic simulation (MDS). Molecular dynamic simulation MDSs were conducted on Hepatitis B core antigen (HBcAg) alone and on three more complex systems, including HBcAg with Heteroaryldihydroprymidine, Cyclopirox, and Sulfamoylbenzamide. HBcAg was used as the receptor in this study. The simulations were performed using the GROMACS simulation software, version 2021.3 34 . Simulations were run on a system equipped with an Intel(R) Core(TM) i7-4510U CPU @ 2.00GHz, with a total of 4 cores and four logical cores. SIMD instructions used were AVX2_256 with a memory model of 64-bit. GPU support was disabled for these simulations. The software was compiled using the GNU 10.2.0 C and C + + compilers with flags -mavx2 -mfma for AVX2 instruction sets and -O3 for optimization. Initial models of HBcAg complexed with each ligand were subjected to energy minimization to remove any close contacts or high-energy conformations. The system was then solvated with explicit water molecules. Any charges present in the system were neutralized using appropriate counter-ions. Accordingly, The HBcAg-Ciclopirox complex was neutralized by 4 Na + ions. Additionally, The HBcAg-Sulfamoylbenzamide compound complex was neutralized with 4 NA + ions. The simulations were carried out under periodic boundary conditions. Each system was equilibrated at 300K and 1 bar using a canonical (NVT) ensemble followed by an isothermal-isobaric (NPT) ensemble. The simulations were run for 100 nanoseconds with a time step of 2 femtoseconds. The analysis of the MD simulation trajectories was performed with GROMACS built-in tools. Trajectory analysis Molecular dynamics (MD) simulation trajectories were subjected to an extensive range of analytical procedures conducted using GROMACS utilities to decipher the system's key structural and dynamic aspects. Structural stability and conformational alterations were evaluated through root mean square deviation (RMSD) calculations ( gmx rms ). Protein flexibility at the residue level was investigated via root mean square fluctuation (RMSF) computations ( gmx rmsf ). System compactness was gauged by determining the radius of gyration (Rg) using gmx gyrate . The strength of receptor-ligand interactions over the simulation period was elucidated by quantifying interaction energies via gmx energy . Hydrogen bonding dynamics within the molecular system were comprehended using the gmx hbond utility. Evolutionary changes in secondary structures were scrutinized employing gmx do_dssp . A principal component analysis (PCA) was performed using the gmx covar and gmx anaeig utilities to identify predominant motion patterns in the system. The solvent-accessible surface area (SASA) was computed to continuously monitor the protein's exposure to the solvent during the simulation. A cluster analysis was conducted to identify the conformational states most frequently adopted by the system. Additionally, radial distribution functions (RDFs) were calculated to characterize the spatial relationships between various entities within the system. All analyzes were performed as reported before 35 – 37 . Declarations Acknowledgements Not applicable. Author Contributions Alireza Mohebbi conceptualized the study, designed the research methodology, and supervised the project. Molecular dynamics simulations were performed by Fatemeh Sana Askari and Alireza Mohebbi . Alireza Mohebbi and Fatemeh Sana Askari contributed to data analysis and interpretation. Alireza Mohebbi assisted in manuscript writing and figure preparation. All authors reviewed and approved the final manuscript. Both authors reviewed and approved the final manuscript. Data availability statement The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Furthermore, the analysis methods of trajectories are provided in the Youtube channel of the corresponding author (https://www.youtube.com/@al1r3z49). Competing interests The author(s) declare no competing interests. Consent for publication Not applicable. References Jeng, W. J., Papatheodoridis, G. V. & Lok, A. S. F. Hepatitis B. The Lancet 401 , 1039–1052 (2023). Naderi, M., Salavatiha, Z., Gogoi, U. & Mohebbi, A. An overview of anti-Hepatitis B virus flavonoids and their mechanisms of action. Front Cell Infect Microbiol 14 , 1356003 (2024). Mohebbi, A., Lorestani, N., Tahamtan, A., Kargar, N. L. & Tabarraei, A. An overview of hepatitis B virus surface antigen secretion inhibitors. Front Microbiol 9 , 1–9 (2018). Kant, R. et al. Small Molecule Assembly Agonist Alters the Dynamics of Hepatitis B Virus Core Protein Dimer and Capsid. J Am Chem Soc (2024) doi:10.1021/jacs.4c08871. Lam, A. M. et al. Preclinical and clinical antiviral characterization of AB-836, a potent capsid assembly modulator against hepatitis B virus. Antiviral Res 231 , 106010–106010 (2024). Iyaniwura, S., Cassidy, T., Ribeiro, R., bioRxiv, A. P.- & 2024, undefined. A multiscale model of the action of a capsid assembly modulator for the treatment of chronic hepatitis B. pmc.ncbi.nlm.nih.govSA Iyaniwura, T Cassidy, RM Ribeiro, AS PerelsonbioRxiv, 2024•pmc.ncbi.nlm.nih.gov . Cole, A. G. et al. Rational Design, Synthesis, and Structure-Activity Relationship of a Novel Isoquinolinone-Based Series of HBV Capsid Assembly Modulators Leading to the Identification of Clinical Candidate AB-836. J Med Chem (2024) doi:10.1021/ACS.JMEDCHEM.4C01568. Mesaros, E. F. et al. Conformationally Constrained Isoquinolinones as Orally Efficacious Hepatitis B Capsid Assembly Modulators. ACS Med Chem Lett 15 , 1627–1634 (2024). Mohebbi, A. et al. Computer-aided drug repurposing & discovery for Hepatitis B capsid protein. In Silico Pharmacology 2025 13:1 13 , 1–22 (2025). Rezanezhadi, M., Mohebbi, A., Askari, F. S., Hosseini, S. D. & Tabarraei, A. Hepatitis B virus reverse transcriptase polymorphisms between treated and treatment-naïve chronically infected patients. Virusdisease 30 , 219–226 (2019). Khani, H., Ghorbani, M., Nojoomi, F. & Mohebbi, A. Honey Bee Dry Venom Reduces Hepatitis B Virus Surface Antigen Secretion in PLC/PRF/5 Cell Line. International Journal of Medical Laboratory (2019) doi:10.18502/ijml.v6i4.2013. Baei, B., Askari, P., Askari, F. S., Kiani, S. J. & Mohebbi, A. Pharmacophore modeling and QSAR analysis of anti-HBV flavonols. PLoS One 20 , e0316765 (2025). Hadden, J. A. et al. All-atom molecular dynamics of the HBV capsid reveals insights into biological function and cryo-EM resolution limits. Elife 7 , (2018). Kim, H., Ko, C., Lee, J. Y. & Kim, M. Current Progress in the Development of Hepatitis B Virus Capsid Assembly Modulators: Chemical Structure, Mode-of-Action and Efficacy. Molecules 2021, Vol. 26, Page 7420 26 , 7420 (2021). Cole, A. G. et al. Rational Design, Synthesis, and Structure-Activity Relationship of a Novel Isoquinolinone-Based Series of HBV Capsid Assembly Modulators Leading to the Identification of Clinical Candidate AB-836. J Med Chem 67 , 16773–16795 (2024). Iyaniwura, S. A., Cassidy, T., Ribeiro, R. M. & Perelson, A. S. A multiscale model of the action of a capsid assembly modulator for the treatment of chronic hepatitis B. bioRxiv (2024) doi:10.1101/2024.07.16.603658. Li, L., Chirapu, S. R., Finn, M. G. & Zlotnick, A. Phase diagrams map the properties of antiviral agents directed against hepatitis B virus core assembly. Antimicrob Agents Chemother 57 , 1505–1508 (2013). Zlotnick, A., Ceres, P., Singh, S. & Johnson, J. M. A Small Molecule Inhibits and Misdirects Assembly of Hepatitis B Virus Capsids. J Virol 76 , 4848 (2002). Ma, Y. et al. Design, synthesis and evaluation of heteroaryldihydropyrimidine analogues bearing spiro ring as hepatitis B virus capsid protein inhibitors. Eur J Med Chem 225 , (2021). Huber, A. D. et al. The Heteroaryldihydropyrimidine Bay 38-7690 Induces Hepatitis B Virus Core Protein Aggregates Associated with Promyelocytic Leukemia Nuclear Bodies in Infected Cells. mSphere 3 , (2018). Pavlova, A. et al. Mechanism of action of HBV capsid assembly modulators can be predicted from binding to early assembly intermediates. J Med Chem 65 , 4854 (2022). Lin, J. et al. Bay41-4109-induced aberrant polymers of hepatitis b capsid proteins are removed via STUB1-promoted p62-mediated macroautophagy. PLoS Pathog 18 , (2022). Pan, T. et al. Design and synthesis of aminothiazole based Hepatitis B Virus (HBV) capsid inhibitors. Eur J Med Chem 166 , 480–501 (2019). Tu, J., Li, J. J., Shan, Z. J. & Zhai, H. L. Exploring the binding mechanism of Heteroaryldihydropyrimidines and Hepatitis B Virus capsid combined 3D-QSAR and molecular dynamics. Antiviral Res 137 , 151–164 (2017). Bourne, C. R., Finn, M. G. & Zlotnick, A. Global structural changes in hepatitis B virus capsids induced by the assembly effector HAP1. J Virol 80 , 11055–11061 (2006). Wang, X. Y. et al. In vitro inhibition of HBV replication by a novel compound, GLS4, and its efficacy against adefovir-dipivoxil-resistant HBV mutations. Antivir Ther 17 , 793–803 (2012). Wynne, S. A., Crowther, R. A. & Leslie, A. G. W. The crystal structure of the human hepatitis B virus capsid. Mol Cell 3 , 771–780 (1999). Motlagh, H. N., Wrabl, J. O., Li, J. & Hilser, V. J. The ensemble nature of allostery. Nature 2014 508:7496 508 , 331–339 (2014). Katen, S. & Zlotnick, A. The thermodynamics of virus capsid assembly. Methods Enzymol 455 , 395–417 (2009). Kim, C. et al. Core Protein-Directed Antivirals and Importin β Can Synergistically Disrupt Hepatitis B Virus Capsids. J Virol 96 , (2022). Kang, J. A. et al. Ciclopirox inhibits Hepatitis B Virus secretion by blocking capsid assembly. Nat Commun 10 , 1–14 (2019). Mohebbi, A., Mohammadi, S. & Memarian, A. Prediction of HBF-0259 interactions with hepatitis B Virus receptors and surface antigen secretory factors. Virusdisease 27 , 234–241 (2016). Mohebbi, A., Askari, F. S., Sammak, A. S., Ebrahimi, M. & Najafimemar, Z. Druggability of cavity pockets within SARS-CoV-2 spike glycoprotein and pharmacophore-based drug discovery. Future Virol 16 , 389–397 (2021). Berendsen, H. J. C., Postma, J. P. M., van Gunsteren, W. F. & Hermans, J. Interaction Models for Water in Relation to Protein Hydration. Intermolecular Forces: Proceedings of the Fourteenth Jerusalem Symposium on Quantum Chemistry and Biochemistry Held in Jerusalem, Israel, April 13--16, 1981 331–342 (1981) doi:10.1007/978-94-015-7658-1_21. Mohebbi, A., Mirarab, A., Shaddel, R., Shafaei Fallah, M. & Memarian, A. Molecular Dynamic Simulation and Docking of Cyclophilin A Mutants with its Potential Inhibitors. Journal of Clinical and Basic Research 5 , 26–41 (2021). Mohebbi, A. Ligand-based 3D pharmacophore modeling, virtual screening, and molecular dynamic simulation of potential smoothened inhibitors. J Mol Model 29 , (2023). Askari, F. S. et al. Digging for the discovery of SARS-CoV-2 nsp12 inhibitors: a pharmacophore-based and molecular dynamics simulation study. Future Virol (2022) doi:10.2217/fvl-2022-0054. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 24 Sep, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 26 Mar, 2025 Editor assigned by journal 18 Mar, 2025 Submission checks completed at journal 18 Mar, 2025 First submitted to journal 17 Mar, 2025 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-6244861","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":430259004,"identity":"4c854016-8a73-43d4-9d9e-8d1cce5f5f76","order_by":0,"name":"Fatemeh Sana Askari","email":"","orcid":"","institution":"Golestan Iran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Fatemeh","middleName":"Sana","lastName":"Askari","suffix":""},{"id":430259005,"identity":"d242bdf2-8444-4bf6-b7d1-74d2840c2dc4","order_by":1,"name":"Alireza Mohebbi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYBACNnbGBjCDn4H5AJCSkCGohZ8ZqkWygS0BpIWHoBbJZijD4ACPAYgmrMXgMHPbhw9/7OQZzp/5/OpGjQUPA/vhoxvwa2FsnjmDJ9mwcUbuNuucY0CH8aSl3SCkhZlHgpmxWYJ3m3EOG1CLBI8ZXi32IC1/DOrt2/jPPDPO+UeEFrAtDAmHE3sYcpgf57YRqYWx58Dx5BkSaWbMuX0SPGwE/XK8/THDjz/VtvvPH378OedbnRw/++FjeLUgAzYJMEmschBg/kCK6lEwCkbBKBg5AADw3kWH/l4SrwAAAABJRU5ErkJggg==","orcid":"","institution":"Iran University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Alireza","middleName":"","lastName":"Mohebbi","suffix":""}],"badges":[],"createdAt":"2025-03-17 13:08:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6244861/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6244861/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-18339-6","type":"published","date":"2025-09-24T15:57:05+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":78887859,"identity":"0973ecea-8af6-43ad-b730-f062a1f95a5c","added_by":"auto","created_at":"2025-03-20 09:54:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":258747,"visible":true,"origin":"","legend":"\u003cp\u003eRMSD analysis of HBc in unbound and CAM-bound states. (A) RMSD of HBc alone and in complex with CAMs over a 100 ns MD simulation. The black dashed line at 15 ns indicates the assumed convergence time, beyond which all systems exhibited stable fluctuations. (B) Ligand RMSD fluctuations within their respective binding pockets, indicating binding stability. Sulfamoylbenzamide exhibited transient dissociation from HBc, suggesting weaker interaction stability\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6244861/v1/1fc19eecd88332f3df75e2e8.png"},{"id":78888471,"identity":"04334873-be84-45f6-a2fb-81ddebe26992","added_by":"auto","created_at":"2025-03-20 10:02:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":155620,"visible":true,"origin":"","legend":"\u003cp\u003eRDF findings for different HBc complexes. RDF analysis illustrates the spatial distribution of ligands around HBcAg, showing the preferential binding regions and cumulative occupancy. Heteroaryldihydropyrimidine remains the most consistently associated with HBcAg, whereas Ciclopirox exhibits transient interactions\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6244861/v1/f5f885d39357ecb1e5d79722.png"},{"id":78887857,"identity":"9902eff2-3491-45e3-b4a7-574274397fd6","added_by":"auto","created_at":"2025-03-20 09:54:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":112607,"visible":true,"origin":"","legend":"\u003cp\u003eRMSF analysis of HBc in unbound and CAM-bound states. RMSF analysis highlights per-residue atomic fluctuations, revealing increased flexibility in specific regions upon ligand binding. The highest fluctuations were observed at the N-terminal (M1-I13) and C-terminal (A131-S141) residues, as well as central domains\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6244861/v1/fcfe5a9fa32abe135924faa2.png"},{"id":78889170,"identity":"6a035ffe-4139-456c-9a16-4aeca4957aa6","added_by":"auto","created_at":"2025-03-20 10:10:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":160917,"visible":true,"origin":"","legend":"\u003cp\u003eLigand atomic fluctuation analysis. RMSF per atom for each ligand in complex with HBc, showing relative stability within the binding pocket. Heteroaryldihydropyrimidine exhibited the lowest atomic fluctuations, suggesting a highly stable binding mode, whereas Sulfamoylbenzamide displayed significant movement, correlating with its transient dissociation\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6244861/v1/a5b2d5c2b497b588a9126fc5.png"},{"id":78887871,"identity":"5b86deb1-a806-4b97-9593-5e3c702c3e87","added_by":"auto","created_at":"2025-03-20 09:54:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":121945,"visible":true,"origin":"","legend":"\u003cp\u003eInteraction energy analysis of HBc-ligand complexes. Two distinct interaction types were observed: (i) van der Waals and electrostatic short-range interactions stabilizing all three complexes and (ii) a strong 1–4 electrostatic interaction in Ciclopirox and Sulfamoylbenzamide complexes, contributing to their binding stability\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6244861/v1/9b6ff335c7c68e9eb2d372cc.png"},{"id":78887876,"identity":"efd43b4d-6f3d-438c-9270-8bf2b4560ee0","added_by":"auto","created_at":"2025-03-20 09:54:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":160845,"visible":true,"origin":"","legend":"\u003cp\u003eSalt bridge formation analysis between CAMs and HBcAg. Ciclopirox exhibited the highest number of salt bridges with positively and negatively charged HBc residues, suggesting strong electrostatic stabilization. Sulfamoylbenzamide formed moderate salt bridges, while Heteroaryldihydropyrimidine relied more on hydrophobic interactions\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6244861/v1/fdaa2811c87c58ea691998f5.png"},{"id":78888486,"identity":"a7e98ab4-58d8-471f-be10-4f0eaf9e76cf","added_by":"auto","created_at":"2025-03-20 10:02:11","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":204240,"visible":true,"origin":"","legend":"\u003cp\u003eSalt bridge and hydrogen bonding analysis between Ciclopirox and HBcAg. (Top) Time evolution of the distances between negatively charged (left) and positively charged (right) residues of HBc and Ciclopirox ligands over 15 to 100 ns of MD simulation. The fluctuations indicate dynamic ligand-residue interactions, with some residues maintaining stable interactions while others exhibit transient binding. (Bottom) Average distances of negatively (left) and positively (right) charged residues from the ligand binding site, highlighting preferential distance of hydrogen bondings\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6244861/v1/836d26ae60299e1025aaeb01.png"},{"id":78887886,"identity":"e3667cd7-cabc-4fab-9629-c45304fad0fa","added_by":"auto","created_at":"2025-03-20 09:54:09","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":195528,"visible":true,"origin":"","legend":"\u003cp\u003eSalt bridge and hydrogen bonding analysis between Sulfamoylbenzamideand and HBcAg. (Top) Time evolution of the distances between negatively charged (left) and positively charged (right) residues of HBc and Sulfamoylbenzamide ligands over 15 to 100 ns of MD simulation\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6244861/v1/e3e90d799395c046fb96724e.png"},{"id":78887872,"identity":"a6d37cb6-9d71-41ce-9242-637004671cec","added_by":"auto","created_at":"2025-03-20 09:54:09","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":199800,"visible":true,"origin":"","legend":"\u003cp\u003eSalt bridge and hydrogen bonding analysis between Heteroaryldihydroprymidine and HBcAg. (Top) Time evolution of the distances between negatively charged (left) and positively charged (right) residues of HBc and Sulfamoylbenzamide ligands over 15 to 100 ns of MD simulation\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6244861/v1/6e26574a78bd4e64b889300b.png"},{"id":78888478,"identity":"ddac60b1-bdc5-43ce-a06d-24db7ff061d2","added_by":"auto","created_at":"2025-03-20 10:02:09","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":211455,"visible":true,"origin":"","legend":"\u003cp\u003eCluster analysis of HBc in unbound and CAM-bound states. The histograms illustrate the distribution of RMS values for HBcAg (blue), Ciclopirox (yellow), Heteroaryldihydropyrimidine (red), and Sulfamoylbenzamide (green) throughout the molecular dynamics simulations. Each peak represents a distinct conformational cluster, indicating the predominant structural states adopted during the simulation. The differences in clustering suggest that ligand binding modulates the structural dynamics of HBc, with Ciclopirox and Sulfamoylbenzamide inducing more flexible conformational transitions\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-6244861/v1/d586259da6ef25a79866a212.png"},{"id":78887878,"identity":"5672f383-bef3-4224-ae33-ea4442105b31","added_by":"auto","created_at":"2025-03-20 09:54:09","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":82604,"visible":true,"origin":"","legend":"\u003cp\u003eSASA analysis of HBcAg and its complexes with CAMs. The graph illustrates the changes in solvent exposure during the MD simulations, with HBcAg (blue) exhibiting the lowest SASA, indicating a compact structure. Sulfamoylbenzamide (green) resulted in the highest SASA, suggesting a more open or destabilized conformation, while Ciclopirox (yellow) and Heteroaryldihydropyrimidine (red) exhibited intermediate effects\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-6244861/v1/3fd6933a6c61015c464cb9a8.png"},{"id":78887863,"identity":"6c89e952-46d2-4c1a-aba7-489451a7c076","added_by":"auto","created_at":"2025-03-20 09:54:08","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":166166,"visible":true,"origin":"","legend":"\u003cp\u003eDSSP-derived secondary structure map of unbonded state of HBcAg and its \u0026nbsp;complexes with Ciclopirox, Sulfamoylbenzamide, and Heteroaryldihydroprymidine from 15 to 100 ns of simulation. Each column represents a simulation snapshot (time progressing from left to right), and each row corresponds to a protein residue. Colors indicate the assigned secondary structure at each snapshot (e.g., red for α-helix, blue for coil, and other hues for turns, bends, or alternative helices)\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-6244861/v1/2087adb7482fa19700373132.png"},{"id":78889169,"identity":"1fad802b-37d5-41e4-8589-eb185388352f","added_by":"auto","created_at":"2025-03-20 10:10:08","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":479968,"visible":true,"origin":"","legend":"\u003cp\u003e(Panel A) Time evolution of the PC projections for unbound HBcAg (black) and HBcAg complexed with Ciclopirox (orange), Sulfamoylbenzamide (green), and Heteroaryldihydropyrimidine (red). The x-axis shows simulation time (ns), and the y-axis is the projection amplitude (nm) onto the indicated PC, illustrating how each system samples that collective mode over time. (Panel B) Per-atom contributions to the PCs for each system. In each subplot, the black line represents the total displacement amplitude, while the red, green, and blue lines show the x, y, and z components, respectively, plotted against the atom index from N- to C-terminus. Peaks indicate regions of the protein exhibiting the largest motions along the corresponding eigenvector.\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-6244861/v1/ba88600536bae1f4b03ee306.png"},{"id":78887899,"identity":"61b1997d-227e-49cc-bc60-e913750ef45a","added_by":"auto","created_at":"2025-03-20 09:54:10","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":284475,"visible":true,"origin":"","legend":"\u003cp\u003eRMSF profiles of HBcAg (top left) and its complexes with Ciclopirox (top right), Sulfamoylbenzamide (bottom left), and Heteroaryldihydropyrimidine (bottom right) along selected PCs. Each row depicts the RMSF of individual atoms (x-axis) in nanometers (y-axis) for a specific eigenvector. Peaks highlight regions of the protein exhibiting the greatest atomic displacements in each collective motion, revealing how ligand binding alters the spatial distribution of fluctuations relative to unbound HBcAg\u003c/p\u003e","description":"","filename":"14.png","url":"https://assets-eu.researchsquare.com/files/rs-6244861/v1/2831436547bbc3ab593739c7.png"},{"id":78887905,"identity":"4eaf6449-6730-4e8e-afab-81db09dd1eeb","added_by":"auto","created_at":"2025-03-20 09:54:11","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":374226,"visible":true,"origin":"","legend":"\u003cp\u003eTwo-dimensional projection of HBcAg conformational substates during MD simulations. Ligand binding influenced conformational space, with Sulfamoylbenzamide introducing additional conformational substates, whereas Heteroaryldihydropyrimidine stabilized HBc with minimal fluctuation\u003c/p\u003e","description":"","filename":"15.png","url":"https://assets-eu.researchsquare.com/files/rs-6244861/v1/526e1627a6eed1e923676d71.png"},{"id":92430473,"identity":"0f140a59-82c5-4ae6-b560-0e039ab4c262","added_by":"auto","created_at":"2025-09-29 16:05:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3958051,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6244861/v1/148882c8-165a-4843-ac6a-cb1138ff28ea.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The dynamic States of Hepatitis B virus Capsid Monomers under the Impact of Different Class of Capsid-Assembly Modulators","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHepatitis B virus (HBV) remains a global health concern, affecting over 296\u0026nbsp;million individuals chronically and leading to approximately 820,000 deaths annually due to liver cirrhosis and hepatocellular carcinoma \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Despite the availability of vaccines and nucleos(t)ide analog therapies, these treatments do not eliminate covalently closed circular DNA (cccDNA) and are challenged by resistance issues \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Therefore, innovative therapeutic strategies are imperative for managing chronic HBV infection. HBV capsid assembly modulators (CAMs) have emerged as a promising class of antiviral agents, targeting the HBV core protein (HBc) essential for viral replication and cccDNA formation. CAMs, such as Heteroaryldihydropyrimidines (HAPs), Isoquinolinone derivatives, and compounds like AB-836, function by inducing defective or empty capsids, thereby hindering the encapsidation of pregenomic RNA (pgRNA) and viral polymerase, critical for viral replication and persistence \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe HBc, an icosahedral structure formed by 120 homodimers of Cp, relies on precise interactions at the dimer-dimer interface for its assembly. Residues such as Val1240, Trp1250, Arg1270, Thr1280, and Pro1340 play critical roles in maintaining capsid stability through hydrophobic and hydrogen bonding interactions. CAMs exploit these interactions by either misdirecting assembly into non-functional capsids or stabilizing incomplete intermediates \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. HAPs, for example, bind to a hydrophobic pocket at the dimer-dimer interface, inducing allosteric changes that accelerate improper assembly or destabilize preformed capsids. HAP-1, in particular, destabilizes mature capsids while promoting the formation of aberrant structures \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Sulfamoylbenzamides (SBAs) and phenylpropenamides (PPAs) offer distinct mechanisms of action. SBAs, such as AB-836, inhibit pgRNA-containing nucleocapsid formation, thereby reducing cccDNA production and viral replication \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Molecular dynamics (MD) simulations and structure-activity relationship (SAR) studies have further elucidated the binding dynamics of these CAMs. Hydrophobic interactions and van der Waals forces are essential for stabilizing inhibitor-protein complexes, while specific substitutions on CAM scaffolds enhance efficacy \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCAMs antiviral effects are multifaceted and it is not completely understood. It has been reprted that CAMs impact capsid stability by inducing structural rearrangements in the spike and base regions of Cp. CAM-A compounds increase dynamics within the spike region, while CAM-E compounds stabilize inter-subunit interactions, promoting tighter capsid assembly \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. These effects are critical for disrupting the HBV lifecycle, as they prevent encapsidation of pgRNA and inhibit reverse transcription. HAP18 disrupts the hydrogen bonding networks within HBV dimers and stabilizes capsid structures, demonstrating allosteric communication that modulates capsid dynamics \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Isoquinolinone-based CAMs have been shown to suppress HBV replication by binding at the dimer:dimer interface of the HBV core protein, locking the preferred conformation and enhancing drug efficacy \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Moreover, AB-836, a potent CAM, inhibits viral replication by preventing the formation of cccDNA, thereby suppressing HBV antigen production and transcription during de novo infection \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Therefore, the therapeutic potential of CAMs extends beyond direct antiviral activity. By targeting HBV capsid assembly, CAMs effectively suppress antigen production, reduce immune evasion, and limit cccDNA replenishment. Their specificity for the HBV core protein minimizes off-target effects, making them suitable for combination therapies with existing antivirals. In the last study of our group \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, it was observed that HAP dynamically stabilize HBc and compound with structurally similar features, like Lovastatin and Simvastatin, also induce identical activities. To further uncover the dynamic of HBc under unbounded and bounded conditions, this study aims to explore the behaviour of the HBV capsid monomers alone or in complexes with HAP, SBA, and PPA (Ciclopirox). Leveraging computational and experimental approaches, we seek to uncover structural determinants of CAM efficacy and contribute to the rational design of next-generation antivirals.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMolecular dynamic simulation\u003c/h2\u003e \u003cp\u003eAn MD simulation study was performed, considering it an efficacious approach for validating the stability of the HBcAg complexes with Ciclopirox and Sulfamoylbenzamide. MD analysis would provide valuable information regarding the dynamic behavior of the Ciclopirox and Sulfamoylbenzamide complexes with HBcAg and ligands' movements within their corresponding binding sites within the receptor. Also, we could see how HBc monomers are conformationally affected by the binding of two classes of HBV CAMs. Therefore, the trajectories were analyzed within 100 ns all-atom MD simulations.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTrajectory analysis of HBcAg apo and holo systems\u003c/h3\u003e\n\u003cp\u003eThe stability of HBcAg-Heteroaryldihydroprymidine, HBcAg-Ciclopirox, and HBcAg-Sulfamoylbenzamide systems was monitored using the GROMACS \u003cem\u003erms module\u003c/em\u003e to estimate their respective RMS values throughout the 100 ns simulation runs. Furthermore, RMSD fluctuations were measured to estimate the convergent time. Accordingly, RMSD fluctuations were measured at the backbone of HBcAg apo or holo systems (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe backbone RMSD fluctuations of HBcAg and its complexes with Ciclopirox, Sulfamoylbenzamide, and Heteroaryldihydroprymidine were analyzed over a 100 ns molecular dynamics (MD) simulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The assumed convergence time was determined at approximately 15 ns (\u003cem\u003esee\u003c/em\u003e the Black dashed line in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), beyond which all systems exhibited stable fluctuations. The HBcAg apo system maintained an average RMSD of 0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 nm. The HBc_Ciclopirox complex exhibited slightly higher structural deviations (0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 nm) than the apo system, implying a stable yet more flexible binding state. The HBc_Sulfamoylbenzamide complex showed the highest RMSD (0.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 nm), indicating notable structural perturbations upon ligand binding. In contrast, the HBc_Heteroaryldihydroprymidine complex showed the lowest HBcAg flexibility ( RMSD of 0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 nm).\u003c/p\u003e \u003cp\u003eLigand stability within the binding pocket was assessed by evaluating the RMSD of individual ligands (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The Heteroaryldihydroprymidine ligand exhibited the lowest RMSD (0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 nm), indicating a highly stable binding mode with minimal fluctuation. Conversely, Sulfamoylbenzamide displayed a higher RMSD (0.195\u0026thinsp;\u0026plusmn;\u0026thinsp;0.039 nm), suggesting moderate movement within the pocket. Ciclopirox exhibited RMSD fluctuations around 0.084\u0026thinsp;\u0026plusmn;\u0026thinsp;0.036 nm, indicating a relatively stable yet dynamic interaction.\u003c/p\u003e \u003cp\u003eThe radial distribution function (RDF) analysis revealed distinct patterns of ligand distribution around HBcAg. Heteroaryldihydropyrimidine exhibits the smallest average radial distance from the protein (1.84 nm) and an average g(r) of 2.70, achieving the highest cumulative number of ligand molecules (24.66) within that radius. By contrast, Ciclopirox showed the largest average g(r) of 4.80 at an distance of 2.06 nm, indicating strong local enrichment at specific separations; however, its cumulative occupancy (14.92) is the lowest among the three ligands, implying that it does not remain as persistently in close proximity to the protein. Sulfamoylbenzamide occupies an intermediate position, with an average distance of 2.21 nm, an average g(r) of 3.73, and a cumulative number of 22.39. This result indicates that Heteroaryldihydropyrimidine remains most consistently near HBcAg, whereas Ciclopirox, despite occasionally forming strong local interactions, spends less total time in close contact, and Sulfamoylbenzamide falls between these two extremes in both distance and occupancy. It was also observed that Sulfamoylbenzamide disassociates transiently and slide on the surface of HBc protein.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eBackbone RMSF analysis\u003c/h3\u003e\n\u003cp\u003eTo see if the lower HBcAg\u0026rsquo;s backbone RMSD was due to the size of the ligands, per-residue (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) and per-atom (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) fluctuations were evaluated using the gromacs \u003cem\u003ermsf\u003c/em\u003e module after system convergence time. Accordingly, The HBcAg apo system (blue line) exhibited the lowest fluctuations, indicating the unbound protein rigidity. The HBcAg complex with the Ciclopirox complex (yellow line) displayed slightly increased fluctuations, particularly in loop regions. The HBcAg-Sulfamoylbenzamide complex (green line) exhibited higher fluctuations, particularly in regions near the ligand binding site. Moreover, The HBcAg-Heteroaryldihydroprymidine system (red line) showed the highest backbone fluctuations, particularly in terminal and loop regions, indicating localized structural flexibility upon ligand binding. It was also observed that all systems exhibited the highest fluctuations at the N-terminal region (M1-I13) and C-terminal residues (A131-S141), which are typically flexible regions of the HBcAg protein.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess ligand stability within the binding pocket, RMSF per atom of each ligand was calculated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Heteroaryldihydroprymidine exhibited the lowest average RMSF (0.07 nm), indicating highly stable binding. The highest fluctuations were observed at C30 (0.0788 nm), O31 (0.1442 nm), and O32 (0.1504 nm), suggesting minor flexibility in functional groups. Sulfamoylbenzamide displayed higher fluctuations (0.195 nm on average), suggesting moderate movement within the binding pocket. Significant fluctuations were observed in C02 (0.2395 nm), F18 (0.2166 nm), and H2 (0.3352 nm), and some degree of instability was observed in these regions. Also, Ciclopirox had relatively stable interactions, with an average RMSF of 0.084 nm. Higher fluctuations were detected at H3 (0.1141 nm), H5 (0.1133 nm), and H8 (0.0946 nm), suggesting that hydrogen atoms in its structure may contribute to flexible interactions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eInteraction energy analysis\u003c/h3\u003e\n\u003cp\u003eMolecular mechanics calculations were performed to investigate the binding interaction energies between HBcAg and three ligands: Ciclopirox, Heteroaryldihydroprymidine, and Sulfamoylbenzamide. The short-range Coulombic (Coul-SR) and Lennard-Jones (LJ-SR) interaction energies, along with the 1\u0026ndash;4 electrostatic (Coul-14) and 1\u0026ndash;4 van der Waals (LJ-14) interactions, were analyzed over the convergence time (15\u0026ndash;100 ns).\u003c/p\u003e \u003cp\u003eThe HBcAg-Ciclopirox complex exhibited a moderate Coulombic short-range interaction energy (-24.76 kJ/mol) and a strong van der Waals stabilization (-79.46 kJ/mol), indicating that hydrophobic interactions contribute significantly to the ligand binding. Interestingly, the Coul-14 interaction energy was highly negative (-102.19 kJ/mol). This could be due to a strong electrostatic attraction, likely facilitated by salt bridge formation between Ciclopirox's charged functional groups and basic HBcAg residues. The LJ-14 component was slightly positive (49.67 kJ/mol), indicating minor steric repulsions within the binding site.\u003c/p\u003e \u003cp\u003eThe Heteroaryldihydroprymidine complex exhibited the most favorable interaction energy profile among the three ligands, with a highly stabilizing Coulombic short-range energy (-52.08 kJ/mol) and strong van der Waals interactions (-128.32 kJ/mol). These findings suggest that the ligand fits well within the binding pocket, forming extensive non-covalent interactions. However, the Coul-14 interaction was highly positive (665.89 kJ/mol), implying the presence of electrostatic repulsion between the ligand and nearby protein residues. Despite this, the negative LJ-SR (-128.32 kJ/mol) and favorable LJ-14 (86.97 kJ/mol) interactions suggest that van der Waals forces help stabilize the complex.\u003c/p\u003e \u003cp\u003eMoreover, the Sulfamoylbenzamide complex showed moderate Coulombic stabilization (-39.19 kJ/mol) but a robust Heteroaryldihydroprymidine -comparable van der Waals interactions (-127.38 kJ/mol), indicating significant hydrophobic contributions to ligand binding. The Coul-14 energy (-1221.26 kJ/mol) was the most negative among all complexes, suggesting extensive charge-charge interactions, possibly involving multiple salt bridges with charged residues in the binding pocket (\u003cem\u003esee\u003c/em\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Table panel).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eSalt bridge analysis\u003c/h3\u003e\n\u003cp\u003eOwing to the meaningful Coulombic 1\u0026ndash;4 interaction energies between ligands and HBcAg, index files containing positively (Lys, Arg, and His) and negatively (Glu and Asp) charged HBcAg residues were generated. The files were used to evaluate the number of salt bridge formations and possible hydrogen bondings between ligands and the receptor through Gromacs \u003cem\u003emindist\u003c/em\u003e and \u003cem\u003ehbond\u003c/em\u003e modules. The salt bridge formation was analyzed at a maximum distance of 3.5 \u0026Aring; and 30\u0026deg; angle.\u003c/p\u003e \u003cp\u003eAs expected, Ciclopirox and Sulfamoylbenzamide form high amounts of salt bridges with positively and negatively charged amino acids of HBcAg (\u003cem\u003esee\u003c/em\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The HBcAg-Ciclopirox complex exhibited the highest salt bridge formation among the three ligands. The average number of salt bridges with negatively charged residues was 13.99\u0026thinsp;\u0026plusmn;\u0026thinsp;7.40, while positive residues formed an even higher number of salt bridges (25.91\u0026thinsp;\u0026plusmn;\u0026thinsp;11.53). This suggests that Ciclopirox forms strong electrostatic interactions with both acidic and basic residues in the binding pocket, likely contributing to its highly negative Coul-14 energy (-102.19 kJ/mol) observed in the interaction energy analysis. The significant fluctuations in the number of contacts over time indicate dynamic interactions, possibly due to ligand flexibility or conformational shifts observed in the ligand RMSD.\u003c/p\u003e \u003cp\u003eThe Sulfamoylbenzamide complex displayed moderate salt bridge formation, but its interactions were more selective. The average number of salt bridges with negatively charged residues was only 0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;1.48, while interactions with positively charged residues were higher (7.80\u0026thinsp;\u0026plusmn;\u0026thinsp;8.84). However, the significantly lower interaction with negatively charged residues indicates a lack of strong anionic interactions, which could explain why its binding was not as stable as Ciclopirox. Interestingly, Heteroaryldihydroprymidine formed no salt bridges with positively charged residues (0 contacts at \u0026gt;\u0026thinsp;3.5 \u0026Aring;), while it displayed weak interactions with negatively charged residues (0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01). This suggests that electrostatic forces are not the primary contributors to the stability of this complex. Instead, van der Waals interactions (as shown by LJ-SR: -128.32 kJ/mol) likely play a dominant role in ligand stabilization. The lack of salt bridge formation may explain why Heteroaryldihydroprymidine exhibited positive Coul-14 interactions (665.89 kJ/mol), indicating repulsion rather than attraction. I have looked into the residue names to make more sense of the interaction at close distances.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Ciclopirox makes high numbers of bind with positively and negatively charged HBc residues. However, it only establishes salt bridges with one negatively charged amino acid residue, Glu42 (0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 nm), three possible positively charged residues, Arg38 (0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 nm), Arg55 (0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 nm), and weakly with Arg27 (0.51\u0026thinsp;\u0026plusmn;\u0026thinsp;21) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). For Sulfamoylbenzamide (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e), only one positively charged residue, Arg149 (0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31 nm), was observed in the range of 3.5 \u0026Aring;. In addition, a weakly possible salt bridge was observed between Heteroaryldihydroprymidine and a negatively charged residue, Asp28 (0.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 nm) (\u003cem\u003esee\u003c/em\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eI have further investigated the hydrogen bond formation between the ligand and the proposed residues in the range of salt bridge formation. Accordingly, an additional index file was generated for each system. For the HBcAg-Ciclopirox complex, the index file was composed of Glu42, Arg38, Arg55, and Arg27. For Sulfamoylbenzamide and Heteroaryldihydroprymidine, the index file comprised Arg149 and Asp28, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCluster analysis\u003c/h2\u003e \u003cp\u003eCluster analysis was performed to evaluate the RMS distributions across unbounded state of HBcAg and its complexes with Ciclopirox, Heteroaryldihydropyrimidine, and Sulfamoylbenzamide. The mean RMS values for the primary clusters (likely representing intra-cluster stability) were relatively low across all groups, ranging from 0.173\u0026thinsp;\u0026plusmn;\u0026thinsp;0.101 nm (Ciclopirox) to 0.227\u0026thinsp;\u0026plusmn;\u0026thinsp;0.132 nm (Sulfamoylbenzamide), with HBcAg (0.178\u0026thinsp;\u0026plusmn;\u0026thinsp;104 nm) and Heteroaryldihydropyrimidine (0.212\u0026thinsp;\u0026plusmn;\u0026thinsp;0.124 nm) falling within this spectrum (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eAlso, the mean number of clusters was similar (0.356 nm) for all groups. However, variability in these clusters was markedly greater, with standard deviations ranging from 0.623 nm (Ciclopirox and Sulfamoylbenzamide) to 0.683 nm (HBcAg).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSASA analysis\u003c/h3\u003e\n\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e, analysis of Solvent Accessible Surface Area (SASA) showed differences in solvent exposure profiles. The average SASA values ranged from 90.64\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67 nm\u0026sup2; (HBcAg) to 101.03\u0026thinsp;\u0026plusmn;\u0026thinsp;2.39 nm\u0026sup2; (Sulfamoylbenzamide-complex), with intermediate values observed for HBc-complexed Heteroaryldihydroprymidine (93.87\u0026thinsp;\u0026plusmn;\u0026thinsp;1.75 nm\u0026sup2;) and HBc-complexed Ciclopirox (99.55 \u0026plusmn; (2.20 nm\u0026sup2;). HBc protein exhibited the highest mean solvent accessibility in complex with Sulfamoylbenzamide. The result showed a relatively compact or shielded surface of HBcAg when it is in complex with Heteroaryldihydroprymidine.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eSecondary structure calculation\u003c/h3\u003e\n\u003cp\u003eAnalysis of the HBcAg simulation from 15 to 100 ns via DSSP indicates that the protein maintains a predominantly α-helical conformation throughout this convergent timeframe. The data reveal a high mean count of α-helix assignments (5143 out of 8502 possible states, corresponding to over half of the residues being helical on average), with minimal to no β-strand content detected. Small proportions of 3\u003csub\u003e10\u003c/sub\u003e​-helix, π-helix, and turns are present, mostly localized to loop or boundary regions. This stable α-helical propensity is also reflected in individual residue frequencies, where many central residues remain in an α-helix for over 90% of the simulation. Only a few segments exhibit noticeable transitions into coil or turn states, suggesting that the overall fold remains stable and does not significantly deviate from its helical core during the 15\u0026ndash;100 ns interval.\u003c/p\u003e \u003cp\u003eCompared to the unbound HBcAg, which retained a predominantly α-helical fold, the HBcAg\u0026ndash;Ciclopirox complex exhibits a marked reduction in regular secondary structure. DSSP analysis reveals a complete loss of α-helix content (mean of 0) and a pronounced increase in bends (\u0026ldquo;S\u0026rdquo;) and coil (\u0026ldquo;~\u0026rdquo;) states. Indeed, bends dominate the conformational ensemble with an average of 7077 out of 8501 possible states, whereas polyproline (\u0026ldquo;P\u0026rdquo;) conformations also appear intermittently. These findings suggest that Ciclopirox disrupts the native α-helical architecture observed in HBcAg alone, instead favoring more less ordered secondary structures as observed by the RMSA comparision, which may reflect alterations in the protein\u0026rsquo;s overall stability or function.\u003c/p\u003e \u003cp\u003eCompared to unbound HBcAg, which retains a predominantly α-helical fold, complexation with Sulfamoylbenzamide leads to a nearly complete loss of α-helix and an increase in more disordered or bend-like (\u0026ldquo;S\u0026rdquo;) states. The DSSP data show that most residues occupy bend (mean of over 7000 out of 8501 possible states) and coil (\u0026ldquo;~\u0026rdquo;) conformations, with occasional polyproline (\u0026ldquo;P\u0026rdquo;) content. This shift away from the stable helical architecture suggests that Sulfamoylbenzamide substantially perturbs the protein\u0026rsquo;s native fold, favoring a more flexible or partially disordered conformation similar to the behavior induced upon Ciclopirox binding, and what was observed in RMSD results. Similarly, Heteroaryldihydroprymidine again leads to the near-complete loss of α-helical content. DSSP analysis reveals a pronounced shift toward bend-like (\u0026ldquo;S\u0026rdquo;) and coil (\u0026ldquo;~\u0026rdquo;) conformations, with an average of over 7000 out of 8501 possible states classified as bends and a smaller fraction of polyproline (\u0026ldquo;P\u0026rdquo;) structures. This disruption of the native α-helix is consistent with the previously observed behavior for the other CAM-bound complexes (Ciclopirox and Sulfamoylbenzamide), suggesting that Heteroaryldihydroprymidine likewise induces an even more flexible or partially disordered secondary structure in HBcAg relative to its unbound form.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePCA analysis\u003c/h2\u003e \u003cp\u003ePCA of the HBcAg systems revealed differences in overall atomic fluctuations and the distribution of essential motions upon ligand binding. The sum of eigenvalues, representing the total variance in atomic displacements, was 13.82 nm\u0026sup2; for unbound HBcAg, 10.70 nm\u0026sup2; for the Ciclopirox complex, 23.90 nm\u0026sup2; for the Sulfamoylbenzamide complex, and 14.89 nm\u0026sup2; for the Heteroaryldihydroprymidine complex. Focusing on the first ten principal components\u0026mdash;which captured over 80% of the variance in the unbound state\u0026mdash;the cumulative variance accounted for was 81.77% for HBcAg alone, 79.05% for the Ciclopirox complex, 87.54% for the Sulfamoylbenzamide complex, and 85.99% for the Heteroaryldihydroprymidine complex. These results indicate that while the essential dynamics of HBcAg are largely confined to the top ten PCs, ligand binding alters the overall fluctuation landscape in a ligand-dependent manner, potentially influencing the protein\u0026rsquo;s functional motions and stability.\u003c/p\u003e \u003cp\u003eThe evolution time of HBcAg\u0026rsquo;s projection onto the first ten eigenvectors showed all PCs are actively contributing in atomic projections. Except one major projection at 50 nm time-point in PC1, the system was stable (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003eA). Similar behaviours were also observed among the HBcAg complexes in the PC5 to PC10. But in the first five PCs the projections were diverse, indicating the involvement of the earlier PCs in reflecting system projections. However, for the sake of riching 80% or higher trajectory data capture by PC, ten components were selected. Furthermore, the mode of motion for different component of HBcAg and its complex with the ligands is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003eB. As a finding, except of PC1, PC2, and PC4, mostly C-terminal of the protein has some peak of total (Black lines) atomic motions mostly in horizontal (x and y) axis. Also, most of the central domain atomic movments were also observed almost in all PCs. For Ciclopirox-complexed HBc protein, the atomic motions were more pronounced in both C-terminal (atoms 370 to 420) and cental domains (atoms 120 to 150 and 200 to 250). For Sulfamoylbenzamide complex, first 20 atoms at the N-terminal domain were significantly mobile mostly in 2D horizontal axis. Here, two other domain of atomic motion were observed excel the ones of N-terminal. In this regard, similar motion like that observed in Ciclopirox complex at central domain was observed but in half of PCs. However, the C-terminal domain was significantly motile at the ending atomes compare to HBc unbounded state and Ciclopirox complex (\u003cem\u003esee\u003c/em\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003eB). In the contrary to these, the complex of HBcAg to Heteroaryldihydroprymidine was very stable at the C-terminal domain. This could also explain the induced flexibility of HBcAg in complex by Heteroaryldihydroprymidine as observed in the RMSD analysis. Neverthless, like other complex the HBcAg was mobile at the N-terminal atomes and central domain similarly.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe RMSF of individual atoms was also evaluated for the given PCs. In accordance to the eigenvalues analysis for atomic motion, the RMSF findings revealed significant atomic peaks at the already mentioned domain. This finding also supporded RMSF of amino acid residues mentioned earlier (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther conformational substates of HBcAg were assessed (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e15\u003c/span\u003e). According to the 2D projection analysis over the first PC with highest motions and the last PC with more stable state, HBcAg spend most of the time of simulation in a stable state at two places with alsmost similar motions as observed in both PCs. The less condensed bridge between these two clusters (black snapshots between two condensed clouds; Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e15\u003c/span\u003e) is also indicates a possible conformational transformation between these two states. This finding was used as a base conformational structure for making the possibility of comparing the unbounded state with conditions that HBcAg is complex with CAMs. Accordingly (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003eA), upon bounding with Ciclopirox, the bridge mentioned between two states is almost disappeared. This possibly confers HBcAg to has a rapid conformational transform state, and structurally rearrange in the second place. Even more interestingly, HBc had a more structurally flexible transitions when it was in complex with Sulfamoylbenzamide, since it was observed that the bridge was extended for almost 2 nm, and this made a new cluster in space. Moreover, the first structure was more condenced in a new space almost 1 nm away from the one observed in unbounded state of HBc. These new conformational states or substates are possibly involve in the structural flexibili induced by Sulfamoylbenzamide. HBcAg was more stable and the trajectory snapshots were more condenced in the first structure in its complex with Heteroaryldihydroprymidine (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e15\u003c/span\u003eC). However, the second place was also nesteded within the second conformation but less dence. Moreover, a new palce in space was also observe that HBc met in this complex. This finding suggested that HBc preserved and stable when bounded to Sulfamoylbenzamide, and also has a new substate (3rd ), which was similar to the one induced by Sulfamoylbenzamide. This supports how Sulfamoylbenzamide reduces the flexibility of HBcAg as observed in RMSD analysis (and also in a movie made for the complexes [data are not provided]).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHBV remains a significant global health concern, with chronic infections leading to severe liver diseases, including cirrhosis and hepatocellular carcinoma \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. A pivotal component in the HBV lifecycle is the capsid, a protein shell that encases the viral genome and plays essential roles in replication and assembly \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Targeting the capsid assembly process has emerged as a promising therapeutic strategy, leading to the development of CAMs \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. In this study it was aimed to dynamically evaluate the HBV capsod monomer in unbounded or complexed states.​ The MD simulations and trajectory analyses in the study provided detailed insights into the structural perturbations induced by distinct classes of CAMs, Heteroaryldihydropyrimidine, Ciclopirox, and Sulfamoylbenzamide. These findings contribute to the growing body of evidence that highlights the efficacy and mechanistic of CAMs in disrupting HBV replication and capsid stability.\u003c/p\u003e \u003cp\u003eHBV capsid assembly is a critical step in the viral lifecycle, with the capsid protein forming an icosahedral structure composed of 120 homodimers \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The stability and structural integrity of these capsids are essential for the encapsidation of pregenomic RNA (pgRNA) and subsequent reverse transcription. Capsid assembly inhibitors function through two primary mechanisms: (1) misdirecting assembly into non-functional structures and (2) stabilizing immature capsids to prevent RNA encapsidation \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Our molecular dynamics simulations reveal distinct effects of CAMs on HBcAg conformation and stability, aligning with previous studies that highlight the potential of CAMs as therapeutic agents \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Accordingly, RMSD analysis indicated differential impacts of CAMs on the HBcAg backbone. Heteroaryldihydropyrimidine exhibited the lowest fluctuations, suggesting a strong stabilizing effect. Conversely, Sulfamoylbenzamide and Ciclopirox induced a relatively higher RMSD, indicating significant structural perturbations. These findings align with previous reports that classify CAMs into different mechanistic categories. Heteroaryldihydropyrimidines, such as BAY 41-4109, are known to stabilize capsids while promoting aberrant structures at high concentrations \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Similarly, the misdirecting effects observed in Sulfamoylbenzamide interactions are consistent with reports on bis-ANS, a small molecule known to misdirect capsid assembly \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe binding affinity and stability of CAMs within the HBcAg binding pocket were assessed through RMSD and radial distribution function (RDF) analysis. Heteroaryldihydropyrimidine remained the most tightly bound ligand, exhibiting the smallest average radial distance and the highest cumulative ligand occupancy. These findings suggest strong and persistent interactions, reinforcing its role as a capsid stabilizer \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Sulfamoylbenzamide, despite moderate binding affinity, exhibited transient dissociation events, consistent with its proposed sliding and surface-exploring behavior. This aligns with studies showing that sulfamoylbenzamides, including NVR 3-778, effectively inhibit pgRNA-containing nucleocapsid formation \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The backbone RMSF analysis demonstrated that ligand binding induced structural flexibility in the HBcAg protein, particularly in the loop and terminal regions. Among the ligands, Heteroaryldihydropyrimidine induced the highest backbone fluctuations, particularly in loop regions. These findings align with previous reports indicating that HBV capsid inhibitors can alter protein flexibility, affecting capsid stability and viral replication \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Further analysis of per-atom ligand RMSF revealed that Heteroaryldihydropyrimidine exhibited the lowest average RMSF, indicating a stable binding within the HBcAg binding pocket. Conversely, Sulfamoylbenzamide displayed higher fluctuations, particularly at functional groups involved in hydrophobic interactions. Ciclopirox maintained relatively stable interactions but showed some flexibility at specific atomic positions. These variations in RMSF suggest that the ligands differ in their ability to anchor within the binding site, a factor that has been previously reported as critical for HBV inhibitor efficacy.\u003c/p\u003e \u003cp\u003eBinding interaction energies provided insight into the stabilizing forces governing CAM-protein interactions. Heteroaryldihydropyrimidine showed the most favorable interaction energy profile, with a strong short-range Coulombic interaction and significant van der Waals forces. However, its highly positive Coul-14 energy suggests electrostatic repulsion, which may affect its overall stability within the binding pocket. In contrast, Sulfamoylbenzamide exhibited moderate Coulombic stabilization but robust van der Waals interactions, comparable to Heteroaryldihydropyrimidine. The moderate interaction energies observed for Ciclopirox suggest that its binding is driven by hydrophobic interactions rather than electrostatic forces. Previous studies indicate that electrostatic interactions play a pivotal role in CAM binding efficacy. BAY 41-4109, for instance, forms extensive electrostatic contacts with positively charged HBcAg residues, mirroring our findings for Sulfamoylbenzamide \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The presence of highly negative Coul-14 interaction energy suggests the formation of electrostatic interactions, potentially via salt bridges. Similar findings have been observed in previous studies where hydrophobic interactions were found to be essential in stabilizing HBV core protein inhibitors \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe salt bridge analysis further elucidated the nature of ligand-protein interactions. Ciclopirox exhibited the highest number of salt bridges, particularly with both positively and negatively charged residues. This finding implies strong electrostatic interactions that enhance its binding stability. The moderate salt bridge formation observed in the Sulfamoylbenzamide complex, particularly with positively charged residues, indicates selective electrostatic interactions. Interestingly, Heteroaryldihydropyrimidine formed no salt bridges with positively charged residues, relying predominantly on van der Waals interactions for stability. These findings are in agreement with previous studies highlighting the role of electrostatic interactions in HBV capsid inhibition. Also, cluster analysis confirmed that all ligand-bound HBcAg systems exhibited similar RMSD, suggesting that the overall structural integrity of the protein remained relatively stable. However, the HBcAg-Heteroaryldihydropyrimidine complex exhibited slightly higher deviations, consistent with the high fluctuations observed in RMSF analysis. SASA analysis further confirmed differences in ligand-induced conformational changes. The HBcAg-Sulfamoylbenzamide complex exhibited the highest solvent accessibility, suggesting a less compact structure, while Heteroaryldihydropyrimidine binding resulted in a more shielded protein surface.\u003c/p\u003e \u003cp\u003eSecondary structure calculations via DSSP analysis revealed that HBcAg maintains a predominantly α-helical conformation throughout the simulation. However, upon ligand binding, Ciclopirox exhibited a marked reduction in regular secondary structure, favoring more flexible conformations. This disruption of α-helical architecture suggests that Ciclopirox binding induces significant structural changes, potentially altering HBcAg functionality. Such ligand-induced structural alterations have been previously reported as a mechanism by which HBV inhibitors misdirect capsid assembly, ultimately disrupting viral replication \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDSSP analysis demonstrated that all ligands destabilized HBeAg\u0026rsquo;s native α-helical fold, favoring bends and coils. Ciclopirox and Sulfamoylbenzamide caused near-complete helix loss, while Heteroaryldihydroprymidine retained marginal order, likely due to its compact binding (SASA: 93.87\u0026thinsp;\u0026plusmn;\u0026thinsp;1.75 nm\u0026sup2;). This structural destabilization implies that ligand binding induces conformational shifts. Also, the increased solvent exposure in the Sulfamoylbenzamide complex (SASA: 101.03\u0026thinsp;\u0026plusmn;\u0026thinsp;2.39 nm\u0026sup2;) suggests partial unfolding or loosened packing, which may enhance proteolytic susceptibility or alter antigenicity\u0026mdash;a phenomenon documented in HBV core protein mutants \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Further conformational state of HBC was further evaluated by PCA. PCA along with cluster analyses revealed ligand-dependent alterations in HBeAg\u0026rsquo;s essential motions. Sulfamoylbenzamide induced the highest variance, reflecting pronounced flexibility, while Ciclopirox restricted motions, likely due to its stabilizing salt bridges. The emergence of distinct conformational substates in ligand-bound systems\u0026mdash;particularly the extended \u0026ldquo;bridge\u0026rdquo; in the Sulfamoylbenzamide complex\u0026mdash;suggests that ligand chemistry dictates accessible conformational ensembles. These findings align with theories positing that ligands act as \u0026ldquo;allosteric modulators\u0026rdquo; by reshaping energy landscapes \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Moreover, it was observed that Heteroaryldihydropyrimidine stabilize HBc in space where the protein was already met, but in a more density manner. It was also observed that the protein meet a place where the protein has not met before that was also bridge by transient transformation between other two structures. This finding suggests that Heteroaryldihydropyrimidine stabilize HBc with more rapid reangments between three stable location in space.\u003c/p\u003e \u003cp\u003eThe destabilization of HBeAg\u0026rsquo;s α-helical structure by all ligands raises questions about functional consequences. While disordered states may impair viral assembly or secretion \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, excessive flexibility (as seen with Sulfamoylbenzamide) could reduce target engagement longevity. Ciclopirox\u0026rsquo;s balanced interaction profile\u0026mdash;combining electrostatic and hydrophobic forces\u0026mdash;positions it as a promising scaffold for optimization. Future work should explore covalent stabilization of salt bridges or incorporation of hydrophobic moieties to enhance Heteroaryldihydroprymidine\u0026rsquo;s efficacy. The findings also provide mechanistic insights into the interactions of HBV core protein inhibitors and their potential antiviral effects. The variations in RMSF, interaction energy, salt bridge formation, and secondary structure disruption suggest that each ligand influences HBcAg differently. The strong electrostatic interactions and stability observed with Ciclopirox suggest its potential as a potent inhibitor. Heteroaryldihydropyrimidine, despite favorable interaction energy, exhibits electrostatic repulsion, which may affect its binding efficiency. Sulfamoylbenzamide, while stable, interacts selectively with HBcAg, which could influence its effectiveness as a capsid assembly inhibitor. These findings contribute to the growing body of research on HBV capsid-targeting antivirals and underscore the importance of molecular dynamics simulations in drug development.\u003c/p\u003e \u003cp\u003eCAMs represent a promising strategy for disrupting HBV replication by interfering with capsid assembly and stability. The results of our study reinforce the therapeutic potential of targeting capsid assembly through distinct molecular mechanisms. Heteroaryldihydropyrimidines appear particularly effective in stabilizing incomplete capsids, making them attractive candidates for antiviral development \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Meanwhile, Sulfamoylbenzamides exhibit misdirecting properties, which could be leveraged to prevent the formation of functional viral particles. But the interaction of Sulfamoylbenzamides was not that stable and visually it was observed that it transiently disassociates HBcAg. The findings of this study have several implications, one of which is combination therapy potential. Given their unique mechanisms, CAMs could be used in combination with nucleos(t)ide analogs to enhance antiviral efficacy while minimizing resistance. Also, personalized medicine approaches. The differential sensitivity of HBV RNA versus DNA to CAMs suggests potential applications in personalized treatment strategies, as proposed in multiscale modeling studies \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Furthermore, future structural modifications. The binding dynamics observed suggest that further modifications to the ligand scaffolds could enhance binding affinity and specificity, an approach successfully employed in aminothiazole-based HBV capsid inhibitors \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe finding of the study demonstrated that different class of anti-HBV CAMs assert their impact either by making stable bounded state through which capsid monomer rapidly rearrange and reduce capsid flexibility. This was observed in Heteroaryldihydroprymidine-complexed HBc. Also, this was supported by secondary structure analysi showing class A CAM, Heteroaryldihydroprymidine, retain α-helical structure that lost by interaction of class B CAMs like Sulfamoylbenzamides. Furthermore, type of interaction energy was differ in two class. Future studies can be done by focusing on repurposing potent CAMs thorough utilizing ligand-based drug discovery approaches. Also, stalization and destabilization of different HBc domain by CAMs could be the focus of future study to discover the important residues affecting stabilization of compounds on HBcAg. This also would help to predict the future potent amino acid substitutions that potentially lead to the rise of CAM-resistant mutants.\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003c/div\u003e \u003c/div\u003e "},{"header":"Methods","content":"\u003ch2\u003eData gathering of HBcAg and Ciclopirox\u003c/h2\u003e\u003cp\u003eThe crystallographic structure of HBcAg of HBV genotype D subtype adw was obtained from a protein data bank (PDB) with a PDB ID of 6j10 and 2.30 Å resolution \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. The structure of HBcAg was cleaned from water molecules and other non-standard fragments, as previously shown \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Furthermore, the chemical structures of Ciclopirox and Sulfamoylbenzamide in complex with the HBcAg were also extracted from the same .pdb file. The complex .pdb files of HBcAg, Ciclopirox, and Sulfamoylbenzamide were used in molecular dynamic simulation (MDS).\u003c/p\u003e\u003ch2\u003eMolecular dynamic simulation\u003c/h2\u003e\u003cp\u003eMDSs were conducted on Hepatitis B core antigen (HBcAg) alone and on three more complex systems, including HBcAg with Heteroaryldihydroprymidine, Cyclopirox, and Sulfamoylbenzamide. HBcAg was used as the receptor in this study. The simulations were performed using the GROMACS simulation software, version 2021.3 \u003csup\u003e34\u003c/sup\u003e. Simulations were run on a system equipped with an Intel(R) Core(TM) i7-4510U CPU @ 2.00GHz, with a total of 4 cores and four logical cores. SIMD instructions used were AVX2_256 with a memory model of 64-bit. GPU support was disabled for these simulations. The software was compiled using the GNU 10.2.0 C and C + + compilers with flags -mavx2 -mfma for AVX2 instruction sets and -O3 for optimization.\u003c/p\u003e\u003cp\u003eInitial models of HBcAg complexed with each ligand were subjected to energy minimization to remove any close contacts or high-energy conformations. The system was then solvated with explicit water molecules. Any charges present in the system were neutralized using appropriate counter-ions. Accordingly, The HBcAg-Ciclopirox complex was neutralized by 4 Na\u003csup\u003e+\u003c/sup\u003e ions. Additionally, The HBcAg-Sulfamoylbenzamide compound complex was neutralized with 4 NA\u003csup\u003e+\u003c/sup\u003e ions. The simulations were carried out under periodic boundary conditions. Each system was equilibrated at 300K and 1 bar using a canonical (NVT) ensemble followed by an isothermal-isobaric (NPT) ensemble. The simulations were run for 100 nanoseconds with a time step of 2 femtoseconds. The analysis of the MD simulation trajectories was performed with GROMACS built-in tools.\u003c/p\u003e\u003ch2\u003eTrajectory analysis\u003c/h2\u003e\u003cp\u003eMolecular dynamics (MD) simulation trajectories were subjected to an extensive range of analytical procedures conducted using GROMACS utilities to decipher the system's key structural and dynamic aspects. Structural stability and conformational alterations were evaluated through root mean square deviation (RMSD) calculations (\u003cem\u003egmx rms\u003c/em\u003e). Protein flexibility at the residue level was investigated via root mean square fluctuation (RMSF) computations (\u003cem\u003egmx rmsf\u003c/em\u003e). System compactness was gauged by determining the radius of gyration (Rg) using \u003cem\u003egmx gyrate\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eThe strength of receptor-ligand interactions over the simulation period was elucidated by quantifying interaction energies via \u003cem\u003egmx energy\u003c/em\u003e. Hydrogen bonding dynamics within the molecular system were comprehended using the \u003cem\u003egmx hbond\u003c/em\u003e utility. Evolutionary changes in secondary structures were scrutinized employing \u003cem\u003egmx do_dssp\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eA principal component analysis (PCA) was performed using the gmx covar and gmx anaeig utilities to identify predominant motion patterns in the system. The solvent-accessible surface area (SASA) was computed to continuously monitor the protein's exposure to the solvent during the simulation. A cluster analysis was conducted to identify the conformational states most frequently adopted by the system. Additionally, radial distribution functions (RDFs) were calculated to characterize the spatial relationships between various entities within the system. All analyzes were performed as reported before \u003csup\u003e\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e–\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAlireza Mohebbi\u003c/strong\u003e conceptualized the study, designed the research methodology, and supervised the project. Molecular dynamics simulations were performed by \u003cstrong\u003eFatemeh Sana Askari\u003c/strong\u003e and \u003cstrong\u003eAlireza Mohebbi\u003c/strong\u003e. \u003cstrong\u003eAlireza Mohebbi\u003c/strong\u003e and \u003cstrong\u003eFatemeh Sana Askari\u003c/strong\u003e contributed to data analysis and interpretation. \u003cstrong\u003eAlireza Mohebbi\u003c/strong\u003e assisted in manuscript writing and figure preparation. All authors reviewed and approved the final manuscript. Both authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Furthermore, the analysis methods of trajectories are provided in the Youtube channel of the corresponding author (https://www.youtube.com/@al1r3z49).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eJeng, W. J., Papatheodoridis, G. V. \u0026amp; Lok, A. S. F. Hepatitis B. \u003cem\u003eThe Lancet\u003c/em\u003e\u003cstrong\u003e401\u003c/strong\u003e, 1039\u0026ndash;1052 (2023).\u003c/li\u003e\n \u003cli\u003eNaderi, M., Salavatiha, Z., Gogoi, U. \u0026amp; Mohebbi, A. 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S. \u003cem\u003eet al.\u003c/em\u003e Digging for the discovery of SARS-CoV-2 nsp12 inhibitors: a pharmacophore-based and molecular dynamics simulation study. \u003cem\u003eFuture Virol\u003c/em\u003e (2022) doi:10.2217/fvl-2022-0054.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Hepatitis B virus, Capsid assembly modulators, Molecular dynamics simulation, Ciclopirox, Heteroaryldihydropyrimidine, Sulfamoylbenzamide","lastPublishedDoi":"10.21203/rs.3.rs-6244861/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6244861/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHepatitis B virus (HBV) remains a global health challenge. Capsid assembly modulators (CAMs) represent a promising class of antiviral agents that disrupt HBV core protein (HBc) function. Understanding the structural and dynamic impact of CAMs on HBc is crucial for the development of next-generation antiviral therapies. This study employed molecular dynamics (MD) simulations to evaluate the conformational behavior of HBc monomers in unbound and ligand-bound states. Different classes of CAMs\u0026mdash;Heteroaryldihydropyrimidine (HAP), Sulfamoylbenzamide (SBA), and Ciclopirox\u0026mdash;were analyzed to assess their impact on HBc stability, flexibility, and interaction energy. RMSD analysis revealed that HAP binding stabilized HBc, reducing backbone fluctuations, whereas SBA and PPA increased HBc flexibility. RMSF calculations demonstrated that CAM interactions influenced loop and terminal region dynamics. PCA suggested ligand-specific alterations in HBc\u0026rsquo;s essential motions, with Sulfamoylbenzamide inducing the highest variance. Salt bridge analysis indicated that Ciclopirox formed the strongest electrostatic interactions, stabilizing its binding. DSSP secondary structure analysis showed that CAMs disrupted α-helical content, with Sulfamoylbenzamide and Ciclopirox exhibiting the most pronounced structural rearrangements. This study provides novel insights into CAM-induced conformational changes in HBc. While HAP stabilizes the core protein, SBA and Ciclopirox increase flexibility, potentially leading to misassembled or destabilized capsids. These findings contribute to the rational design of CAM-based antiviral therapies and highlight key structural determinants for future drug optimization.\u003c/p\u003e","manuscriptTitle":"The dynamic States of Hepatitis B virus Capsid Monomers under the Impact of Different Class of Capsid-Assembly Modulators","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-20 09:54:03","doi":"10.21203/rs.3.rs-6244861/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-03-26T09:54:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-18T05:12:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-18T05:10:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-03-17T12:53:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9892644e-6e6c-4f67-ae34-aeed4ad7e706","owner":[],"postedDate":"March 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":45824445,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":45824446,"name":"Biological sciences/Microbiology/Virology/Hepatitis b virus"}],"tags":[],"updatedAt":"2025-09-29T16:00:31+00:00","versionOfRecord":{"articleIdentity":"rs-6244861","link":"https://doi.org/10.1038/s41598-025-18339-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-09-24 15:57:05","publishedOnDateReadable":"September 24th, 2025"},"versionCreatedAt":"2025-03-20 09:54:03","video":"","vorDoi":"10.1038/s41598-025-18339-6","vorDoiUrl":"https://doi.org/10.1038/s41598-025-18339-6","workflowStages":[]},"version":"v1","identity":"rs-6244861","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6244861","identity":"rs-6244861","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}