Structure based design of Benzimidazole derivative inhibitors targeting Streptococcus pneumoniae FtsZ: an integrated computational framework

Structure based design of Benzimidazole derivative inhibitors targeting Streptococcus pneumoniae FtsZ: an integrated computational framework | 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 Research Article Structure based design of Benzimidazole derivative inhibitors targeting Streptococcus pneumoniae FtsZ: an integrated computational framework Anisha Kumari, Priyanka Kataria, Rajni Khan, Pradeep Kumar Singh, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9342737/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The ongoing increase in bacterial antibiotic resistance, especially against Streptococcus pneumoniae , is a global cause for concern and demands the exploration of new antibacterial agents. FtsZ, a prokaryotic homologue of tubulin, is essential for orchestrating cytokinesis in bacteria. Similar to tubulin, which is the core target of cancer chemotherapy, FtsZ provides a promising target to be explored for the development of a new class of antibiotics due to its pivotal role in cell division. Here, based on the structure-function relationship, we have designed a library of 50 novel benzimidazole inhibitors targeting Streptococcus pneumoniae FtsZ (SpFtsZ). Molecular docking analysis identified three lead candidates: RR26, RR27, and RR33 as promising inhibitors against SpFtsZ, having binding affinities: -9.0, -9.1 and − 9.4 kcal/mol, respectively. The docking analysis revealed that these inhibitors occupy the interdomain cleft of SpFtsZ, engaging crucial H7 helix and T7 loop regions responsible for the protofilament interaction and GTP hydrolysis. Furthermore, the molecular dynamics (MD) simulation results suggest stable SpFtsZ-inhibitors interactions. The binding free estimation applying MM-PBSA further confirmed favourable spatial binding of inhibitors at the binding cavity of protein, yielding ∆G bind values of − 28.18 ± 3.60 kcal/mol (RR26), − 39.88 ± 3.60 kcal/mol (RR27), and − 36.40 ± 4.05 kcal/mol (RR33). Notably, RR27 exhibited the lowest binding free energy, suggesting the strongest affinity and highest inhibitory potential among the three compounds. Thus, this study provides a rational basis for the design of SpFtsZ-targeted inhibitors and identifies lead compounds with significant potential for subsequent in vitro and in vivo studies in the future. Cytokinesis Molecular Docking antibacterial agents Z-ring FtsZ polymerization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Infectious diseases account for 33% of global mortality, of which mortality caused by bacteria alone accounts for 30–50% (1, 2). In addition, the emergence of antibiotic resistance to pre-existing drugs has become a major hindrance to antibacterial treatment for diseases such as pneumonia, sepsis, and tuberculosis (2, 3). To overcome this problem, identifying novel drug targets and developing new antibacterial moieties has become the need of the hour. In recent times, FtsZ has become one of the promising drug candidates (4–6). FtsZ is a highly conserved protein and a key regulator of bacterial cell division (7, 8). Several studies have reinforced FtsZ as a novel antibacterial target. FtsZ is a GTPase that orchestrates bacterial cytokinesis (9–11). FtsZ assembly is highly dynamic in nature, and it is precisely regulated by its interacting proteins known as regulators of FtsZ assembly, such as FtsA, ZapA-D, ZipA, SepF, etc (7, 12–15). FtsZ shows both structural and functional similarities with the mammalian cytoskeleton protein tubulin, which forms microtubules in the eukaryotic systems. Both exhibit GTP-binding sites and form filamentous structures (10, 16, 17). Several anticancer drugs such as vinblastine, vincristine, paclitaxel, and estramustine are known to target microtubule dynamics and are successfully used in cancer chemotherapy and other diseases (18–20). Since FtsZ shows homology to tubulin, it can also be developed as an antimicrobial drug target (4, 10, 16). Any abnormalities in FtsZ assembly lead to the formation of a non-functional Z-ring, which inhibits the bacterial cytokinesis without perturbing DNA replication and nucleoid segregation. This resulted in bacterial cell elongation and formation of filamentous morphology, which ultimately leads to cell death (9, 11, 21). Therefore, the inhibitors that either block the polymerization of FtsZ or block the interaction of regulatory proteins with FtsZ produce a defect in cell division and are lethal for bacteria (4, 5, 22). Identifying such compounds may lead to the development of novel antibacterial therapeutic agents. Although FtsZ has been considered as a suitable target for novel antibacterial, the area has remained only partially explored (4, 5, 23). These demands target a highly conserved protein FtsZ and its interacting proteins which will lead to the development of new anti-pneumococcal drug therapeutics (24). There are several phytochemicals, synthetic compounds, and natural peptides that are known to inhibit bacterial cell division by targeting FtsZ assembly. For example, Totarol, barbarine, curcumin, chrysophaentin A, coumarins, dichamanetin, cinnamaldehyde are important phytochemicals which are known to inhibit the growth of several bacterial species by perturbing cytokinesis (25–28). Synthetic compounds like PC190723, TXA707, TXA709, zantrins, 3-methoxybenzamide (3-MBA), viriditoxin, quinazolinones, benzamides, arylthiazoles, phenoxyacetamides, diarylureas, benzofuroquinolines and arylthiazoles are found to inhibit the growth of a wide range of bacterial species by targeting FtsZ assembly (22, 29–33). Natural peptide like CRAMP (16–33) was also reported to inhibit bacterial cell division by targeting FtsZ assembly (34). The benzimidazole moiety is the privileged nitrogen-containing heterocycle with a wide range of biological activity, including anticancer, antibacterial, antifungal, anthelminthic, analgesic, antidiabetic, etc (35, 36). The potency of benzimidazole analogs against a wide range of microbes is primarily due to the disruption of crucial cellular processes, such as binding to tubulin proteins that interfere with the formation of microtubules in parasites and fungi, halting the cell division machinery (37–39). Benzimidazoles also displayed their dominance as anticancer agents, mainly as tubulin inhibitors, PARP inhibitors, kinase inhibitors, and alkylating agents (40–42). BT-benzo-29, a benzimidazole derivative, was found to inhibit the growth of Bacillus. Subtilis , Mycobacterium smegmatis by depolymerizing the FstZ polymers (35). It showed a minimal effect on the growth of HeLa cell proliferation and did not affect tubulin polymerization. In this study, we designed and screened a library of 50 novel benzimidazole in silico to target SpFtsZ dynamics. We found three compounds: (R)-5-amino-N-(1-(1-((1-(2-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-5,6-dimethyl-1H benzo[d]imidazol-2-yl)-2-(3-hydroxyphenyl)ethyl)picolinamide ( RR26 ), (R)-5-amino-N-(2-(3-fluorophenyl)-1-(1-((1-(2-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-5,6-dimethyl-1H-benzo[d]imidazol-2-yl)ethyl)picolinamide ( RR27 ) and (R)-6-((1-(6-methyl-1-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-1H-benzo[d]imidazol-2-yl)-2-phenylethyl)carbamoyl)pyridin-3-yl acetate ( RR33 ) exhibited promising lowest binding affinity. Binding kinetics indicate that RR26, RR27, and RR33 bind near the H7 helix and T7 loop region of FtsZ, which play an important role in connecting the protofilaments for forming the FtsZ ring at the mid-cell position and GTP hydrolysis for providing force for the constriction of septa for division. Material and Methods Ligand Preparation To study the effect of substituents and obtain the maximum binding affinity, structural diversity achieved by modifying six positions (R 1 -R 6 , Table 1 ). The different substituents display varied electronic and steric effects. The R 1 position often features bulky groups like benzyl (Bn) and functionalized benzyl groups, including 4-methoxybenzyl, hydroxybenzyl, 3-carboxybenzyl, etc. These bulky groups likely orient the inhibitor within a hydrophobic pocket of the SpFtsZ receptor. Substituents ranging from electron-donating (e.g., 4-methoxybenzyl) to electron-withdrawing (e.g., p-chlorobenzyl or 3-carboxybenzyl) alter the electron density of the aromatic ring, affecting the π-π stacking interactions with aromatic ring-containing amino acid residues. R 2 and R 3 positions represent the variations on the benzimidazole ring, which vary from simple hydrogens, methyl, halogens, to strongly polar substituents, including NH 2 , OH, NO 2 , COOH, etc. These groups act as hydrogen bond donors and acceptors, potentially forming critical anchors with the active site of SpFtsZ receptor. R 4 represents the substituent variation in the aromatic ring of triazole. The triazole is customized with phenyl and aryl substituents featuring NH 2 , OH, NO 2 , halogens, etc. It significantly increases the lipophilicity and electron-withdrawing properties of this region, potentially enhancing binding affinity through stronger electrostatic interactions. R 5 and R 6 positions represent the variation on the pyridine ring. While R 5 is frequently hydrogen, R 6 is frequently a bulkier group such as methyl, amine, or halogen. These groups can prevent off-target interactions by blocking specific metabolic sites or forcing the pyridine ring into a specific bioactive conformation. Generic structure of designed novel benzimidazole-triazole compounds are shown in Fig. 1 . The two-dimensional (2D) structures of 50 novel benzimidazole-based hybrid molecules were sketched using ChemBioDraw Ultra 14.0® software and saved as SDF format. Further, SDF files of the compounds were converted into three-dimensional (3D) structures and PDB (Protein Data Bank) format using cactus.nci.nih.gov, followed by conversion into AutoDock pdbqt format using Open Babel® software (43, 44). The detailed stricture of all compounds with their IUPAC name is listed in Table 1 . SpFtsZ Receptor preparation : In silico computations were performed using the 3D structure of the protein SpFtsZ with Uniprot primary accession number A0A0H2ZNE0-F1. The amino acid sequence was obtained from the UniProt server ( https://www.uniprot.org/ ) and processed in the AlphaFold database to obtain the 3D model of the protein (45, 46). The structure was initially saved in an image format and then converted into a PDB format using PyMol for further analysis(47). Molecular docking experiment : As the standard procedure of AutoDock protocol, the PDBQT format files for the ligand, receptor, and configuration were saved for processing in AutoDock Vina (44, 48). A grid box with dimensions of 60 Å × 60 Å × 60 Å, with a grid spacing value of 1.0 Å, was used to accept the binding pocket of SpFtsZ protein. The grid centres were located at the following coordinates: x = 6.231, y = 0.723 and z = 7.412. Molecular Dynamics Simulation : All atoms simulation were performed with the co-ordinates of protein, SpFtsZ (A0A0H2ZNE0-F1) and its docked complexes with ligands RR26, RR27, and RR33 employing GROMACS v2024.5(44, 49). The force field chosen as CHARMm36 and the water model TIP3P used to solvate each system in a cubic simulation box of dimension 80 Å 3 with a minimum distance of 1.0 nm between the protein surface and the box boundary (50). Charges on the systems were neutralized by adding the counterions (Na + Cl - ). The prepared systems were subjected to minimization process, till the convergence to approximately 1000 kJ mol − 1 nm − 1 to remove steric clashes and unfavorable contacts, opting the algorithms steepest descent followed by conjugate gradient. The equilibration phase done in two steps: NVT ensemble followed by NPT, each carried out for up to 1000 ps. The temperature (300 K) and pressure (1 bar) while the simulation maintained using the velocity-rescaling thermostat, and Parrinello–Rahman barostat (51, 52). The long-range electrostatic interactions were treated using the Particle Mesh Ewald (PME) method with a cutoff of 1.2 nm, and the same cutoff distance was applied for van der Waals interactions (53). All covalent bonds involving hydrogen atoms were constrained using the LINCS algorithm, allowing a simulation time step of 2 fs (54). Following equilibration, production MD simulations were performed under periodic boundary conditions for 100 ns for each system(55). Trajectory analyses, including RMSD, radius of gyration (Rg), solvent-accessible surface area (SASA), hydrogen-bond occupancy, and free-energy landscape calculations, were conducted using standard GROMACS utilities (49, 56–59). Results and discussion Molecular docking analysis Cheminformatics tools helps us to design the new target-based inhibitors (60). Molecular docking study provides a way to find out the lead potential inhibitors based on the binding interactions of receptor protein and inhibitors (61, 62). In this study, to perform the molecular docking analysis, we used AutoDock vina to validate the interaction of 50 novel benzimidazole-based hybrid molecules with SpFtsZ by using their binding energy of interaction. It was observed that most of the compounds fitted well into the binding cavity of SpFtsZ protein. For each compound, 8 distinct protein-ligand interaction conformations were generated and visually observed. Among all these compounds which were docked with SpFtsZ protein, three compounds ( RR26, RR27 and RR33 ) were found to strongly interact with the SpFtsZ protein with the binding affinities of -9.0, -9.1 and − 9.4 Kcal/mol, respectively. The compound RR26 showed a binding affinity of -9.0 Kcal/mol with the SpFtsZ protein (Fig. 2 A), it forms two conventional hydrogen bonds with the active-site residues ARG 144 and SER 110. The compound interacts with its halogen, fluorine, with GLU 140. The π-cation and π-anion bond formed at ARG 144, while the stacking interaction was observed with GLY 73 and π-alkyl formed with LEU 180 and LEU 184 (Fig. 2 D). Compound RR27 (Fig. 2 B), demonstrated three hydrogen-bond interactions with the active-site residues GLY 23, ASN 26, and SER 110. While ASN 167 residues interacted with a pi-donor hydrogen bond. Apart from this, it also forms one pi-sigma bond with LEU 184 and two pi-alkyl bonds with LEU 181 and ALA 187, the active residue (Fig. 2 E). Compound RR33 (Fig. 2 C) that showed the best binding affinity − 9.3 Kcal/mol as it forms five hydrogen bonds with GLY 23, GLY 73 and GLY74, ASN 26 and SER 110. LEU 184 involved in pi-sigma bond and alkyl bond was observed with LEU 180, LEU 181, LEU 184 and ALA 187 (Fig. 2 F). Molecular docking analysis reveals that these inhibitors bind at the interdomain cleft of SpFtsZ. The interdomain cleft contains very crucial region of SpFtsZ which includes H7 helix and T7 loop. Protofilaments interaction which needs for FtsZ assembly and GTP hydrolysis requires for providing energy for FstZ assembly dynamics and remodeling (9, 29, 63). Similar to these inhibitors several FtsZ based inhibitors like PC190723, Zantrin Z3, benzamide derivates and berberine and its derivates are reported to binds with the interdomain cleft of Staphylococcus aureus FtsZ (SaFtsZ), Escherichia coli FtsZ (EcFtsZ), SaFtsZ, respectively, which includes residues from the H7 helix and the T7 loop (64–68). Similar to these known inhibitors, RR26, RR27 and RR33 have potential to target FtsZ assembly. Conformational stabilities of protein-ligand complexes To examine the binding stability of ligands interacting with FtsZ, MD simulations were performed in water for the period of 100 ns at 300K. The simulations allowed assessment of the dynamic behavior and stability of the ligand–SpFtsZ complexes at the atomic resolutions, providing insights into their conformational stability and interaction persistence under physiological conditions. The structural stability of the protein–ligand complexes was evaluated using root-mean-square deviation (RMSD), radius of gyration (Rg), and solvent-accessible surface area (SASA) analyses and the average values during the simulations are enumerated in Table 1 . Table 1 Average RMSD, radius of gyration (Rg), and solvent-accessible surface area (SASA) values for SpFtsZ and its complexes during the MD simulation System RMSD Rg SASA SpFtsZ 0.16 ± 0.01 nm 1.90 ± 0.01 nm 173.70 ± 1.36 nm² RR26 0.19 ± 0.03 nm 1.87 ± 0.01 nm 172.84 ± 1.41 nm² RR27 0.23 ± 0.02 nm 1.87 ± 0.01 nm 172.66 ± 1.31 nm² RR33 0.22 ± 0.03 nm 1.87 ± 0.01 nm 173.90 ± 1.25 nm² In addition, hydrogen bond (H-bond) interactions and ligand RMSD were examined to elucidate the spatial stability and binding persistence of ligands RR26, RR27, and RR33 within the SpFtsZ binding pocket. The time evolution plots of Cα-RMSD of FtsZ and FtsZ-docked complexes are shown in Fig. 3 . In the Fig. 3 , it can be seen that the structure of SpFtsZ optimized quickly around the average RMSD 0.16 ± 0.01 nm. Upon the binding of ligands, a slight agitation in RMSD was observed, reflecting minor conformational adjustments of the protein. The SpFtsZ– RR26 complex stabilized at an average RMSD of 0.22 ± 0.03 nm, whereas SpFtsZ– RR27 and SpFtsZ– RR33 equilibrated around 0.23 ± 0.02 nm and 0.19 ± 0.03 nm, respectively. These results suggest that ligand binding induces moderate but stable conformational rearrangements in SpFtsZ without compromising the overall conformational stability of the protein. Figure 4 shows the radius of gyration (Rg), which measures protein structural compactness. The protein, SpFtsZ reached equilibrium at 1.90 ± 0.01 nm, indicating a stable global fold during the simulation. In comparison, the ligand-bound complexes SpFtsZ– RR26, SpFtsZ– RR27, and SpFtsZ– RR33 exhibited slightly lower and consistent Rg values of 1.87 ± 0.01 nm, suggesting that the compactness of protein get increased upon binding with ligand molecule. The reduced Rg observed for the complexes implies that ligand interaction contributes to structural stabilization of SpFtsZ without inducing major conformational rearrangements. Similarly, the solvent-accessible surface area (SASA) analysis revealed marginal variation between SpFtsZ and the ligand-bound complexes (Fig. 5 ). All systems stabilized around an average SASA value of 173 ± 1.50 nm², indicating that ligand binding does not significantly alter the solvent exposure of the protein. The comparable SASA profiles across the systems suggest preservation of the overall surface topology and support the structural stability of SpFtsZ upon complex formation. Furthermore, the root mean square fluctuation (RMSF) of the protein backbone residues was calculated to evaluate the dynamic behavior and flexibility of individual residues during simulation (Fig. 6 ). The low RMSF values observed for residues belonging to stable secondary conformations of α-helices and β-sheets, and the higher RMSF values were primarily belonging to the N- and C-terminal regions and loops. The active site has been found to be present in the N-terminal globular domain that contain a central parallel β-sheet core, a flanking α-helices (H1–H7) and T7 loop which is highly conserved across bacterial genera and is important for GTP-hydrolysis. The ≤ 1.0 Å average atomic fluctuation reflects structural stability of SpFtsZ is important for GTP hydrolysis. The RMSF analysis revealed reduced average fluctuations of active-site polar residues, GLY21, GLY22, GLY23, ASN26, ASP47, SER110, THR134, GLU140, ARG144, and ASP188 in the docked complexes indicate enhanced structural stabilization of SpFtsZ, upon ligand binding. These residues are crucial for catalytic activity and play a significant role in electrostatic interactions and hydrogen bonding, thereby contributing to the stability of the protein-ligand complexes. Similarly, the amino acid residues LEU180, LEU181, LEU184, ALA187, PHE137, VAL98, and MET106 belonging to hydrophobic cavity also shows stable RMSF. These residues appeared to form π–π stacking, π–alkyl and van der Waals interactions which helps for the stabilization of planer benzimidazole scaffold inside the binding cleft of SpFtsZ. The reduced flexibility of residues at the binding cleft of SpFtsZ indicates spatially stable protein-ligand interactions. The T7 loop (Gly73–Ser110), which is highly conserved in tubulin and critically involved in GTP hydrolysis, also exhibited reduced flexibility in the docked complexes. These results indicate that the Benzimidazole analogs ligands were spatially well accommodated within the GTP binding domain of SpFtsZ while the simulation period. Hydrogen bond interactions Furthermore, the spatial binding stability of ligands within the SpFtsZ binding pocket was evaluated by analyzing the occupancy of protein–ligand hydrogen bonds (H-bonds) throughout the simulation (Fig. 7 ). The SpFtsZ– RR26 complex exhibited the formation of three to four H-bonds during the initial ~ 0–15 ns, however, this interaction weakened to a single intermittent H-bond between ~ 20–80 ns, followed by the re-appearance of three H-bonds during the ~ 80–100 ns. Whereas the SpFtsZ– RR27 complex displayed initially four to five H-bonds, among them four H-bonds observed consistently maintained throughout the entire simulation, indicating strong and persistent binding. Similarly, SpFtsZ– RR33 showed four to five H-bonds during the initial ~ 0–20 ns, which stabilized to three persistent H-bonds for the remaining period of simulation. These results indicate the higher occupancy of hydrogen-bonding between the protein and ligands for SpFtsZ– RR27 and SpFtsZ– RR33 complexes contribute to greater binding stability within the SpFtsZ active site as compared to SpFtsZ– RR26. Free-energy landscape In addition, to investigate the conformational dynamics of SpFtsZ and docked complexes we also constructed the free-energy landscape (FEL) analysis, from the joint probability distribution of RMSD and Rg (Fig. 8 ). Figure 8 A shows that the SpFtsZ protein predominantly occupied to a well-defined and consolidated energy basin, indicating conformational stability and restricted structural fluctuations. Upon ligand binding, the protein explores a comparatively broader conformational space, reflecting ligand-induced modulation of its dynamic behavior. Notably, among the complexes, SpFtsZ– RR26 and SpFtsZ– RR33 display relatively shallow and dispersed energy minima, indicative of enhanced conformational flexibility and the presence of multiple metastable states (Fig. 8 B and 8 D). This behavior correlates with the fluctuating hydrogen-bond occupancies observed for these ligands and suggests comparatively weaker or less persistent stabilization of the binding pocket. Moreover, the SpFtsZ– RR27 complex displays a well-defined and densely populated energy basin, indicative of a more stable and energetically favorable conformational ensemble (Fig. 8 C). These observations further corroborate the molecular docking results, suggesting stable binding of ligands RR26, RR27, and RR33 with SpFtsZ, respectively. Binding free energy estimation Finally, to quantify the binding energetics of the selected benzimidazole derivatives in complex with SpFtsZ, the binding free energies were estimated using the MM-PBSA approach. The total binding free energy (ΔG bind ) was decomposed into van der Waals (ΔE vdW ), electrostatic (ΔE eel ), polar solvation (ΔG pol ), and non-polar solvation (ΔG np ) components to elucidate the physicochemical determinants governing the spatial binding of benzimidazole derivatives at the SpFtsZ active site (Table 2 ). Results show that all three complexes exhibited favourable total binding free energies, however, RR27 exhibited higher ΔG bind = − 39.88 ± 3.60 kcal/mol, followed by RR33 (− 36.40 ± 4.05 kcal/mol) and RR26 (− 28.18 ± 3.60 kcal/mol), suggesting a comparatively stronger binding affinity of RR27 toward SpFtsZ. The decomposition analysis of energetic terms reveals that binding affinity of compounds is predominantly driven by van der Waals interactions. RR27 displays the most favourable ΔE vdW contribution − 49.30 ± 1.33 kcal/mol, followed by RR33 (− 36.66 ± 1.25 kcal/mol) and RR26 (− 32.43 ± 0.73 kcal/mol), suggesting optimal hydrophobic packing and efficient shape complementarity within the binding pocket. Whereas the component, ΔE eel is observed strongly positive for all systems (RR26: 322.37 ± 20.07; RR27: 283.95 ± 4.11; RR33: 202.79 ± 12.81 kcal/mol), indicating unfavourable gas-phase electrostatics. However, this effect is largely compensated by the polar solvation energy term, ΔG pol which contributes negatively (RR26: −294.79 ± 15.22; RR27: −241.13 ± 4.47; RR33: −178.09 ± 0.59 kcal/mol), reflecting solvent stabilization of the charged states. It can be seen in residue-wise energy decomposition plots that crucial active site residues, GLU140, ARG144, ASP188 contribute substantially (> 20 kcal/mol) to both ΔE eel as well as ΔG pol ( Supplementary Figure S1 -3 ). This pattern reflects strong electrostatic interactions between these charged residues and the ligands, however, these contributions are largely offset by opposing polar solvation effects, resulting in a minimal net electrostatic contribution to the overall binding free energy. Table 2 The binding free energy (kcal/mol) estimation of compounds (RR26, RR27 and RR33) using MM-PBSA. Complex ∆E vdW ∆E eel ∆G pol ∆G np ∆G bind RR26 -32.43 ± 0.73 322.37 ± 20.07 -294.79 ± 15.22 -23.33 ± 0.34 -28.18 ± 3.60 RR27 -49.30 ± 1.33 283.95 ± 4.11 -241.13 ± 4.47 -33.40 ± 1.62 -39.88 ± 3.60 RR33 -36.66 ± 1.25 202.79 ± 12.81 -178.09 ± 0.59 -24.44 ± 0.49 -36.40 ± 4.05 Furthermore, the energetic term, ΔG np , which correlates with solvent-accessible surface burial, further stabilizes the complexes. Among the three compounds, RR27 exhibited the most favourable contribution of ΔG np = − 33.40 ± 1.62 kcal/mol as compared to RR33 (− 24.44 ± 0.49 kcal/mol) and RR26 (− 23.33 ± 0.34 kcal/mol) which observed consistent with the dominant role of hydrophobic interactions in stabilizing ligand binding within the interdomain cleft of SpFtsZ. Thus, the enhanced van der Waals and non-polar contributions outweigh the electrostatic penalty, resulting in the lowest overall ΔG bind for RR27. The compound RR33, while exhibiting weaker van der Waals interactions than RR27, benefits from a comparatively lower electrostatic penalty, yielding a competitive binding free energy. Whereas RR26 displays the least favourable binding, primarily due to weaker dispersion interactions and less favourable hydrophobic contributions. These findings are consistent with molecular docking and MD simulation results, which indicate stable ligand accommodation within the H7 helix–T7 loop region of SpFtsZ. The superior energetic profile of RR27 further supports its identification as a promising lead candidate for subsequent experimental validation. Conclusion In this study, we employed a structure-based drug design strategy to develop benzimidazole derivatives as potential inhibitors targeting the polymerization dynamics of SpFtsZ. Using an integrated computational approach, including molecular docking, MD simulations, and MM-PBSA binding free energy calculations, we systematically designed and characterized a library of novel compounds. Out of 50 designed inhibitors, RR26, RR27, and RR33 exhibited the highest binding affinities toward SpFtsZ. Molecular docking analyses revealed stable ligand–protein interactions, with binding localized at the interdomain cleft involving the H7 helix and T7 loop, a functionally critical region associated with protofilament formation and GTP hydrolysis. Furthermore, Molecular dynamics simulations, followed by MM-PBSA analysis, further substantiated the stability of all these complexes. Among these, RR27 exhibited the most favorable energetic profile, demonstrating superior stability and the lowest binding free energy, thereby identifying it as the most promising candidate among the three compounds. FtsZ is a GTP-dependent cytoskeletal protein that polymerizes upon GTP binding to form protofilaments and higher-order bundles, which assemble into the Z-ring required for septum formation during bacterial cell division. GTP hydrolysis plays a crucial role in regulating the dynamic turnover of FtsZ protofilaments; impairment of this process leads to hyperstabilization of FtsZ polymers, ultimately disrupting Z-ring constriction and inhibiting successful cell division. Any aberrations in GTP hydrolysis leads to hamper the bacterial cytokinesis by over stabilizing the FtsZ protofilaments (12, 69–71). The known inhibitors of FtsZ like PC190723, Zantrin Z3 and berberine binds to the interdomain cleft near T7 loop and known to inhibit the bacterial cell division by targeting FtsZ assembly (64–68). Our findings indicate that the binding of RR26, RR27, and RR33, preferentially localize within the interdomain cleft of SpFtsZ, in proximity to the H7 helix and T7 loop, could be a plausible shared mechanism of action. This interaction is likely to modulate GTP hydrolysis and perturb the intrinsic polymerization–depolymerization dynamics of FtsZ, thereby compromising Z-ring assembly and ultimately impairing bacterial cytokinesis. In summary, this study provides strong evidence supporting the potential of benzimidazole derivatives as FtsZ-targeting inhibitors and underscores the utility of rational, structure-based drug design approaches in advancing therapeutic strategies against bacterial diseases. Declarations Conflict of interest: The authors declare no competing interests. Funding AKS and SR thanks Mahatma Gandhi Central University Motihari, Bihar for providing seed money under research promotion scheme. Author Contribution Anisha Kumari : Methodology, Software, Formal analysis, Investigation, Visualization, Writing & Editing; Priyanka Kataria : Methodology, Investigation, Writing & Editing; Rajni Khan : Methodology, Software, Formal analysis, Investigation, Visualization, Reviewing and Editing; Pradeep Kumar Singh : Conceptualization, Methodology, Validation, Resources, Writing- Reviewing and Editing; Basant Narain Singh: Conceptualization, Methodology, Validation, Resources, Writing- Reviewing and Editing; Ankit Rai: Conceptualization, Methodology, Validation, Resources, Writing- Original draft preparation, Writing- Reviewing and Editing, and Supervision. Anil Kumar Singh: Conceptualization, Methodology, Validation, Resources, Writing- Original draft preparation, Writing- Reviewing and Editing, and Supervision. Amresh Prakash: Conceptualization, Methodology, Validation, Resources, Writing- Original draft preparation, Writing- Reviewing and Editing, and Supervision.; Shashikant Ray: Conceptualization, Methodology, Validation, Resources, Writing- Original draft preparation, Writing- Reviewing and Editing, and Supervision. Acknowledgement AKS and SR thanks Mahatma Gandhi Central University Motihari, Bihar for providing seed money under research promotion scheme. References Fauci AS, Morens DM. The perpetual challenge of infectious diseases. N Engl J Med. 2012;366(5):454 − 61. Antimicrobial Resistance C. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet. 2022;399(10325):629 − 55. Ventola CL. The antibiotic resistance crisis: part 1: causes and threats. P T. 2015;40(4):277 − 83. Lock RL, Harry EJ. Cell-division inhibitors: new insights for future antibiotics. Nat Rev Drug Discov. 2008;7(4):324 − 38. 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TXA709, an FtsZ-Targeting Benzamide Prodrug with Improved Pharmacokinetics and Enhanced In Vivo Efficacy against Methicillin-Resistant Staphylococcus aureus. Antimicrob Agents Chemother. 2015;59(8):4845-55. Sogawa H, Sato R, Suzuki K, Tomioka S, Shinzato T, Karpov P, et al. Binding sites of Zantrin inhibitors to the bacterial cell division protein FtsZ: Molecular docking and ab initio molecular orbital calculations. Chemical Physics. 2020;530:110603. Nepomuceno GM, Chan KM, Huynh V, Martin KS, Moore JT, O’Brien TE, et al. Synthesis and Evaluation of Quinazolines as Inhibitors of the Bacterial Cell Division Protein FtsZ. ACS Medicinal Chemistry Letters. 2015;6(3):308 − 12. Wang J, Galgoci A, Kodali S, Herath KB, Jayasuriya H, Dorso K, et al. Discovery of a small molecule that inhibits cell division by blocking FtsZ, a novel therapeutic target of antibiotics. J Biol Chem. 2003;278(45):44424-8. Ray S, Dhaked HP, Panda D. Antimicrobial peptide CRAMP (16–33) stalls bacterial cytokinesis by inhibiting FtsZ assembly. Biochemistry. 2014;53(41):6426-9. Ray S, Jindal B, Kunal K, Surolia A, Panda D. BT-benzo-29 inhibits bacterial cell proliferation by perturbing FtsZ assembly. Febs j. 2015;282(20):4015-33. Rai A, Gupta TK, Kini S, Kunwar A, Surolia A, Panda D. CXI-benzo-84 reversibly binds to tubulin at colchicine site and induces apoptosis in cancer cells. Biochem Pharmacol. 2013;86(3):378 − 91. Lacey E. Mode of action of benzimidazoles. Parasitol Today. 1990;6(4):112-5. Davidse LC. Benzimidazole Fungicides: Mechanism of Action and Biological Impact. Annual Review of Phytopathology. 1986;24(Volume 24, 1986):43–65. Lacey E, Gill JH. Biochemistry of benzimidazole resistance. Acta Tropica. 1994;56(2):245 − 62. Bansal Y, Silakari O. The therapeutic journey of benzimidazoles: A review. Bioorganic & Medicinal Chemistry. 2012;20(21):6208-36. El Rashedy AA, Aboul-Enein HY. Benzimidazole derivatives as potential anticancer agents. Mini Rev Med Chem. 2013;13(3):399–407. Refaat HM. Synthesis and anticancer activity of some novel 2-substituted benzimidazole derivatives. European Journal of Medicinal Chemistry. 2010;45(7):2949-56. Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, et al. AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J Comput Chem. 2009;30(16):2785-91. Pandey P, Rane JS, Chatterjee A, Kumar A, Khan R, Prakash A, et al. Targeting SARS-CoV-2 spike protein of COVID-19 with naturally occurring phytochemicals: an in silico study for drug development. J Biomol Struct Dyn. 2021;39(16):6306-16. Varadi M, Anyango S, Deshpande M, Nair S, Natassia C, Yordanova G, et al. AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Res. 2022;50(D1):D439-D44. UniProt C. UniProt: the Universal Protein Knowledgebase in 2023. Nucleic Acids Res. 2023;51(D1):D523-D31. Delano WL, editor The PyMOL Molecular Graphics System2002. Trott O, Olson AJ. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem. 2010;31(2):455 − 61. Abraham MJ, Murtola T, Schulz R, Páll S, Smith JC, Hess B, et al. GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX. 2015;1–2:19–25. Joung IS, Cheatham TE, 3rd. Determination of alkali and halide monovalent ion parameters for use in explicitly solvated biomolecular simulations. J Phys Chem B. 2008;112(30):9020-41. Bussi G, Donadio D, Parrinello M. Canonical sampling through velocity rescaling. J Chem Phys. 2007;126(1):014101. Parrinello M, Rahman A. Crystal Structure and Pair Potentials: A Molecular-Dynamics Study. Physical Review Letters. 1980;45(14):1196-9. Wilczynski NL, Haynes RB, Hedges T. Developing optimal search strategies for detecting clinically sound prognostic studies in MEDLINE: an analytic survey. BMC Med. 2004;2:23. Hess B. P-LINCS: A Parallel Linear Constraint Solver for Molecular Simulation. J Chem Theory Comput. 2008;4(1):116 − 22. Rane JS, Pandey P, Chatterjee A, Khan R, Kumar A, Prakash A, et al. Targeting virus-host interaction by novel pyrimidine derivative: an in silico approach towards discovery of potential drug against COVID-19. J Biomol Struct Dyn. 2021;39(15):5768-78. Van Der Spoel D, Lindahl E, Hess B, Groenhof G, Mark AE, Berendsen HJ. GROMACS: fast, flexible, and free. J Comput Chem. 2005;26(16):1701-18. Miller BR, 3rd, McGee TD, Jr., Swails JM, Homeyer N, Gohlke H, Roitberg AE. MMPBSA.py: An Efficient Program for End-State Free Energy Calculations. J Chem Theory Comput. 2012;8(9):3314-21. Singh R, Kumar A, Rane JS, Khan R, Tripathi G, Ajay AK, et al. Arylcoumarin perturbs SARS-CoV-2 pathogenesis by targeting the S-protein/ACE2 interaction. Sci Rep. 2022;12(1):17038. Kumar P, Khan R, Singh BN, Kumari A, Rai A, Singh AK, et al. Hydroxyethylamine based analog targets microtubule assembly: an in silico study for anti-cancerous drug development. Sci Rep. 2024;14(1):31381. Xu J, Hagler A. Chemoinformatics and Drug Discovery. Molecules. 2002;7(8):566–600. Hughes JP, Rees S, Kalindjian SB, Philpott KL. Principles of early drug discovery. Br J Pharmacol. 2011;162(6):1239-49. Raj S, Sasidharan S, Dubey VK, Saudagar P. Identification of lead molecules against potential drug target protein MAPK4 from L. donovani: An in-silico approach using docking, molecular dynamics and binding free energy calculation. PLoS One. 2019;14(8):e0221331. Matsui T, Yamane J, Mogi N, Yamaguchi H, Takemoto H, Yao M, et al. Structural Reorganization of the Bacterial Cell-Division Protein FtsZ from Staphylococcus aureus. Acta Crystallogr, Sect D: Biol Crystallogr. 2012;68:1175. Sun N, Chan FY, Lu YJ, Neves MA, Lui HK, Wang Y, et al. Rational design of berberine-based FtsZ inhibitors with broad-spectrum antibacterial activity. PLoS One. 2014;9(5):e97514. Matsui T, Yamane J, Mogi N, Yamaguchi H, Takemoto H, Yao M, et al. Structural reorganization of the bacterial cell-division protein FtsZ from Staphylococcus aureus. Acta Crystallogr D Biol Crystallogr. 2012;68(Pt 9):1175-88. Tan CM, Therien AG, Lu J, Lee SH, Caron A, Gill CJ, et al. Restoring methicillin-resistant Staphylococcus aureus susceptibility to β-lactam antibiotics. Sci Transl Med. 2012;4(126):126ra35. Haydon DJ, Stokes NR, Ure R, Galbraith G, Bennett JM, Brown DR, et al. An inhibitor of FtsZ with potent and selective anti-staphylococcal activity. Science. 2008;321(5896):1673-5. Anderson DE, Kim MB, Moore JT, O'Brien TE, Sorto NA, Grove CI, et al. Comparison of small molecule inhibitors of the bacterial cell division protein FtsZ and identification of a reliable cross-species inhibitor. ACS Chem Biol. 2012;7(11):1918-28. Erickson HP, Anderson DE, Osawa M. FtsZ in bacterial cytokinesis: cytoskeleton and force generator all in one. Microbiol Mol Biol Rev. 2010;74(4):504 − 28. Romberg L, Levin PA. Assembly dynamics of the bacterial cell division protein FTSZ: poised at the edge of stability. Annu Rev Microbiol. 2003;57:125 − 54. Leckie F, Mattei B, Capodicasa C, Hemmings A, Nuss L, Aracri B, et al. The specificity of polygalacturonase-inhibiting protein (PGIP): a single amino acid substitution in the solvent-exposed beta-strand/beta-turn region of the leucine-rich repeats (LRRs) confers a new recognition capability. EMBO J. 1999;18(9):2352-63. Additional Declarations No competing interests reported. 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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-9342737","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":633366247,"identity":"c99998d1-6a92-4bb3-90c6-c3710af64bc1","order_by":0,"name":"Anisha Kumari","email":"","orcid":"","institution":"Mahatma Gandhi Central University","correspondingAuthor":false,"prefix":"","firstName":"Anisha","middleName":"","lastName":"Kumari","suffix":""},{"id":633366249,"identity":"1de9cf33-489c-4abf-8b29-fce83da803c2","order_by":1,"name":"Priyanka Kataria","email":"","orcid":"","institution":"Amity University Haryana","correspondingAuthor":false,"prefix":"","firstName":"Priyanka","middleName":"","lastName":"Kataria","suffix":""},{"id":633366250,"identity":"29fff316-d346-4eb9-b471-1e91dfa502ad","order_by":2,"name":"Rajni Khan","email":"","orcid":"","institution":"Pandit Deendayal Upadhyaya Shekhawati University","correspondingAuthor":false,"prefix":"","firstName":"Rajni","middleName":"","lastName":"Khan","suffix":""},{"id":633366251,"identity":"c2c873a5-2854-4639-82d3-5469a97ff482","order_by":3,"name":"Pradeep Kumar Singh","email":"","orcid":"","institution":"Silicon University","correspondingAuthor":false,"prefix":"","firstName":"Pradeep","middleName":"Kumar","lastName":"Singh","suffix":""},{"id":633366252,"identity":"2cfd2aeb-73ce-40b9-8d67-a6a81c6517b2","order_by":4,"name":"Basant Narain Singh","email":"","orcid":"","institution":"Pandit Deendayal Upadhyaya Shekhawati University","correspondingAuthor":false,"prefix":"","firstName":"Basant","middleName":"Narain","lastName":"Singh","suffix":""},{"id":633366253,"identity":"7bdbd58a-9f2b-42b0-a638-72f947d3d150","order_by":5,"name":"Ankit Rai","email":"","orcid":"","institution":"Gujrat Biotechnology University","correspondingAuthor":false,"prefix":"","firstName":"Ankit","middleName":"","lastName":"Rai","suffix":""},{"id":633366254,"identity":"c962c2bb-4bde-4a7c-b3d7-fbcb658b4477","order_by":6,"name":"Anil Kumar Singh","email":"","orcid":"","institution":"Mahatma Gandhi Central University","correspondingAuthor":false,"prefix":"","firstName":"Anil","middleName":"Kumar","lastName":"Singh","suffix":""},{"id":633366255,"identity":"27ab78e1-1ff2-46fe-b5a3-4abedc11f953","order_by":7,"name":"Amresh Prakash","email":"","orcid":"","institution":"Amity University Haryana","correspondingAuthor":false,"prefix":"","firstName":"Amresh","middleName":"","lastName":"Prakash","suffix":""},{"id":633366256,"identity":"d404319f-3902-4ed1-bee2-eb668c09e223","order_by":8,"name":"Shashikant Ray","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDUlEQVRIiWNgGAWjYDACCQYGZgaDBCivAkTwMCBIvFrYQLwzRGthgGphbMOvGAzkZzc//lxQkCZvLt/78DPvvG12/TNyDzD8qGGQMcehxeDOMTPpGQY5hjvb2I2lebfdTp5xIy+BsecYA49lAw4tEglmzDwGFYwbjrExgLUYSOQYMPA2MPAYHMDhsBnpnz8DtdgDtTD/5p0D0cL4F48Whhs5BtI8BjmJQC1s0rwNt+1AWpjx2WJwI6cMqCUtecOxNDbLOcduJ0iceZdwWOaYBD6Hbf7M8yfZdsPhY8w33tTctudvzz348E2NjT1OhyEDJmCMJDYAGQfA8UUMYPzBwGBPnNJRMApGwSgYSQAATxxXA5CVoHQAAAAASUVORK5CYII=","orcid":"","institution":"Mahatma Gandhi Central University","correspondingAuthor":true,"prefix":"","firstName":"Shashikant","middleName":"","lastName":"Ray","suffix":""}],"badges":[],"createdAt":"2026-04-07 09:38:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9342737/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9342737/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108804775,"identity":"0df159ce-e4c7-41c1-b82d-ab1549072393","added_by":"auto","created_at":"2026-05-08 15:23:28","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":441806,"visible":true,"origin":"","legend":"\u003cp\u003eShowing generic structure of designed novel benzimidazole-triazole compounds.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9342737/v1/b41c39fc742159cc09fa1907.jpg"},{"id":108805101,"identity":"49b896fe-8184-4c77-b590-bf52cc6085ec","added_by":"auto","created_at":"2026-05-08 15:24:49","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":113799,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular docking analysis of RR26, RR27, and RR33 with SpFtsZ. A, B and C represents the top hit inhibitors in stick model binds in the binding pocket of the 3-dimenisonal ribbon structure of SpFtsZ. D, E and F represents the key interacting amino acid residues of SpFtsZ with RR26, RR27 and RR28, respectively.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9342737/v1/3e3dd64b401a5add21bd43d1.jpg"},{"id":108539997,"identity":"84f4624c-4c7f-4d73-9f09-67e308984816","added_by":"auto","created_at":"2026-05-05 18:21:33","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":63594,"visible":true,"origin":"","legend":"\u003cp\u003eTime evolution of RMSD plots of SpFtsZ and its complexes with RR26, RR27, and RR33, demonstrating ligand-induced stabilization and convergence of protein structures during the MD simulation. The color scheme representing the protein and docked complexes is defined in the inset of the figure.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9342737/v1/91f6e6c360f3cb25e971affa.jpg"},{"id":108540000,"identity":"8123152c-396e-4da9-8dec-41fafc54a0ed","added_by":"auto","created_at":"2026-05-05 18:21:33","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":67872,"visible":true,"origin":"","legend":"\u003cp\u003eRadius of gyration (Rg) plots of protein, SpFtsZ and its ligand-bound complexes with RR26, RR27, and RR33), demonstrating ligand-induced stabilization and structural compactness during the simulation, respectively. The color scheme representing the protein and docked complexes is defined in the inset of the figure.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9342737/v1/c938e8f71e5ff5ae02e42ba3.jpg"},{"id":108804314,"identity":"e8c9281a-59c9-4e98-9908-2bb2f1143295","added_by":"auto","created_at":"2026-05-08 15:19:14","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":63136,"visible":true,"origin":"","legend":"\u003cp\u003eTime evolution of solvent-accessible surface area (SASA) of SpFtsZ and its docked complexes with ligands RR26, RR27, and RR33, reflecting changes in protein surface exposure upon ligand binding.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9342737/v1/4c90635edbab94ea3a73b7ab.jpg"},{"id":108540002,"identity":"581cecc7-8b3d-45ff-8201-672372928e1e","added_by":"auto","created_at":"2026-05-05 18:21:33","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":35809,"visible":true,"origin":"","legend":"\u003cp\u003eRoot mean square fluctuation (RMSF) plots showing residue-level flexibility of SpFtsZ in the absence and presence of ligands RR26, RR27, and RR33, highlighting regions stabilized upon ligand binding.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9342737/v1/39e68d64f303681930f57281.jpg"},{"id":108804142,"identity":"aa240099-1a1e-4d04-b625-d1dd46ce3bca","added_by":"auto","created_at":"2026-05-08 15:16:34","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":62588,"visible":true,"origin":"","legend":"\u003cp\u003eThe occupancy of hydrogen bond (H-bond) interactions between the protein SpFtsZ and the ligands RR26, RR27, and RR33, respectively. The color scheme representing the docked complexes is defined in the inset of the figure.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9342737/v1/e2bef6a78d64991031e71d6d.jpg"},{"id":108540005,"identity":"8bd4ed49-5b85-440c-976e-9eab7821d9b7","added_by":"auto","created_at":"2026-05-05 18:21:33","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":125958,"visible":true,"origin":"","legend":"\u003cp\u003eFree energy landscape (FEL) of the protein and docked complexes. (A) SpFtsZ, (B) SpFtsZ–RR26, (C) SpFtsZ–RR27, and (D) SpFtsZ–RR33. The color-coded energy basins depict the conformational dynamics of the protein and its docked complexes. Free energy values are represented in kcal/mol, as indicated by the color bar shown in the right panel of the plots.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9342737/v1/f7608fee4d5eb6ec58901cc1.jpg"},{"id":108809607,"identity":"10ae6c52-dea2-4245-bcdd-e35ec39bc8a0","added_by":"auto","created_at":"2026-05-08 15:54:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1256221,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9342737/v1/6b0d3efd-a2fb-4ecd-80c9-f6dced5fb58a.pdf"},{"id":108804996,"identity":"ecf260d0-021a-4639-98e2-3cca8002933c","added_by":"auto","created_at":"2026-05-08 15:24:28","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":752612,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-9342737/v1/f687abc8d9ec3bab5535674f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Structure based design of Benzimidazole derivative inhibitors targeting Streptococcus pneumoniae FtsZ: an integrated computational framework","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInfectious diseases account for 33% of global mortality, of which mortality caused by bacteria alone accounts for 30\u0026ndash;50% (1, 2). In addition, the emergence of antibiotic resistance to pre-existing drugs has become a major hindrance to antibacterial treatment for diseases such as pneumonia, sepsis, and tuberculosis (2, 3). To overcome this problem, identifying novel drug targets and developing new antibacterial moieties has become the need of the hour. In recent times, FtsZ has become one of the promising drug candidates (4\u0026ndash;6). FtsZ is a highly conserved protein and a key regulator of bacterial cell division (7, 8). Several studies have reinforced FtsZ as a novel antibacterial target. FtsZ is a GTPase that orchestrates bacterial cytokinesis (9\u0026ndash;11). FtsZ assembly is highly dynamic in nature, and it is precisely regulated by its interacting proteins known as regulators of FtsZ assembly, such as FtsA, ZapA-D, ZipA, SepF, etc (7, 12\u0026ndash;15). FtsZ shows both structural and functional similarities with the mammalian cytoskeleton protein tubulin, which forms microtubules in the eukaryotic systems. Both exhibit GTP-binding sites and form filamentous structures (10, 16, 17). Several anticancer drugs such as vinblastine, vincristine, paclitaxel, and estramustine are known to target microtubule dynamics and are successfully used in cancer chemotherapy and other diseases (18\u0026ndash;20). Since FtsZ shows homology to tubulin, it can also be developed as an antimicrobial drug target (4, 10, 16). Any abnormalities in FtsZ assembly lead to the formation of a non-functional Z-ring, which inhibits the bacterial cytokinesis without perturbing DNA replication and nucleoid segregation. This resulted in bacterial cell elongation and formation of filamentous morphology, which ultimately leads to cell death (9, 11, 21). Therefore, the inhibitors that either block the polymerization of FtsZ or block the interaction of regulatory proteins with FtsZ produce a defect in cell division and are lethal for bacteria (4, 5, 22). Identifying such compounds may lead to the development of novel antibacterial therapeutic agents. Although FtsZ has been considered as a suitable target for novel antibacterial, the area has remained only partially explored (4, 5, 23). These demands target a highly conserved protein FtsZ and its interacting proteins which will lead to the development of new anti-pneumococcal drug therapeutics (24).\u003c/p\u003e \u003cp\u003eThere are several phytochemicals, synthetic compounds, and natural peptides that are known to inhibit bacterial cell division by targeting FtsZ assembly. For example, Totarol, barbarine, curcumin, chrysophaentin A, coumarins, dichamanetin, cinnamaldehyde are important phytochemicals which are known to inhibit the growth of several bacterial species by perturbing cytokinesis (25\u0026ndash;28). Synthetic compounds like PC190723, TXA707, TXA709, zantrins, 3-methoxybenzamide (3-MBA), viriditoxin, quinazolinones, benzamides, arylthiazoles, phenoxyacetamides, diarylureas, benzofuroquinolines and arylthiazoles are found to inhibit the growth of a wide range of bacterial species by targeting FtsZ assembly (22, 29\u0026ndash;33). Natural peptide like CRAMP (16\u0026ndash;33) was also reported to inhibit bacterial cell division by targeting FtsZ assembly (34).\u003c/p\u003e \u003cp\u003eThe benzimidazole moiety is the privileged nitrogen-containing heterocycle with a wide range of biological activity, including anticancer, antibacterial, antifungal, anthelminthic, analgesic, antidiabetic, etc (35, 36). The potency of benzimidazole analogs against a wide range of microbes is primarily due to the disruption of crucial cellular processes, such as binding to tubulin proteins that interfere with the formation of microtubules in parasites and fungi, halting the cell division machinery (37\u0026ndash;39). Benzimidazoles also displayed their dominance as anticancer agents, mainly as tubulin inhibitors, PARP inhibitors, kinase inhibitors, and alkylating agents (40\u0026ndash;42). BT-benzo-29, a benzimidazole derivative, was found to inhibit the growth of \u003cem\u003eBacillus. Subtilis\u003c/em\u003e, \u003cem\u003eMycobacterium smegmatis\u003c/em\u003e by depolymerizing the FstZ polymers (35). It showed a minimal effect on the growth of HeLa cell proliferation and did not affect tubulin polymerization. In this study, we designed and screened a library of 50 novel benzimidazole \u003cem\u003ein silico\u003c/em\u003e to target SpFtsZ dynamics. We found three compounds: (R)-5-amino-N-(1-(1-((1-(2-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-5,6-dimethyl-1H benzo[d]imidazol-2-yl)-2-(3-hydroxyphenyl)ethyl)picolinamide (\u003cb\u003eRR26\u003c/b\u003e), (R)-5-amino-N-(2-(3-fluorophenyl)-1-(1-((1-(2-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-5,6-dimethyl-1H-benzo[d]imidazol-2-yl)ethyl)picolinamide (\u003cb\u003eRR27\u003c/b\u003e) and (R)-6-((1-(6-methyl-1-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-1H-benzo[d]imidazol-2-yl)-2-phenylethyl)carbamoyl)pyridin-3-yl acetate (\u003cb\u003eRR33\u003c/b\u003e) exhibited promising lowest binding affinity. Binding kinetics indicate that RR26, RR27, and RR33 bind near the H7 helix and T7 loop region of FtsZ, which play an important role in connecting the protofilaments for forming the FtsZ ring at the mid-cell position and GTP hydrolysis for providing force for the constriction of septa for division.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003e \u003cstrong\u003eLigand Preparation\u003c/strong\u003e \u003cp\u003eTo study the effect of substituents and obtain the maximum binding affinity, structural diversity achieved by modifying six positions (R\u003csup\u003e1\u003c/sup\u003e-R\u003csup\u003e6\u003c/sup\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The different substituents display varied electronic and steric effects. The R\u003csup\u003e1\u003c/sup\u003e position often features bulky groups like benzyl (Bn) and functionalized benzyl groups, including 4-methoxybenzyl, hydroxybenzyl, 3-carboxybenzyl, etc. These bulky groups likely orient the inhibitor within a hydrophobic pocket of the SpFtsZ receptor. Substituents ranging from electron-donating (e.g., 4-methoxybenzyl) to electron-withdrawing (e.g., p-chlorobenzyl or 3-carboxybenzyl) alter the electron density of the aromatic ring, affecting the π-π stacking interactions with aromatic ring-containing amino acid residues. R\u003csup\u003e2\u003c/sup\u003e and R\u003csup\u003e3\u003c/sup\u003e positions represent the variations on the benzimidazole ring, which vary from simple hydrogens, methyl, halogens, to strongly polar substituents, including NH\u003csub\u003e2\u003c/sub\u003e, OH, NO\u003csub\u003e2\u003c/sub\u003e, COOH, etc. These groups act as hydrogen bond donors and acceptors, potentially forming critical anchors with the active site of SpFtsZ receptor. R\u003csup\u003e4\u003c/sup\u003e represents the substituent variation in the aromatic ring of triazole. The triazole is customized with phenyl and aryl substituents featuring NH\u003csub\u003e2\u003c/sub\u003e, OH, NO\u003csub\u003e2\u003c/sub\u003e, halogens, etc. It significantly increases the lipophilicity and electron-withdrawing properties of this region, potentially enhancing binding affinity through stronger electrostatic interactions. R\u003csup\u003e5\u003c/sup\u003e and R\u003csup\u003e6\u003c/sup\u003e positions represent the variation on the pyridine ring. While R\u003csup\u003e5\u003c/sup\u003e is frequently hydrogen, R\u003csup\u003e6\u003c/sup\u003e is frequently a bulkier group such as methyl, amine, or halogen. These groups can prevent off-target interactions by blocking specific metabolic sites or forcing the pyridine ring into a specific bioactive conformation. Generic structure of designed novel benzimidazole-triazole compounds are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe two-dimensional (2D) structures of 50 novel benzimidazole-based hybrid molecules were sketched using ChemBioDraw Ultra 14.0\u0026reg; software and saved as SDF format. Further, SDF files of the compounds were converted into three-dimensional (3D) structures and PDB (Protein Data Bank) format using cactus.nci.nih.gov, followed by conversion into AutoDock pdbqt format using Open Babel\u0026reg; software (43, 44). The detailed stricture of all compounds with their IUPAC name is listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e \u003cb\u003eSpFtsZ Receptor preparation\u003c/b\u003e: \u003cem\u003eIn silico\u003c/em\u003e computations were performed using the 3D structure of the protein SpFtsZ with Uniprot primary accession number A0A0H2ZNE0-F1. The amino acid sequence was obtained from the UniProt server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and processed in the AlphaFold database to obtain the 3D model of the protein (45, 46). The structure was initially saved in an image format and then converted into a PDB format using PyMol for further analysis(47).\u003c/p\u003e\u003cp\u003e \u003cb\u003eMolecular docking experiment\u003c/b\u003e: As the standard procedure of AutoDock protocol, the PDBQT format files for the ligand, receptor, and configuration were saved for processing in AutoDock Vina (44, 48). A grid box with dimensions of 60 \u0026Aring; \u0026times; 60 \u0026Aring; \u0026times; 60 \u0026Aring;, with a grid spacing value of 1.0 \u0026Aring;, was used to accept the binding pocket of SpFtsZ protein. The grid centres were located at the following coordinates: x\u0026thinsp;=\u0026thinsp;6.231, y\u0026thinsp;=\u0026thinsp;0.723 and z\u0026thinsp;=\u0026thinsp;7.412.\u003c/p\u003e\u003cp\u003e \u003cb\u003eMolecular Dynamics Simulation\u003c/b\u003e: All atoms simulation were performed with the co-ordinates of protein, SpFtsZ (A0A0H2ZNE0-F1) and its docked complexes with ligands RR26, RR27, and RR33 employing GROMACS v2024.5(44, 49). The force field chosen as CHARMm36 and the water model TIP3P used to solvate each system in a cubic simulation box of dimension 80 \u0026Aring;\u003csup\u003e3\u003c/sup\u003e with a minimum distance of 1.0 nm between the protein surface and the box boundary (50). Charges on the systems were neutralized by adding the counterions (Na\u003csup\u003e+\u003c/sup\u003eCl\u003csup\u003e-\u003c/sup\u003e). The prepared systems were subjected to minimization process, till the convergence to approximately 1000 kJ mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e nm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to remove steric clashes and unfavorable contacts, opting the algorithms steepest descent followed by conjugate gradient. The equilibration phase done in two steps: NVT ensemble followed by NPT, each carried out for up to 1000 ps. The temperature (300 K) and pressure (1 bar) while the simulation maintained using the velocity-rescaling thermostat, and Parrinello\u0026ndash;Rahman barostat (51, 52). The long-range electrostatic interactions were treated using the Particle Mesh Ewald (PME) method with a cutoff of 1.2 nm, and the same cutoff distance was applied for van der Waals interactions (53). All covalent bonds involving hydrogen atoms were constrained using the LINCS algorithm, allowing a simulation time step of 2 fs (54). Following equilibration, production MD simulations were performed under periodic boundary conditions for 100 ns for each system(55). Trajectory analyses, including RMSD, radius of gyration (Rg), solvent-accessible surface area (SASA), hydrogen-bond occupancy, and free-energy landscape calculations, were conducted using standard GROMACS utilities (49, 56\u0026ndash;59).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e \u003cstrong\u003eMolecular docking analysis\u003c/strong\u003e \u003cp\u003eCheminformatics tools helps us to design the new target-based inhibitors (60). Molecular docking study provides a way to find out the lead potential inhibitors based on the binding interactions of receptor protein and inhibitors (61, 62). In this study, to perform the molecular docking analysis, we used AutoDock vina to validate the interaction of 50 novel benzimidazole-based hybrid molecules with SpFtsZ by using their binding energy of interaction. It was observed that most of the compounds fitted well into the binding cavity of SpFtsZ protein. For each compound, 8 distinct protein-ligand interaction conformations were generated and visually observed. Among all these compounds which were docked with SpFtsZ protein, three compounds (\u003cb\u003eRR26, RR27\u003c/b\u003e and \u003cb\u003eRR33\u003c/b\u003e) were found to strongly interact with the SpFtsZ protein with the binding affinities of -9.0, -9.1 and \u0026minus;\u0026thinsp;9.4 Kcal/mol, respectively.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eThe compound \u003cb\u003eRR26\u003c/b\u003e showed a binding affinity of -9.0 Kcal/mol with the SpFtsZ protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), it forms two conventional hydrogen bonds with the active-site residues ARG 144 and SER 110. The compound interacts with its halogen, fluorine, with GLU 140. The π-cation and π-anion bond formed at ARG 144, while the stacking interaction was observed with GLY 73 and π-alkyl formed with LEU 180 and LEU 184 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Compound \u003cb\u003eRR27\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), demonstrated three hydrogen-bond interactions with the active-site residues GLY 23, ASN 26, and SER 110. While ASN 167 residues interacted with a pi-donor hydrogen bond. Apart from this, it also forms one pi-sigma bond with LEU 184 and two pi-alkyl bonds with LEU 181 and ALA 187, the active residue (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Compound \u003cb\u003eRR33\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) that showed the best binding affinity\u0026thinsp;\u0026minus;\u0026thinsp;9.3 Kcal/mol as it forms five hydrogen bonds with GLY 23, GLY 73 and GLY74, ASN 26 and SER 110. LEU 184 involved in pi-sigma bond and alkyl bond was observed with LEU 180, LEU 181, LEU 184 and ALA 187 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Molecular docking analysis reveals that these inhibitors bind at the interdomain cleft of SpFtsZ. The interdomain cleft contains very crucial region of SpFtsZ which includes H7 helix and T7 loop. Protofilaments interaction which needs for FtsZ assembly and GTP hydrolysis requires for providing energy for FstZ assembly dynamics and remodeling (9, 29, 63). Similar to these inhibitors several FtsZ based inhibitors like PC190723, Zantrin Z3, benzamide derivates and berberine and its derivates are reported to binds with the interdomain cleft of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e FtsZ (SaFtsZ), \u003cem\u003eEscherichia coli\u003c/em\u003e FtsZ (EcFtsZ), SaFtsZ, respectively, which includes residues from the H7 helix and the T7 loop (64\u0026ndash;68). Similar to these known inhibitors, RR26, RR27 and RR33 have potential to target FtsZ assembly.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConformational stabilities of protein-ligand complexes\u003c/strong\u003e \u003cp\u003eTo examine the binding stability of ligands interacting with FtsZ, MD simulations were performed in water for the period of 100 ns at 300K. The simulations allowed assessment of the dynamic behavior and stability of the ligand\u0026ndash;SpFtsZ complexes at the atomic resolutions, providing insights into their conformational stability and interaction persistence under physiological conditions. The structural stability of the protein\u0026ndash;ligand complexes was evaluated using root-mean-square deviation (RMSD), radius of gyration (Rg), and solvent-accessible surface area (SASA) analyses and the average values during the simulations are enumerated in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAverage RMSD, radius of gyration (Rg), and solvent-accessible surface area (SASA) values for SpFtsZ and its complexes during the MD simulation\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSystem\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRMSD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRg\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSASA\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpFtsZ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 nm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 nm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e173.70\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36 nm\u0026sup2;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRR26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 nm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 nm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e172.84\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41 nm\u0026sup2;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRR27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 nm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 nm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e172.66\u0026thinsp;\u0026plusmn;\u0026thinsp;1.31 nm\u0026sup2;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRR33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 nm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 nm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e173.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25 nm\u0026sup2;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn addition, hydrogen bond (H-bond) interactions and ligand RMSD were examined to elucidate the spatial stability and binding persistence of ligands RR26, RR27, and RR33 within the SpFtsZ binding pocket. The time evolution plots of Cα-RMSD of FtsZ and FtsZ-docked complexes are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. In the Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, it can be seen that the structure of SpFtsZ optimized quickly around the average RMSD 0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 nm. Upon the binding of ligands, a slight agitation in RMSD was observed, reflecting minor conformational adjustments of the protein. The SpFtsZ\u0026ndash; RR26 complex stabilized at an average RMSD of 0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 nm, whereas SpFtsZ\u0026ndash; RR27 and SpFtsZ\u0026ndash; RR33 equilibrated around 0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 nm and 0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 nm, respectively. These results suggest that ligand binding induces moderate but stable conformational rearrangements in SpFtsZ without compromising the overall conformational stability of the protein. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows the radius of gyration (Rg), which measures protein structural compactness. The protein, SpFtsZ reached equilibrium at 1.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 nm, indicating a stable global fold during the simulation. In comparison, the ligand-bound complexes SpFtsZ\u0026ndash; RR26, SpFtsZ\u0026ndash; RR27, and SpFtsZ\u0026ndash; RR33 exhibited slightly lower and consistent Rg values of 1.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 nm, suggesting that the compactness of protein get increased upon binding with ligand molecule. The reduced Rg observed for the complexes implies that ligand interaction contributes to structural stabilization of SpFtsZ without inducing major conformational rearrangements. Similarly, the solvent-accessible surface area (SASA) analysis revealed marginal variation between SpFtsZ and the ligand-bound complexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). All systems stabilized around an average SASA value of 173\u0026thinsp;\u0026plusmn;\u0026thinsp;1.50 nm\u0026sup2;, indicating that ligand binding does not significantly alter the solvent exposure of the protein. The comparable SASA profiles across the systems suggest preservation of the overall surface topology and support the structural stability of SpFtsZ upon complex formation. Furthermore, the root mean square fluctuation (RMSF) of the protein backbone residues was calculated to evaluate the dynamic behavior and flexibility of individual residues during simulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The low RMSF values observed for residues belonging to stable secondary conformations of α-helices and β-sheets, and the higher RMSF values were primarily belonging to the N- and C-terminal regions and loops. The active site has been found to be present in the N-terminal globular domain that contain a central parallel β-sheet core, a flanking α-helices (H1\u0026ndash;H7) and T7 loop which is highly conserved across bacterial genera and is important for GTP-hydrolysis. The \u0026le;\u0026thinsp;1.0 \u0026Aring; average atomic fluctuation reflects structural stability of SpFtsZ is important for GTP hydrolysis. The RMSF analysis revealed reduced average fluctuations of active-site polar residues, GLY21, GLY22, GLY23, ASN26, ASP47, SER110, THR134, GLU140, ARG144, and ASP188 in the docked complexes indicate enhanced structural stabilization of SpFtsZ, upon ligand binding. These residues are crucial for catalytic activity and play a significant role in electrostatic interactions and hydrogen bonding, thereby contributing to the stability of the protein-ligand complexes. Similarly, the amino acid residues LEU180, LEU181, LEU184, ALA187, PHE137, VAL98, and MET106 belonging to hydrophobic cavity also shows stable RMSF. These residues appeared to form π\u0026ndash;π stacking, π\u0026ndash;alkyl and van der Waals interactions which helps for the stabilization of planer benzimidazole scaffold inside the binding cleft of SpFtsZ. The reduced flexibility of residues at the binding cleft of SpFtsZ indicates spatially stable protein-ligand interactions. The T7 loop (Gly73\u0026ndash;Ser110), which is highly conserved in tubulin and critically involved in GTP hydrolysis, also exhibited reduced flexibility in the docked complexes. These results indicate that the Benzimidazole analogs ligands were spatially well accommodated within the GTP binding domain of SpFtsZ while the simulation period.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eHydrogen bond interactions\u003c/strong\u003e \u003cp\u003eFurthermore, the spatial binding stability of ligands within the SpFtsZ binding pocket was evaluated by analyzing the occupancy of protein\u0026ndash;ligand hydrogen bonds (H-bonds) throughout the simulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The SpFtsZ\u0026ndash; RR26 complex exhibited the formation of three to four H-bonds during the initial\u0026thinsp;~\u0026thinsp;0\u0026ndash;15 ns, however, this interaction weakened to a single intermittent H-bond between ~\u0026thinsp;20\u0026ndash;80 ns, followed by the re-appearance of three H-bonds during the ~\u0026thinsp;80\u0026ndash;100 ns. Whereas the SpFtsZ\u0026ndash; RR27 complex displayed initially four to five H-bonds, among them four H-bonds observed consistently maintained throughout the entire simulation, indicating strong and persistent binding. Similarly, SpFtsZ\u0026ndash; RR33 showed four to five H-bonds during the initial\u0026thinsp;~\u0026thinsp;0\u0026ndash;20 ns, which stabilized to three persistent H-bonds for the remaining period of simulation. These results indicate the higher occupancy of hydrogen-bonding between the protein and ligands for SpFtsZ\u0026ndash; RR27 and SpFtsZ\u0026ndash; RR33 complexes contribute to greater binding stability within the SpFtsZ active site as compared to SpFtsZ\u0026ndash; RR26.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eFree-energy landscape\u003c/strong\u003e \u003cp\u003eIn addition, to investigate the conformational dynamics of SpFtsZ and docked complexes we also constructed the free-energy landscape (FEL) analysis, from the joint probability distribution of RMSD and Rg (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Figure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA shows that the SpFtsZ protein predominantly occupied to a well-defined and consolidated energy basin, indicating conformational stability and restricted structural fluctuations. Upon ligand binding, the protein explores a comparatively broader conformational space, reflecting ligand-induced modulation of its dynamic behavior. Notably, among the complexes, SpFtsZ\u0026ndash; RR26 and SpFtsZ\u0026ndash; RR33 display relatively shallow and dispersed energy minima, indicative of enhanced conformational flexibility and the presence of multiple metastable states (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). This behavior correlates with the fluctuating hydrogen-bond occupancies observed for these ligands and suggests comparatively weaker or less persistent stabilization of the binding pocket. Moreover, the SpFtsZ\u0026ndash; RR27 complex displays a well-defined and densely populated energy basin, indicative of a more stable and energetically favorable conformational ensemble (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). These observations further corroborate the molecular docking results, suggesting stable binding of ligands RR26, RR27, and RR33 with SpFtsZ, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eBinding free energy estimation\u003c/strong\u003e \u003cp\u003eFinally, to quantify the binding energetics of the selected benzimidazole derivatives in complex with SpFtsZ, the binding free energies were estimated using the MM-PBSA approach. The total binding free energy (ΔG\u003csub\u003ebind\u003c/sub\u003e) was decomposed into van der Waals (ΔE\u003csub\u003evdW\u003c/sub\u003e), electrostatic (ΔE\u003csub\u003eeel\u003c/sub\u003e), polar solvation (ΔG\u003csub\u003epol\u003c/sub\u003e), and non-polar solvation (ΔG\u003csub\u003enp\u003c/sub\u003e) components to elucidate the physicochemical determinants governing the spatial binding of benzimidazole derivatives at the SpFtsZ active site (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Results show that all three complexes exhibited favourable total binding free energies, however, RR27 exhibited higher ΔG\u003csub\u003ebind\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;39.88\u0026thinsp;\u0026plusmn;\u0026thinsp;3.60 kcal/mol, followed by RR33 (\u0026minus;\u0026thinsp;36.40\u0026thinsp;\u0026plusmn;\u0026thinsp;4.05 kcal/mol) and RR26 (\u0026minus;\u0026thinsp;28.18\u0026thinsp;\u0026plusmn;\u0026thinsp;3.60 kcal/mol), suggesting a comparatively stronger binding affinity of RR27 toward SpFtsZ.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe decomposition analysis of energetic terms reveals that binding affinity of compounds is predominantly driven by van der Waals interactions. RR27 displays the most favourable ΔE\u003csub\u003evdW\u003c/sub\u003e contribution\u0026thinsp;\u0026minus;\u0026thinsp;49.30\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33 kcal/mol, followed by RR33 (\u0026minus;\u0026thinsp;36.66\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25 kcal/mol) and RR26 (\u0026minus;\u0026thinsp;32.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73 kcal/mol), suggesting optimal hydrophobic packing and efficient shape complementarity within the binding pocket. Whereas the component, ΔE\u003csub\u003eeel\u003c/sub\u003e is observed strongly positive for all systems (RR26: 322.37\u0026thinsp;\u0026plusmn;\u0026thinsp;20.07; RR27: 283.95\u0026thinsp;\u0026plusmn;\u0026thinsp;4.11; RR33: 202.79\u0026thinsp;\u0026plusmn;\u0026thinsp;12.81 kcal/mol), indicating unfavourable gas-phase electrostatics. However, this effect is largely compensated by the polar solvation energy term, ΔG\u003csub\u003epol\u003c/sub\u003e which contributes negatively (RR26: \u0026minus;294.79\u0026thinsp;\u0026plusmn;\u0026thinsp;15.22; RR27: \u0026minus;241.13\u0026thinsp;\u0026plusmn;\u0026thinsp;4.47; RR33: \u0026minus;178.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59 kcal/mol), reflecting solvent stabilization of the charged states. It can be seen in residue-wise energy decomposition plots that crucial active site residues, GLU140, ARG144, ASP188 contribute substantially (\u0026gt;\u0026thinsp;20 kcal/mol) to both ΔE\u003csub\u003eeel\u003c/sub\u003e as well as ΔG\u003csub\u003epol\u003c/sub\u003e (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-3\u003c/b\u003e). This pattern reflects strong electrostatic interactions between these charged residues and the ligands, however, these contributions are largely offset by opposing polar solvation effects, resulting in a minimal net electrostatic contribution to the overall binding free energy.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe binding free energy (kcal/mol) estimation of compounds (RR26, RR27 and RR33) using MM-PBSA.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eComplex\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e∆E\u003csub\u003evdW\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e∆E\u003csub\u003eeel\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e∆G\u003csub\u003epol\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e∆G\u003csub\u003enp\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e∆G\u003csub\u003ebind\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRR26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e-32.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e322.37\u0026thinsp;\u0026plusmn;\u0026thinsp;20.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e-294.79\u0026thinsp;\u0026plusmn;\u0026thinsp;15.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e-23.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e-28.18\u0026thinsp;\u0026plusmn;\u0026thinsp;3.60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRR27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e-49.30\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e283.95\u0026thinsp;\u0026plusmn;\u0026thinsp;4.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e-241.13\u0026thinsp;\u0026plusmn;\u0026thinsp;4.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e-33.40\u0026thinsp;\u0026plusmn;\u0026thinsp;1.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e-39.88\u0026thinsp;\u0026plusmn;\u0026thinsp;3.60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRR33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e-36.66\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e202.79\u0026thinsp;\u0026plusmn;\u0026thinsp;12.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e-178.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e-24.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e-36.40\u0026thinsp;\u0026plusmn;\u0026thinsp;4.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFurthermore, the energetic term, ΔG\u003csub\u003enp\u003c/sub\u003e, which correlates with solvent-accessible surface burial, further stabilizes the complexes. Among the three compounds, RR27 exhibited the most favourable contribution of ΔG\u003csub\u003enp\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;33.40\u0026thinsp;\u0026plusmn;\u0026thinsp;1.62 kcal/mol as compared to RR33 (\u0026minus;\u0026thinsp;24.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49 kcal/mol) and RR26 (\u0026minus;\u0026thinsp;23.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34 kcal/mol) which observed consistent with the dominant role of hydrophobic interactions in stabilizing ligand binding within the interdomain cleft of SpFtsZ. Thus, the enhanced van der Waals and non-polar contributions outweigh the electrostatic penalty, resulting in the lowest overall ΔG\u003csub\u003ebind\u003c/sub\u003e for RR27. The compound RR33, while exhibiting weaker van der Waals interactions than RR27, benefits from a comparatively lower electrostatic penalty, yielding a competitive binding free energy. Whereas RR26 displays the least favourable binding, primarily due to weaker dispersion interactions and less favourable hydrophobic contributions. These findings are consistent with molecular docking and MD simulation results, which indicate stable ligand accommodation within the H7 helix\u0026ndash;T7 loop region of SpFtsZ. The superior energetic profile of RR27 further supports its identification as a promising lead candidate for subsequent experimental validation.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we employed a structure-based drug design strategy to develop benzimidazole derivatives as potential inhibitors targeting the polymerization dynamics of SpFtsZ. Using an integrated computational approach, including molecular docking, MD simulations, and MM-PBSA binding free energy calculations, we systematically designed and characterized a library of novel compounds. Out of 50 designed inhibitors, RR26, RR27, and RR33 exhibited the highest binding affinities toward SpFtsZ. Molecular docking analyses revealed stable ligand\u0026ndash;protein interactions, with binding localized at the interdomain cleft involving the H7 helix and T7 loop, a functionally critical region associated with protofilament formation and GTP hydrolysis. Furthermore, Molecular dynamics simulations, followed by MM-PBSA analysis, further substantiated the stability of all these complexes. Among these, RR27 exhibited the most favorable energetic profile, demonstrating superior stability and the lowest binding free energy, thereby identifying it as the most promising candidate among the three compounds. FtsZ is a GTP-dependent cytoskeletal protein that polymerizes upon GTP binding to form protofilaments and higher-order bundles, which assemble into the Z-ring required for septum formation during bacterial cell division. GTP hydrolysis plays a crucial role in regulating the dynamic turnover of FtsZ protofilaments; impairment of this process leads to hyperstabilization of FtsZ polymers, ultimately disrupting Z-ring constriction and inhibiting successful cell division.\u003c/p\u003e \u003cp\u003eAny aberrations in GTP hydrolysis leads to hamper the bacterial cytokinesis by over stabilizing the FtsZ protofilaments (12, 69\u0026ndash;71). The known inhibitors of FtsZ like PC190723, Zantrin Z3 and berberine binds to the interdomain cleft near T7 loop and known to inhibit the bacterial cell division by targeting FtsZ assembly (64\u0026ndash;68).\u003c/p\u003e \u003cp\u003eOur findings indicate that the binding of RR26, RR27, and RR33, preferentially localize within the interdomain cleft of SpFtsZ, in proximity to the H7 helix and T7 loop, could be a plausible shared mechanism of action. This interaction is likely to modulate GTP hydrolysis and perturb the intrinsic polymerization\u0026ndash;depolymerization dynamics of FtsZ, thereby compromising Z-ring assembly and ultimately impairing bacterial cytokinesis. In summary, this study provides strong evidence supporting the potential of benzimidazole derivatives as FtsZ-targeting inhibitors and underscores the utility of rational, structure-based drug design approaches in advancing therapeutic strategies against bacterial diseases.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eConflict of interest:\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eAKS and SR thanks Mahatma Gandhi Central University Motihari, Bihar for providing seed money under research promotion scheme.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAnisha Kumari : Methodology, Software, Formal analysis, Investigation, Visualization, Writing \u0026amp; Editing; Priyanka Kataria : Methodology, Investigation, Writing \u0026amp; Editing; Rajni Khan : Methodology, Software, Formal analysis, Investigation, Visualization, Reviewing and Editing; Pradeep Kumar Singh : Conceptualization, Methodology, Validation, Resources, Writing- Reviewing and Editing; Basant Narain Singh: Conceptualization, Methodology, Validation, Resources, Writing- Reviewing and Editing; Ankit Rai: Conceptualization, Methodology, Validation, Resources, Writing- Original draft preparation, Writing- Reviewing and Editing, and Supervision. Anil Kumar Singh: Conceptualization, Methodology, Validation, Resources, Writing- Original draft preparation, Writing- Reviewing and Editing, and Supervision. Amresh Prakash: Conceptualization, Methodology, Validation, Resources, Writing- Original draft preparation, Writing- Reviewing and Editing, and Supervision.; Shashikant Ray: Conceptualization, Methodology, Validation, Resources, Writing- Original draft preparation, Writing- Reviewing and Editing, and Supervision.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eAKS and SR thanks Mahatma Gandhi Central University Motihari, Bihar for providing seed money under research promotion scheme.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFauci AS, Morens DM. The perpetual challenge of infectious diseases. N Engl J Med. 2012;366(5):454 − 61.\u003c/li\u003e\n\u003cli\u003eAntimicrobial Resistance C. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. 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EMBO J. 1999;18(9):2352-63.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cytokinesis, Molecular Docking, antibacterial agents, Z-ring, FtsZ polymerization","lastPublishedDoi":"10.21203/rs.3.rs-9342737/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9342737/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe ongoing increase in bacterial antibiotic resistance, especially against \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e, is a global cause for concern and demands the exploration of new antibacterial agents. FtsZ, a prokaryotic homologue of tubulin, is essential for orchestrating cytokinesis in bacteria. Similar to tubulin, which is the core target of cancer chemotherapy, FtsZ provides a promising target to be explored for the development of a new class of antibiotics due to its pivotal role in cell division. Here, based on the structure-function relationship, we have designed a library of 50 novel benzimidazole inhibitors targeting \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e FtsZ (SpFtsZ). Molecular docking analysis identified three lead candidates: RR26, RR27, and RR33 as promising inhibitors against SpFtsZ, having binding affinities: -9.0, -9.1 and \u0026minus;\u0026thinsp;9.4 kcal/mol, respectively. The docking analysis revealed that these inhibitors occupy the interdomain cleft of SpFtsZ, engaging crucial H7 helix and T7 loop regions responsible for the protofilament interaction and GTP hydrolysis. Furthermore, the molecular dynamics (MD) simulation results suggest stable SpFtsZ-inhibitors interactions. The binding free estimation applying MM-PBSA further confirmed favourable spatial binding of inhibitors at the binding cavity of protein, yielding ∆G\u003csub\u003ebind\u003c/sub\u003e values of \u0026minus;\u0026thinsp;28.18\u0026thinsp;\u0026plusmn;\u0026thinsp;3.60 kcal/mol (RR26), \u0026minus;\u0026thinsp;39.88\u0026thinsp;\u0026plusmn;\u0026thinsp;3.60 kcal/mol (RR27), and \u0026minus;\u0026thinsp;36.40\u0026thinsp;\u0026plusmn;\u0026thinsp;4.05 kcal/mol (RR33). Notably, RR27 exhibited the lowest binding free energy, suggesting the strongest affinity and highest inhibitory potential among the three compounds. Thus, this study provides a rational basis for the design of SpFtsZ-targeted inhibitors and identifies lead compounds with significant potential for subsequent \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies in the future.\u003c/p\u003e","manuscriptTitle":"Structure based design of Benzimidazole derivative inhibitors targeting Streptococcus pneumoniae FtsZ: an integrated computational framework","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-05 18:21:24","doi":"10.21203/rs.3.rs-9342737/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2aa73ddf-8341-4a96-8b18-e26d29b9e234","owner":[],"postedDate":"May 5th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-05T18:21:25+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-05 18:21:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9342737","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9342737","identity":"rs-9342737","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}