Elucidating the Isorhamnetin-3-O-glucoside-iNOS Interaction via Molecular Dynamics and Hirshfeld Surface Analyses

Elucidating the Isorhamnetin-3-O-glucoside-iNOS Interaction via Molecular Dynamics and Hirshfeld Surface Analyses | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Elucidating the Isorhamnetin-3-O-glucoside-iNOS Interaction via Molecular Dynamics and Hirshfeld Surface Analyses oussama khibech, Haytham Bouammali, Yousra Hammouti, Mohamed bouhrim, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7400020/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 Inducible nitric oxide synthase (iNOS) overproduction drives chronic inflammation and oncogenic signaling, yet selective small-molecule modulation remains elusive. We interrogated isorhamnetin-3-O-glucoside (I3OG), a dietary flavonol glycoside, against murine (3E6T) and human (3E7G) iNOS oxygenase domains using a validated in silico pipeline spanning redocking, explicit-solvent molecular dynamics (100 ns), and MM/GBSA free-energy analysis. Redocking reproduced co-crystal poses (RMSD 1.21/1.25 Å), and Vina ranked I3OG favorably (-10.1/-9.7 kcal·mol⁻¹). MD revealed confined ligand motions and intact protein compaction; 3E6T displayed tighter RMSD variability and a denser hydrogen-bond network (≈4-6 persistent bonds) than 3E7G (≈1-3), with damped local flexibility around the pocket. MM/GBSA from equilibrated frames yielded ΔGbind = -44.9 ± 3.9 kcal·mol⁻¹ (3E6T) versus -36.1 ± 3.7 kcal·mol⁻¹ (3E7G), driven by favorable gas-phase van der Waals/electrostatics that outweigh polar desolvation. Hot-spot residues (3E6T: Trp188, Cys194, Trp366, Phe363; 3E7G: Trp194, Phe369, Trp372) rationalize species-dependent stabilization. Complementary Hirshfeld analysis of I3OG crystals highlights dominant O···H/H···O contacts and ancillary π-stacking, mirroring solution-phase recognition. Collectively, I3OG emerges as a mechanism-aware, tractable scaffold for iNOS attenuation, with the murine domain offering a more avid binding environment and a quantitative benchmark for future selective inhibitor design. Biological sciences/Biochemistry Biological sciences/Biophysics Biological sciences/Chemical biology Physical sciences/Chemistry Biological sciences/Computational biology and bioinformatics Biological sciences/Drug discovery Hirshfeld surface analysis iNOS I3OG MM/GBSA MD Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 1. Introduction Inducible nitric oxide synthase (iNOS) is a central enzymatic source of high-output nitric oxide (NO) in innate and adaptive immunity, where dysregulated NO contributes to chronic inflammation, metabolic and cardiovascular disorders, and cancer 1 – 4 . Therapeutic modulation of iNOS remains attractive yet challenging because selectivity and on-target efficacy must be reconciled with complex redox biochemistry and multi-domain conformational control. Structural and biochemical studies have mapped the oxygenase domain, heme-pterin chemistry, and dimerization interfaces that govern activity, offering tractable footholds for ligand design and mechanism-guided inhibition 5 . Natural products especially dietary flavonoids have long been recognized for anti-inflammatory actions that include dampening NF-κB signaling and suppressing COX-2 and iNOS expression 6 – 8 . Within this class, isorhamnetin and its glycosides (notably isorhamnetin-3-O-glucoside, I3OG) show antioxidant and anti-inflammatory effects and reduce NO overproduction in cellular models, making them compelling chemical probes for iNOS modulation 9 – 11 . Recent surveys of isorhamnetin glycosides underscore their prevalence, pharmacology, and relevance to human health, while broader analyses emphasize the enduring role of natural products as leads for drug discovery 12 . Computational pipelines that combine carefully validated docking with explicit-solvent molecular dynamics (MD) and end-point binding free-energy calculations have become standard to interrogate protein-ligand recognition at atomistic resolution. Docking engines such as AutoDock Vina guide pose generation and enrichment 13 , 14 , and learning-augmented rescoring further improves pose quality while retaining the classical RMSD ≤ 2.0 Å benchmark for pose fidelity 15 , 16 . Production MD implemented in modern GPU-accelerated codes captures conformational adaptation, ligand stability, and the time-dependent behavior of key observables (RMSD, RMSF, hydrogen bonds, and radius of gyration) 17 – 21 . Post-processing with MM/GBSA consolidates nonbonded interactions and solvation to estimate relative binding affinities and helps prioritize poses consistent with the dynamics 22 , 23 . Crucially, experimental crystal-packing analyses complement solution-phase simulations by mapping short-range contacts, π-stacking, and hydrogen-bonding motifs that underlie solid-state stability. Hirshfeld surface analysis and fingerprint plotting (as implemented in CrystalExplorer) provide a quantitative picture of intermolecular interactions that can be related to recognition patterns seen in complexes 24 , 25 . In this context, we investigate I3OG against iNOS oxygenase domains represented by the mouse (PDB 3E6T) and human (PDB 3E7G) structures 5 , 26 . We pair validated docking with long-timescale MD to monitor structural stability (RMSD), residue-level flexibility (RMSF), hydrogen-bond persistence, and global compaction (Rg), and we quantify binding via MM/GBSA using extensive trajectory snapshots to reduce statistical noise. Our structural choices leverage high-resolution templates and contemporary simulation protocols to minimize methodological bias and to ensure that free-energy trends are grounded in physically realistic dynamics. iNOS is a validated yet challenging target in chronic inflammation and oncology, so robust, mechanism-aware leads are needed. We focus on I3OG because flavonol glycosides combine a favorable safety profile, tractability, and anti-inflammatory potential. Our objectives are to define stable binding modes of I3OG in murine and human iNOS oxygenase domains, quantify their relative binding free energies with MM/GBSA using dense sampling, and relate dynamic observables (RMSD, RMSF, hydrogen bonds, Rg) to energetic trends to strengthen biological plausibility. We also integrate crystal-packing insights (Hirshfeld) to cross-validate interaction motifs across solution and solid phases. 2. Materials and Methods 2.1. Hirshfeld Surface Analysis with CrystalExplorer In this study, we limited the Hirshfeld surface analysis to our crystallized compound only, Isorhamnetin-3-O-glucoside (I3OG). CrystalExplorer 21.5 was applied to the compound’s CIF file to compute the descriptors d norm , d i , d e , Shape Index, and Curvedness, and to generate 2D fingerprint plots, allowing us to identify close contacts within the crystal packing and quantify the contributions of H···H, O···H/H···O, C···H/H···C interactions 24 . This crystallographic analysis complements our solution-phase results (MD/MM-GBSA), providing a coherent, multiscale picture of interaction patterns and system stability. 2.2. AutoDock Vina Molecular docking was carried out with AutoDock Vina (via the ADT 1.5.7 interface) 27 . Protein structures (PDB IDs 3E6T and 3E7G) were preprocessed by removing crystallographic waters, adding polar hydrogens, and assigning charges following the AutoDock recommendations; Discovery Studio was used for inspection and minor corrections before and after docking. Ligands were sketched and geometry-optimized in ChemDraw, then converted to PDBQT with ADT to define torsions and charges. For the search space, protein-specific grid boxes were used: for 3E6T, the box was centered at (124.31, 111.55, 30.43) with dimensions 15.87 × 14.40 × 14.12 Å; for 3E7G, the box was centered at (56.96, 20.24, 84.71) with dimensions 13.10 × 15.74 × 14.55 Å. Vina parameters were exhaustiveness = 8, num_modes = 8, and energy_range = 4; all other settings were left at defaults. Poses were ranked by Vina affinity and analyzed in ADT and Discovery Studio to characterize H-bonds and hydrophobic contacts. Protocol validation was performed by redocking the co-crystallized ligand into each receptor using the same settings. The best redocked pose reproduced the experimental conformation with RMSD = 1.210 Å (3E6T ) and RMSD = 1.253 Å (3E7G) both well below the 2.0 Å acceptance threshold confirming the reliability of the docking setup (RMSD computed in PyMOL) (Fig. 1 ). 2.3. Implementation of Molecular Dynamics Simulations Using GROMACS Molecular-dynamics simulations were performed with GROMACS 2021.3 28 . Using gmx pdb2gmx with the AMBER99SB-ILDN force field, missing protein hydrogens were added and protonation states adjusted. The ligand was parameterised separately; the validated .itp and .gro files were then merged with the protein to build the full complex. This complex was centred in a cubic TIP3P water box, neutralised with counter-ions, and relaxed via steepest-descent energy minimisation. Sequential NVT and NPT equilibration phases stabilised temperature and pressure, respectively. Finally, a 100 ns production run was conducted, saving coordinates and velocities at regular intervals to characterise stability, conformational dynamics, and key intermolecular interactions under near-physiological conditions. 2.4. MM/GBSA Calculation Binding free energies were calculated with AmberTools23 (MMPBSA.py, parallel mode) using 100 snapshots sampled every 0.4 ns from the 60–100 ns window of each GROMACS trajectory. The HCT generalized-Born model (igb = 5) was applied with dielectric constants ε_in = 1.0 and ε_out = 80.0 and an ionic strength of 0.15 M. The nonpolar solvation term was estimated from the solvent-accessible surface area. Temporary files were not retained (keep_files = 0). Per-residue decomposition (idecomp = 1) was enabled to resolve van der Waals, electrostatic, polar, and nonpolar contributions for each residue. 3.1. HirshfeldSurface 3.3.1. Hirshfeld Surface Metrics and Lattice-Water-Mediated Crystal Packing of I3OG Hirshfeld surface analysis provides a standardized way to connect the crystal structure to the nature of intermolecular contacts: it highlights short contacts on the d norm map, describes surface topography via shape index and curvedness, and supplies concise morphological descriptors (surface volume, area, globularity, asphericity) that help rationalize crystal packing and interaction diversity. For isorhamnetin-3-O-glucoside, the calculations yielded a volume of 514.30 ų and a surface area of 440.60 Ų, reflecting a relatively large molecular envelope for an aryl glycoside and indicating ample regions available for intermolecular contact 29 . A globularity of 0.705 (where 1 denotes a perfect sphere) points to a moderately globular shape neither rod-like nor flat so contacts are expected to be distributed over the surface rather than concentrated at one end. The asphericity of 0.114 (zero for a perfect sphere) is low, indicating only a modest departure from sphericity with slight elongation along the aromatic core and sugar 30 . Taken together, these descriptors suggest that the I3OG surface is well suited to form a mixed network of polar (H···O/O···H) and hydrophobic contacts, consistent with the picture to be refined by detailed Hirshfeld maps and the docking results. The Fig. 2 shows a hydrogen-bond bridge (green) linking neighboring isorhamnetin-3-O-glucoside molecules, mediated by lattice water. Dashed contacts indicate O-H···O interactions between ligand oxygen donors/acceptors that connect adjacent molecules. These intermolecular H-bonds help stabilize the crystal packing and anticipate strong O···H/H···O contributions in the Hirshfeld analysis. 3.3.2. Intermolecular Contacts in I3OG Crystals Revealed by Hirshfeld Analysis To contextualize the crystal packing of I3OG, we first computed and visualized its Hirshfeld surface. Table 1 quantifies the surface metrics and contact percentages, while Fig. 2 displays the corresponding maps (d i , dₑ, d norm , fragment patches, shape index, and curvedness). As summarized in Table 1 , the Hirshfeld metrics reveal pronounced close contacts: the d_norm minimum − 0.746 (with d i /d e minima ≈ 0.64/0.70 Å) flags red hotspots shorter than the vdW sum, while the shape index range (~-1 to + 1) and the low mean curvedness (-1.02) indicate extended, relatively flat patches suitable for π-stacking alongside H-bonding sites 30 . The fingerprint decomposition in Table 1 shows H···H = 27.7%, but the dominant directional interactions are O···H/H···O = 42.5% (24.8% + 17.7%), consistent with a strong hydrogen-bond network; hydrophobic stabilization is moderate via C···H/H···C = 12.3%, with minor C···C (5.9%) and C···O/O···C (≈ 6.5%) contributions. Table 1 Hirshfeld surface metrics and fingerprint contact contributions (%) for I3OG. Interaction Mode Minimum Mean Maximum d norm -0.7461 0.4408 1.5878 d i 0.6392 1.6778 2.4636 d e 0.7011 1.6897 2.5084 Shape Index (SI) -0.9925 0.1724 0.9996 Curvedness (Cr) -4.0100 -1.0181 0.3573 Fingerprint% via total surface area for closed contact between atoms inside and outside the surface Outside Atom% Inside Atoms C H O C 5.9 6.6 3.4 H 5.7 27.7 24.8 O 3.1 17.7 5.1 Figure 3 compiles six complementary Hirshfeld surfaces for I3OG. The d i and dₑ maps locate the nearest internal and external neighbors; localized blue/cyan patches around phenolic and sugar O-H groups mark the shortest approaches. The d norm surface gathers these into red hotspots contacts shorter than the vdW sum identifying the main H-bond nodes that knit adjacent molecules 31 . The FP (fragment patches) view segments the surface by contact type and visually confirms the dominance of O···H/H···O and H···H interactions. The shape index shows complementary red/blue triangular motifs on the flavonoid faces, diagnostic of π-π stacking, while the predominantly green curvedness map with limited blue ridges indicates extended flat regions separated by edges. Together, the six maps depict a surface that blends directional hydrogen bonding with aromatic stacking and moderate hydrophobic contacts, consistent with the packing features inferred from the quantitative metrics. Figure 4 presents Hirshfeld fingerprint plots resolved by atom identity (inside vs. outside). Inside the surface, H atoms dominate the contacts (58.2%), followed by O (25.9%) and C (16.0%); for outside atoms the shares are H 52.0%, O 33.3%, and C 14.7%. The sharp spikes at low d i /d e in the O(in)/H(out) and H(in)/O(out) maps diagnose directional O···H/H···O hydrogen bonds as the principal motif, in line with Table 1 and the red hotspots on d norm . The broader wings in the H-resolved maps indicate numerous H···H contacts, whereas the smaller carbon fractions point to a secondary hydrophobic component (C···H/H···C) rather than dominant face-to-face π-π stacking. Overall, Fig. 4 shows a hydrogen-bond-led packing network complemented by moderate hydrophobic interactions. 3.2. Molecular docking Molecular docking offers a mechanistic bridge between chemical structure and target modulation by predicting low-energy poses and the noncovalent interaction network within a protein active site. For Isorhamnetin-3-O-glucoside (I3OG) a polyphenolic glycoside with multiple H-bond donors/acceptors and an extended π system docking is particularly informative because it tests whether the flavonoid core and sugar hydroxyls can cooperatively engage the polar access channel and the heme-proximal pocket of inducible nitric-oxide synthase (iNOS). We selected the oxygenase domain from mouse (PDB 3E6T, 2.50 Å) and human (PDB 3E7G, 2.20 Å) to capture species-conserved recognition features and ensure translational relevance 5 , 26 ; both entries contain the catalytic heme prosthetic group and co-bound reference inhibitors, which permits grid definition around the native ligand and preservation of the correct Fe-heme environment. Pharmacologically, iNOS inhibition is a validated anti-inflammatory strategy because pathological NO overproduction drives oxidative stress and tissue injury; thus, evaluating I3OG against these two high-quality structures tests whether a natural antioxidant scaffold can sterically and electronically complement the iNOS active site. As shown in Fig. 5 , panel A (3E6T) and panel B (3E7G) depict Isorhamnetin-3-O-glucoside (red) occupying a well-defined binding pocket (grey) on the protein surface (blue). In both structures, the flavonoid core sits deeper along a hydrophobic corridor, while the glucose moiety projects toward the pocket rim where polar donors/acceptors enable plausible hydrogen bonds, yielding minor pose differences but a conserved recognition pattern across mouse and human iNOS oxygenase domains. To account for the docking scores (-10.1 kcal/mol with 3E6T and − 9.7 kcal/mol with 3E7G), Figs. 6 and 7 reveal a coherent interaction network that explains pose stability within the iNOS oxygenase pocket. In Fig. 6 (3E6T), three conventional hydrogen bonds with Gly365, Asn364, and Thr184 anchor the ligand’s orientation in the polar channel and cooperate with four π-π stacking contacts two with Trp188 and two with Phe363 to bury the aromatic core and reinforce shape complementarity 32 , 33 ; these are complemented by five alkyl/π-alkyl contacts, providing hydrophobic support that lowers the desolvation cost. In Fig. 7 (3E7G), the ligand retains three conventional hydrogen bonds (two with Trp372 and one with Cys200), together with three π-π stacking interactions involving Phe369, Asn370, and Trp194, two π-σ contacts with Gly202 and Gly371, and a π-sulfur contact with Met434; six additional alkyl/π-alkyl contacts further tighten hydrophobic burial 34 , 35 . This balance of hydrogen bonding, aromatic stacking, and hydrophobic contacts across both structures explains the slight advantage for 3E6T and supports realistic shape-electrostatic complementarity for the ligand, motivating follow-up explicit-solvent MD and MM/GBSA to verify pose stability and estimate binding free energy. 3.3. Molecular Dynamics Simulation Molecular dynamics (MD) is the critical stress-test that turns a static docking pose into a time-resolved, solvent-aware assessment of binding stability. Across a 100-ns trajectory, four standard readouts provide complementary evidence: the ligand RMSD (pose persistence relative to the protein frame), the protein backbone RMSF (local flexibility of residues surrounding the pocket), the time-series of hydrogen bonds (polar anchoring and persistence), and the protein’s radius of gyration, Rg (global compactness). Together they verify whether the docked pose for I3OG, is both kinetically stable and structurally sensible before proceeding to MM/GBSA 36 . Figure 8 (ligand RMSD after least-squares fit to the protein) shows that I3OG remains well confined in both complexes. The 3E6T system (red) stabilizes rapidly within a narrow band of ~ 0.17–0.22 nm, whereas 3E7G (black) plateaus around ~ 0.07–0.17 nm but with a broader spread and occasional short-lived excursions toward ~ 0.20 nm 37 . The absence of long-term drift in either trace indicates stable poses; however, the tighter RMSD fluctuations in 3E6T despite its slightly higher mean RMSD than 3E7G point to a more consistently anchored ligand. In short, lower RMSD variability (not just a lower mean) signals greater target stability, so I3OG appears more stable with 3E6T. Figure 9 (backbone RMSF) shows low baseline fluctuations (~ 0.05–0.15 nm) across most residues for both proteins, consistent with a rigid catalytic core; 3E6T exhibits an isolated spike at the N-terminus and a modest rise near the C-terminus typical for solvent-exposed termini whereas 3E7G displays higher and more frequent loop mobilities (several peaks ~ 0.25–0.35 nm), suggesting a slightly softer pocket environment around the ligand in the human isoform 38 . In Fig. 10 , the hydrogen-bond trace reveals a clear difference in polar retention: I3OG forms a persistent network of ~ 4–6 H-bonds in 3E6T (frequent bursts to 7–8), versus a thinner, mostly 1–3 H-bond pattern in 3E7G; in parallel, Rg remains flat in both trajectories (≈ 2.23–2.29 nm) with an early, gentle compaction in 3E6T, confirming the complexes maintain global structural integrity without unfolding. Overall, MD corroborates the docking results for I3OG in both systems and favors 3E6T as the more stable complex, supported by lower ligand RMSD, damped backbone fluctuations near the binding site, a denser H-bonding network, and slightly greater compactness features that foreshadow a more favorable MM/GBSA binding free energy for the mouse iNOS oxygenase domain. 3.4. MM/GBSA Free-Energy Analysis MM/GBSA complements docking and MD by estimating the binding free energy from equilibrated MD frames while decomposing the driving forces into van der Waals and electrostatics in the gas phase (ΔG gas ) and the polar/non-polar solvation response (ΔG solv = ΔE GB + ΔE surf ). Using 100 snapshots extracted from the 60–100 ns window to ensure well-equilibrated sampling, Fig. 11 shows that both complexes are favorable in water (negative ΔG bind ). For I3OG-3E6T, the interaction is strongly driven by gas-phase terms (ΔE vdW ≈ -50.5 kcal/mol and ΔE elec ≈ -62.2 kcal/mol; ΔG gas ≈ -112.7), partly offset by a polar desolvation penalty (ΔE GB ≈ + 74.6) with a small non-polar gain (ΔE surf ≈ -6.8), yielding ΔG bind ≈ -44.9 ± 3.9 kcal/mol (SEM ≈ 0.44) 39 . For I3OG-3E7G, the pattern is similar but weaker electrostatics (ΔE vdW ≈ -54.2; ΔE elec ≈ -19.7; ΔG gas ≈ -73.9) and a smaller polar penalty (ΔE GB ≈ + 44.2; ΔE surf ≈ -6.4) lead to ΔG bind ≈ -36.1 ± 3.7 kcal/mol (SEM ≈ 0.31). Thus, both proteins stabilize I3OG, but 3E6T is favored by ~ 9 kcal/mol because its stronger gas-phase attraction especially electrostatics more than compensates its larger desolvation cost. This energetic profile is fully consistent with the MD readouts (lower ligand RMSD and denser H-bonding in 3E6T), reinforcing the conclusion that I3OG binds more stably to 3E6T. Figure 12 presents the residue-wise MM/GBSA decomposition from 100 snapshots taken between 60–100 ns, mapping the “hot spots” that anchor I3OG in the active site; residues with per-residue contributions < -1 kcal/mol are considered stabilizing. In I3OG-3E6T, Trp188, Cys194, Trp366, and Phe363 show < -2 kcal/mol and thus act as the primary anchors fully consistent with the docking contacts and additional pocket residues just below − 1 kcal/mol form a hydrophobic belt that helps keep the ligand seated. In I3OG-3E7G, three key residues dominate (Trp194, Phe369, Trp372), each < -2 kcal/mol; notably, Trp372 was flagged in docking for two conventional hydrogen bonds, which agrees with the MD hydrogen-bond trace (Fig. 10) where two H-bonds persist from ~ 60 ns to the end of the run. Overall, Fig. 12 confirms that the ligand is well stabilized in the active site of both proteins, with deeper hot spots in 3E6T, in line with its more favorable ΔG bind and MD stability readouts. Figure 13 is a time-resolved heat map of the per-residue MM/GBSA interaction energies over the 60-100 ns window (blue = favorable/negative; pale = weak or transient). In I3OG–3E6T, the darkest, most continuous bands sit on Phe363 (dominant hotspot) and Trp366, with persistent contributions from Trp188 and Cys194 exactly the anchors highlighted by Figure 12 while loop residues show intermittent, lighter stripes indicative of fleeting contacts. In I3OG-3E7G, Trp372 is the chief hotspot, followed by Phe369 and Trp194; the sustained dark-blue signal on Trp372 agrees with docking (two conventional H-bonds) and with the MD H-bond trace in Figure 10 , where two hydrogen bonds persist from ~60 ns to the end. The bottom LIG (I3OG) row remains strongly negative throughout in both panels, confirming a stable net attraction. Overall, Figure 13 corroborates Figure 12 : the key hotspots stay engaged across the trajectory, with more continuous strong contacts in 3E6T, consistent with its more favorable ΔG bind . Conclusion This work identifies isorhamnetin-3-O-glucoside (I3OG) as a credible, mechanism-aligned scaffold for attenuating iNOS activity. Consistent docking, restrained ligand dynamics, and favorable MM/GBSA estimates converge to support stable engagement of the iNOS oxygenase domain, with stronger stabilization in the murine (3E6T) than the human (3E7G) construct and clear residue-level hot spots that rationalize this species dependence. These results argue that I3OG and closely related chromone/flavonol chemotypes merit progression beyond in silico screening. Immediate priorities are orthogonal biophysical validation (SPR/ITC), enzyme inhibition and NO-suppression assays in relevant macrophage models, and selectivity profiling against eNOS/nNOS. Given the permeability liabilities of glycosides, medicinal chemistry should explore aglycone analogs, sugar bioisosteres, and prodrug strategies while preserving the π-stacking and hydrogen-bonding motifs highlighted here. Finally, longer simulations and models incorporating full cofactor/dimer contexts will refine translatability to the human enzyme. Collectively, our data provide a quantitative blueprint for structure-guided iNOS inhibitor design and a tractable starting point for anti-inflammatory lead optimization. Abbreviations RMSD Root Mean Square Deviation RMSF Root Mean Square Fluctuation iNOS Inducible nitric oxide synthase ADME Absorption, Distribution, Metabolism, and Excretion I3OG Isorhamnetin-3-O-Glucoside Declarations Author Contributions: O, K; H, B; Y, H; M, B; M, M Methodology and Writing original draft. S, A; B,B ;A, C Supervision and Project administration. O, K Docking and dynamic simulation. O, K; H,B; Y,H; M, B; M,M; S,A; M, AL z; F A.N; A, A.Q; B,B; A, C Writing review and editing. Funding: Biology Department, College of Science, Imam Mohammad Ibn Saud Islamic University (IMSIU) (grant number IMSIU-DDRSP2501), Riyadh, 11623, Saudi Arabia. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: All datasets generated and/or analyzed in this study are publicly available at the open-access repository https://github.com/khibech/I3OG-to-iNOS. The repository includes: the crystallographic .CIF file of the compound together with the CrystalExplorer project (.cxp) and output files; AutoDock Vina input/output files for both targets (3E6T and 3E7G); GROMACS system/parameter files (.mdp, .itp, .gro); and MM/GBSA input and result files. No additional data are required to support the findings of this work. Acknowledgements: This work was supported and funded by the Deanship of Scientific Research at Imam Mohammad Ibn Saud Islamic University (IMSIU) (grant number IMSIU-DDRSP2501). We gratefully acknowledge the HPC Marwan team for granting us privileged access to their high-performance computing resources. 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Theory Comput. 8 , 3314–3321 (2012). Rahimi, A. et al. ADMET-Guided Docking and GROMACS Molecular Dynamics of Ziziphus lotus Phytochemicals Uncover Mutation-Agnostic Allosteric Stabilisers of the KRAS Switch-I / II Groove. 1–20 (2025). Spackman, P. R. et al. CrystalExplorer: a program for Hirshfeld surface analysis, visualization and quantitative analysis of molecular crystals. Appl. Crystallogr. 54 , 1006–1011 (2021). Spackman, M. A. & Jayatilaka, D. Hirshfeld surface analysis. CrystEngComm 11 , 19–32 (2009). Garcin, E. D. et al. HHS Public Access. 4 , 700–707 (2010). Trott, O. & Olson, A. J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 31 , 455–461 (2010). Abraham, M. J. et al. Gromacs: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1 – 2 , 19–25 (2015). Zubir, M. Z. M., Jamaludin, N. S. & Halim, S. N. A. Hirshfeld surface analysis of some new heteroleptic Copper (I) complexes. J. Mol. Struct. 1193 , 141–150 (2019). Ramalingam, A. Hirshfeld surface analysis, interaction energy calculation and spectroscopical study of 3-chloro-3-methyl-r(2),c(6)-bis(p-tolyl)piperidin-4-one using DFT approaches. J. Mol. Struct. 1248 , 131483 (2022). Nbili, W. et al. X-ray diffraction, structural analysis, quantum reactivity studies, molecular interactions, spectroscopic insights and antiepileptic activities of [Cu(C7N4H8O2)2(NO3)(H2O)2]NO3 hybrid material by molecular docking. J. Mol. Struct. 1307 , (2024). Merzouki, M., Khibech, O., Bouammali, H., Farh, L. El & Bouammali, B. Chromone – Thiophene Hybrids as Non-Peptidic Inhibitors of SARS-CoV-2 Mpro : Integrated ADME , Docking , and Molecular Dynamics Approach. 16 , 1–10 (2025). Et-Tazy, L. et al. Effects of Monoterpene-Based Biostimulants on Chickpea (Cicer arietinum L.) Plants: Functional and Molecular Insights. Biology (Basel). 14 , 657 (2025). Amala, M., Rajamanikandan, S., Prabhu, D., Surekha, K. & Jeyakanthan, J. Identification of anti-filarial leads against aspartate semialdehyde dehydrogenase of Wolbachia endosymbiont of Brugia malayi: combined molecular docking and molecular dynamics approaches. J. Biomol. Struct. Dyn. 37 , 394–410 (2019). Touam, S., Nacer, H., Ziani, N., Lebrazi, S. & Kertiou, N. Prediction of Surface Tension of Propane Derivatives Using QSPR Approach and Artificial Neural Networks Prediction of Surface Tension of Propane Derivatives Using QSPR Approach and Artificial Neural Networks. (2025) doi:10.48317/IMIST.PRSM/morjchem-v13i2.54664. Camargo, P. G. et al. In silico evaluation of N-aryl-1,10-phenanthroline-2-amines as potential inhibitors of T. cruzi GP63 zinc-metalloprotease by docking and molecular dynamics simulations. Sci. Rep. 15 , 6036 (2025). Ahmed, B., Khan, S., Nouroz, F., Farooq, U. & Khalid, S. Exploring multi-target inhibitors using in silico approach targeting cell cycle dysregulator–CDK proteins. J. Biomol. Struct. Dyn. 40 , 8825–8839 (2022). Imran, M. et al. Exploring therapeutic paradigm focusing on genes, proteins, and pathways to combat leprosy and tuberculosis: A network medicine and drug repurposing approach. J. Infect. Public Health 18 , 102763 (2025). Wang, E. et al. End-Point Binding Free Energy Calculation with MM/PBSA and MM/GBSA: Strategies and Applications in Drug Design. Chem. Rev. 119 , 9478–9508 (2019). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-7400020","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":509329686,"identity":"5e2daad6-96d4-4f2b-a470-1af5dd7bf14e","order_by":0,"name":"oussama khibech","email":"data:image/png;base64,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","orcid":"","institution":"University Mohammed Premier, Faculty of Science, Laboratory of Applied Chemistry and Environment","correspondingAuthor":true,"prefix":"","firstName":"oussama","middleName":"","lastName":"khibech","suffix":""},{"id":509329687,"identity":"16ceba59-170f-4f82-8f17-58cd5fe9c506","order_by":1,"name":"Haytham Bouammali","email":"","orcid":"","institution":"University Mohammed Premier, Faculty of Science, Laboratory of Applied Chemistry and Environment","correspondingAuthor":false,"prefix":"","firstName":"Haytham","middleName":"","lastName":"Bouammali","suffix":""},{"id":509329688,"identity":"485eac46-f479-461f-bfd2-1c9afe0fe3df","order_by":2,"name":"Yousra Hammouti","email":"","orcid":"","institution":"Université Mohammed Premier","correspondingAuthor":false,"prefix":"","firstName":"Yousra","middleName":"","lastName":"Hammouti","suffix":""},{"id":509329689,"identity":"b4bdb713-75e8-4e77-b56a-8b6604c592a3","order_by":3,"name":"Mohamed bouhrim","email":"","orcid":"","institution":"Laboratoires TBC, Laboratory of Pharmacology, Pharmacokinetics, and Clinical Pharmacy, Faculty of Pharmaceutical and Biological Sciences, Lille, France","correspondingAuthor":false,"prefix":"","firstName":"Mohamed","middleName":"","lastName":"bouhrim","suffix":""},{"id":509329690,"identity":"ad47715a-176f-4203-9d90-70907cd80acb","order_by":4,"name":"Mohammed Merzouki","email":"","orcid":"","institution":"University Mohammed Premier, Faculty of Science, Laboratory of Applied Chemistry and Environment","correspondingAuthor":false,"prefix":"","firstName":"Mohammed","middleName":"","lastName":"Merzouki","suffix":""},{"id":509329691,"identity":"b0ba78f6-13c3-4de9-ac2d-f1b9172268cd","order_by":5,"name":"Said Abadi","email":"","orcid":"","institution":"University Mohammed Premier, Faculty of Science, Laboratory of Applied Chemistry and Environment","correspondingAuthor":false,"prefix":"","firstName":"Said","middleName":"","lastName":"Abadi","suffix":""},{"id":509329693,"identity":"fbd7d6be-d9ef-436a-a9b6-9aa00f1caabd","order_by":6,"name":"Mohammed Al-zahrani","email":"","orcid":"","institution":"4 Biology Department, College of Science, Imam Mohammad Ibn Saud Islamic University (IMSIU), Riyadh, 11623, Saudi Arabia","correspondingAuthor":false,"prefix":"","firstName":"Mohammed","middleName":"","lastName":"Al-zahrani","suffix":""},{"id":509329695,"identity":"74495f87-ccf7-4eb5-a827-3db28388b496","order_by":7,"name":"Fahd A. Nasr","email":"","orcid":"","institution":"4 Biology Department, College of Science, Imam Mohammad Ibn Saud Islamic University (IMSIU), Riyadh, 11623, Saudi Arabia","correspondingAuthor":false,"prefix":"","firstName":"Fahd","middleName":"A.","lastName":"Nasr","suffix":""},{"id":509329697,"identity":"7619d693-08e7-466a-8f51-8a8869a4ef3f","order_by":8,"name":"Ashraf Ahmed Qurtam","email":"","orcid":"","institution":"4 Biology Department, College of Science, Imam Mohammad Ibn Saud Islamic University (IMSIU), Riyadh, 11623, Saudi Arabia","correspondingAuthor":false,"prefix":"","firstName":"Ashraf","middleName":"Ahmed","lastName":"Qurtam","suffix":""},{"id":509329699,"identity":"d87da3c8-fb89-44e0-9cbe-ae9cc8ecbd5d","order_by":9,"name":"Boufelja Bouammali","email":"","orcid":"","institution":"4 Biology Department, College of Science, Imam Mohammad Ibn Saud Islamic University (IMSIU), Riyadh, 11623, Saudi Arabia","correspondingAuthor":false,"prefix":"","firstName":"Boufelja","middleName":"","lastName":"Bouammali","suffix":""},{"id":509329701,"identity":"50f3f918-3a8d-4be6-95d6-861fbe2f1aa9","order_by":10,"name":"Allal Challioui","email":"","orcid":"","institution":"University Mohammed Premier, Faculty of Science, Laboratory of Applied Chemistry and Environment","correspondingAuthor":false,"prefix":"","firstName":"Allal","middleName":"","lastName":"Challioui","suffix":""}],"badges":[],"createdAt":"2025-08-18 13:08:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7400020/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7400020/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90939099,"identity":"33558c44-93d5-4fbd-811e-36d73a6c30ee","added_by":"auto","created_at":"2025-09-09 17:42:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":265064,"visible":true,"origin":"","legend":"\u003cp\u003eRedocking validation of the docking protocol: superposition of co-crystallized vs re-docked ligand in 3E6T and 3E7G (RMSD = 1.210 Å and 1.253 Å, 43 atoms aligned).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7400020/v1/41f5374ee512a9247e337261.png"},{"id":90939098,"identity":"3251efbd-71a4-4fea-a863-c0cd2ee159a3","added_by":"auto","created_at":"2025-09-09 17:42:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":74802,"visible":true,"origin":"","legend":"\u003cp\u003eLattice-water-mediated hydrogen bonding between neighboring isorhamnetin-3-O-glucoside molecules in the crystal.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7400020/v1/07c69dbc88407433393823ab.png"},{"id":90939536,"identity":"9c07257a-81f0-4717-b3c4-7515c198fb92","added_by":"auto","created_at":"2025-09-09 17:50:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":323028,"visible":true,"origin":"","legend":"\u003cp\u003eHirshfeld surface maps of isorhamnetin-3-O-glucoside (I3OG): dᵢ, dₑ, d\u003csub\u003enorm\u003c/sub\u003e, fragment patches, shape index, and curvedness.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7400020/v1/94dccb2ab2d467b0821a5d78.png"},{"id":90939102,"identity":"28553ec0-30be-461c-b763-203fcdecaec1","added_by":"auto","created_at":"2025-09-09 17:42:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":390779,"visible":true,"origin":"","legend":"\u003cp\u003eHirshfeld surface of isorhamnetin-3-O-glucoside: 3D views (d\u003csub\u003enorm\u003c/sub\u003e) and 2D fingerprint plots resolved by atom type (C, H, O).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7400020/v1/fe49343cdf734e92d17a7921.png"},{"id":90939101,"identity":"b010a53c-69ba-4fac-9c3f-53c575ab539c","added_by":"auto","created_at":"2025-09-09 17:42:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":162331,"visible":true,"origin":"","legend":"\u003cp\u003eI3OG binding in iNOS oxygenase: 3E6T(A) and 3E7G(B).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7400020/v1/7b88bf1de60aa406eb2b4bec.png"},{"id":90940330,"identity":"a7686eba-94ed-4161-b5ee-3207b8a594a9","added_by":"auto","created_at":"2025-09-09 17:58:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":211490,"visible":true,"origin":"","legend":"\u003cp\u003eI3OG in iNOS (mouse, 3E6T): 3D/2D visualization.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7400020/v1/c8fb756472aeb5d7c0d9b7e1.png"},{"id":90939115,"identity":"f7ddfee9-6a94-4ae1-a03e-3bfcb6c778c7","added_by":"auto","created_at":"2025-09-09 17:42:58","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":242530,"visible":true,"origin":"","legend":"\u003cp\u003eI3OG in iNOS (human, 3E7G): 3D/2D visualization.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7400020/v1/dfd26d58ac92b3fc32e30c44.png"},{"id":90939544,"identity":"71f300df-e6b5-434f-b6ed-6632fc6a7b85","added_by":"auto","created_at":"2025-09-09 17:50:58","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":63611,"visible":true,"origin":"","legend":"\u003cp\u003eTime evolution of ligand RMSD (I3OG) after protein fit in the 3E6T and 3E7G complexes (100-ns MD).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7400020/v1/e2341805afbaa27d9fdf66c3.png"},{"id":90941051,"identity":"fd3274dd-a7fc-4267-9d35-e2fd067b36fa","added_by":"auto","created_at":"2025-09-09 18:14:58","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":56470,"visible":true,"origin":"","legend":"\u003cp\u003eResidue-wise backbone RMSF (Cα) for 3E6T and 3E7G in complex with I3OG.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7400020/v1/2f19b305a274f15fce126d04.png"},{"id":90939118,"identity":"3374531a-df6c-488b-ab11-c26053fae3d5","added_by":"auto","created_at":"2025-09-09 17:42:58","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":89528,"visible":true,"origin":"","legend":"\u003cp\u003eLeft: Protein radius of gyration (Rg). Right: Protein–ligand hydrogen-bond count for I3OG-bound 3E6T and 3E7G (100-ns MD).\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-7400020/v1/250df894a3f7da63a90b107e.png"},{"id":90940337,"identity":"53f2ce44-04fb-4654-a8dd-c2bdc57c2b31","added_by":"auto","created_at":"2025-09-09 17:58:58","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":37766,"visible":true,"origin":"","legend":"\u003cp\u003eMM/GBSA binding free-energy decomposition (GGAS, GSOLV, TOTAL) for I3OG in complexes 3E6T and 3E7G (100 snapshots, 60-100 ns).\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-7400020/v1/11637aa4dc13634573a82089.png"},{"id":90939549,"identity":"65c0457d-22e4-47af-8452-d0f82060fcf0","added_by":"auto","created_at":"2025-09-09 17:50:58","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":48985,"visible":true,"origin":"","legend":"\u003cp\u003eMM/GBSA per-residue binding free-energy contributions (ΔG\u003csub\u003ebind\u003c/sub\u003e; mean ± SEM) for I3OG in complexes 3E6T and 3E7G (100 snapshots, 60-100 ns).\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-7400020/v1/4444c0c278e78fae092f4bbc.png"},{"id":90939128,"identity":"9b4c2506-65a6-48b4-93e6-99a20fef51bb","added_by":"auto","created_at":"2025-09-09 17:42:58","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":84238,"visible":true,"origin":"","legend":"\u003cp\u003eTime-resolved heatmaps of per-residue MM/GBSA interaction energies over 60–100 ns for I3OG bound to 3E6T (left) and 3E7G (right).\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-7400020/v1/ed8e7e124204187f6ff43ecc.png"},{"id":92194427,"identity":"bc9f27a0-bf94-4c9d-9f89-4dc7c4b57741","added_by":"auto","created_at":"2025-09-25 15:39:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2821825,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7400020/v1/8f0384ad-d166-48b4-8baa-6119e197ca4e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Elucidating the Isorhamnetin-3-O-glucoside-iNOS Interaction via Molecular Dynamics and Hirshfeld Surface Analyses","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eInducible nitric oxide synthase (iNOS) is a central enzymatic source of high-output nitric oxide (NO) in innate and adaptive immunity, where dysregulated NO contributes to chronic inflammation, metabolic and cardiovascular disorders, and cancer\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Therapeutic modulation of iNOS remains attractive yet challenging because selectivity and on-target efficacy must be reconciled with complex redox biochemistry and multi-domain conformational control. Structural and biochemical studies have mapped the oxygenase domain, heme-pterin chemistry, and dimerization interfaces that govern activity, offering tractable footholds for ligand design and mechanism-guided inhibition\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eNatural products especially dietary flavonoids have long been recognized for anti-inflammatory actions that include dampening NF-κB signaling and suppressing COX-2 and iNOS expression \u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Within this class, isorhamnetin and its glycosides (notably isorhamnetin-3-O-glucoside, I3OG) show antioxidant and anti-inflammatory effects and reduce NO overproduction in cellular models, making them compelling chemical probes for iNOS modulation\u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Recent surveys of isorhamnetin glycosides underscore their prevalence, pharmacology, and relevance to human health, while broader analyses emphasize the enduring role of natural products as leads for drug discovery\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eComputational pipelines that combine carefully validated docking with explicit-solvent molecular dynamics (MD) and end-point binding free-energy calculations have become standard to interrogate protein-ligand recognition at atomistic resolution. Docking engines such as AutoDock Vina guide pose generation and enrichment\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, and learning-augmented rescoring further improves pose quality while retaining the classical RMSD\u0026thinsp;\u0026le;\u0026thinsp;2.0 \u0026Aring; benchmark for pose fidelity\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Production MD implemented in modern GPU-accelerated codes captures conformational adaptation, ligand stability, and the time-dependent behavior of key observables (RMSD, RMSF, hydrogen bonds, and radius of gyration)\u003csup\u003e\u003cspan additionalcitationids=\"CR18 CR19 CR20\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Post-processing with MM/GBSA consolidates nonbonded interactions and solvation to estimate relative binding affinities and helps prioritize poses consistent with the dynamics\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eCrucially, experimental crystal-packing analyses complement solution-phase simulations by mapping short-range contacts, π-stacking, and hydrogen-bonding motifs that underlie solid-state stability. Hirshfeld surface analysis and fingerprint plotting (as implemented in CrystalExplorer) provide a quantitative picture of intermolecular interactions that can be related to recognition patterns seen in complexes\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn this context, we investigate I3OG against iNOS oxygenase domains represented by the mouse (PDB 3E6T) and human (PDB 3E7G) structures\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. We pair validated docking with long-timescale MD to monitor structural stability (RMSD), residue-level flexibility (RMSF), hydrogen-bond persistence, and global compaction (Rg), and we quantify binding via MM/GBSA using extensive trajectory snapshots to reduce statistical noise. Our structural choices leverage high-resolution templates and contemporary simulation protocols to minimize methodological bias and to ensure that free-energy trends are grounded in physically realistic dynamics.\u003c/p\u003e\u003cp\u003eiNOS is a validated yet challenging target in chronic inflammation and oncology, so robust, mechanism-aware leads are needed. We focus on I3OG because flavonol glycosides combine a favorable safety profile, tractability, and anti-inflammatory potential. Our objectives are to define stable binding modes of I3OG in murine and human iNOS oxygenase domains, quantify their relative binding free energies with MM/GBSA using dense sampling, and relate dynamic observables (RMSD, RMSF, hydrogen bonds, Rg) to energetic trends to strengthen biological plausibility. We also integrate crystal-packing insights (Hirshfeld) to cross-validate interaction motifs across solution and solid phases.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. Hirshfeld Surface Analysis with CrystalExplorer\u003c/h2\u003e\n \u003cp\u003eIn this study, we limited the Hirshfeld surface analysis to our crystallized compound only, Isorhamnetin-3-O-glucoside (I3OG). CrystalExplorer 21.5 was applied to the compound\u0026rsquo;s CIF file to compute the descriptors d\u003csub\u003enorm\u003c/sub\u003e, d\u003csub\u003ei\u003c/sub\u003e, d\u003csub\u003ee\u003c/sub\u003e, Shape Index, and Curvedness, and to generate 2D fingerprint plots, allowing us to identify close contacts within the crystal packing and quantify the contributions of H\u0026middot;\u0026middot;\u0026middot;H, O\u0026middot;\u0026middot;\u0026middot;H/H\u0026middot;\u0026middot;\u0026middot;O, C\u0026middot;\u0026middot;\u0026middot;H/H\u0026middot;\u0026middot;\u0026middot;C interactions\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. This crystallographic analysis complements our solution-phase results (MD/MM-GBSA), providing a coherent, multiscale picture of interaction patterns and system stability.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. AutoDock Vina\u003c/h2\u003e\n \u003cp\u003eMolecular docking was carried out with AutoDock Vina (via the ADT 1.5.7 interface)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Protein structures (PDB IDs 3E6T and 3E7G) were preprocessed by removing crystallographic waters, adding polar hydrogens, and assigning charges following the AutoDock recommendations; Discovery Studio was used for inspection and minor corrections before and after docking. Ligands were sketched and geometry-optimized in ChemDraw, then converted to PDBQT with ADT to define torsions and charges.\u003c/p\u003e\n \u003cp\u003eFor the search space, protein-specific grid boxes were used: for 3E6T, the box was centered at (124.31, 111.55, 30.43) with dimensions 15.87 \u0026times; 14.40 \u0026times; 14.12 \u0026Aring;; for 3E7G, the box was centered at (56.96, 20.24, 84.71) with dimensions 13.10 \u0026times; 15.74 \u0026times; 14.55 \u0026Aring;. Vina parameters were exhaustiveness\u0026thinsp;\u003cstrong\u003e=\u003c/strong\u003e\u0026thinsp;8, num_modes\u0026thinsp;=\u0026thinsp;8, and energy_range\u0026thinsp;=\u0026thinsp;4; all other settings were left at defaults. Poses were ranked by Vina affinity and analyzed in ADT and Discovery Studio to characterize H-bonds and hydrophobic contacts.\u003c/p\u003e\n \u003cp\u003eProtocol validation was performed by redocking the co-crystallized ligand into each receptor using the same settings. The best redocked pose reproduced the experimental conformation with RMSD\u0026thinsp;=\u0026thinsp;1.210 \u0026Aring; (3E6T\u003cstrong\u003e)\u003c/strong\u003e and RMSD\u0026thinsp;\u003cstrong\u003e=\u003c/strong\u003e\u0026thinsp;1.253 \u0026Aring; (3E7G) both well below the 2.0 \u0026Aring; acceptance threshold confirming the reliability of the docking setup (RMSD computed in PyMOL) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3. Implementation of Molecular Dynamics Simulations Using GROMACS\u003c/h2\u003e\n \u003cp\u003eMolecular-dynamics simulations were performed with GROMACS 2021.3\u003csup\u003e28\u003c/sup\u003e. Using gmx pdb2gmx with the AMBER99SB-ILDN force field, missing protein hydrogens were added and protonation states adjusted. The ligand was parameterised separately; the validated .itp and .gro files were then merged with the protein to build the full complex. This complex was centred in a cubic TIP3P water box, neutralised with counter-ions, and relaxed via steepest-descent energy minimisation. Sequential NVT and NPT equilibration phases stabilised temperature and pressure, respectively. Finally, a 100 ns production run was conducted, saving coordinates and velocities at regular intervals to characterise stability, conformational dynamics, and key intermolecular interactions under near-physiological conditions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4. MM/GBSA Calculation\u003c/h2\u003e\n \u003cp\u003eBinding free energies were calculated with AmberTools23 (MMPBSA.py, parallel mode) using 100 snapshots sampled every 0.4 ns from the 60\u0026ndash;100 ns window of each GROMACS trajectory. The HCT generalized-Born model (igb\u0026thinsp;=\u0026thinsp;5) was applied with dielectric constants \u0026epsilon;_in\u0026thinsp;=\u0026thinsp;1.0 and \u0026epsilon;_out\u0026thinsp;=\u0026thinsp;80.0 and an ionic strength of 0.15 M. The nonpolar solvation term was estimated from the solvent-accessible surface area. Temporary files were not retained (keep_files\u0026thinsp;=\u0026thinsp;0). Per-residue decomposition (idecomp\u0026thinsp;=\u0026thinsp;1) was enabled to resolve van der Waals, electrostatic, polar, and nonpolar contributions for each residue.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1. HirshfeldSurface\u003c/h2\u003e\n \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.1. Hirshfeld Surface Metrics and Lattice-Water-Mediated Crystal Packing of I3OG\u003c/h2\u003e\n \u003cp\u003eHirshfeld surface analysis provides a standardized way to connect the crystal structure to the nature of intermolecular contacts: it highlights short contacts on the d\u003csub\u003enorm\u003c/sub\u003e map, describes surface topography via shape index and curvedness, and supplies concise morphological descriptors (surface volume, area, globularity, asphericity) that help rationalize crystal packing and interaction diversity. For isorhamnetin-3-O-glucoside, the calculations yielded a volume of 514.30 \u0026Aring;\u0026sup3; and a surface area of 440.60 \u0026Aring;\u0026sup2;, reflecting a relatively large molecular envelope for an aryl glycoside and indicating ample regions available for intermolecular contact\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. A globularity of 0.705 (where 1 denotes a perfect sphere) points to a moderately globular shape neither rod-like nor flat so contacts are expected to be distributed over the surface rather than concentrated at one end. The asphericity of 0.114 (zero for a perfect sphere) is low, indicating only a modest departure from sphericity with slight elongation along the aromatic core and sugar\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Taken together, these descriptors suggest that the I3OG surface is well suited to form a mixed network of polar (H\u0026middot;\u0026middot;\u0026middot;O/O\u0026middot;\u0026middot;\u0026middot;H) and hydrophobic contacts, consistent with the picture to be refined by detailed Hirshfeld maps and the docking results.\u003c/p\u003e\n \u003cp\u003eThe Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e shows a hydrogen-bond bridge (green) linking neighboring isorhamnetin-3-O-glucoside molecules, mediated by lattice water. Dashed contacts indicate O-H\u0026middot;\u0026middot;\u0026middot;O interactions between ligand oxygen donors/acceptors that connect adjacent molecules. These intermolecular H-bonds help stabilize the crystal packing and anticipate strong O\u0026middot;\u0026middot;\u0026middot;H/H\u0026middot;\u0026middot;\u0026middot;O contributions in the Hirshfeld analysis.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.2. Intermolecular Contacts in I3OG Crystals Revealed by Hirshfeld Analysis\u003c/h2\u003e\n \u003cp\u003eTo contextualize the crystal packing of I3OG, we first computed and visualized its Hirshfeld surface. Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e quantifies the surface metrics and contact percentages, while Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e displays the corresponding maps (d\u003csub\u003ei\u003c/sub\u003e, dₑ, d\u003csub\u003enorm\u003c/sub\u003e, fragment patches, shape index, and curvedness).\u003c/p\u003e\n \u003cp\u003eAs summarized in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, the Hirshfeld metrics reveal pronounced close contacts: the d_norm minimum \u0026minus;\u0026thinsp;0.746 (with d\u003csub\u003ei\u003c/sub\u003e/d\u003csub\u003ee\u003c/sub\u003e minima\u0026thinsp;\u0026asymp;\u0026thinsp;0.64/0.70 \u0026Aring;) flags red hotspots shorter than the vdW sum, while the shape index range (~-1 to +\u0026thinsp;1) and the low mean curvedness (-1.02) indicate extended, relatively flat patches suitable for \u0026pi;-stacking alongside H-bonding sites\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The fingerprint decomposition in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e shows H\u0026middot;\u0026middot;\u0026middot;H\u0026thinsp;=\u0026thinsp;27.7%, but the dominant directional interactions are O\u0026middot;\u0026middot;\u0026middot;H/H\u0026middot;\u0026middot;\u0026middot;O\u0026thinsp;=\u0026thinsp;42.5% (24.8% + 17.7%), consistent with a strong hydrogen-bond network; hydrophobic stabilization is moderate via C\u0026middot;\u0026middot;\u0026middot;H/H\u0026middot;\u0026middot;\u0026middot;C\u0026thinsp;=\u0026thinsp;12.3%, with minor C\u0026middot;\u0026middot;\u0026middot;C (5.9%) and C\u0026middot;\u0026middot;\u0026middot;O/O\u0026middot;\u0026middot;\u0026middot;C (\u0026asymp;\u0026thinsp;6.5%) contributions.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eHirshfeld surface metrics and fingerprint contact contributions (%) for I3OG.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eInteraction Mode\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMinimum\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMean\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMaximum\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ed\u003csub\u003e\u003cem\u003enorm\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.7461\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.4408\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.5878\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ed\u003csub\u003ei\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.6392\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.6778\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.4636\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ed\u003csub\u003ee\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.7011\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.6897\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.5084\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eShape Index (SI)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.9925\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.1724\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.9996\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCurvedness (Cr)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-4.0100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1.0181\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.3573\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eFingerprint% via total surface area for closed contact between atoms inside and outside the surface\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eOutside Atom%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eInside Atoms\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eO\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e compiles six complementary Hirshfeld surfaces for I3OG. The d\u003csub\u003ei\u003c/sub\u003e and dₑ maps locate the nearest internal and external neighbors; localized blue/cyan patches around phenolic and sugar O-H groups mark the shortest approaches. The d\u003csub\u003enorm\u003c/sub\u003e surface gathers these into red hotspots contacts shorter than the vdW sum identifying the main H-bond nodes that knit adjacent molecules\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. The FP (fragment patches) view segments the surface by contact type and visually confirms the dominance of O\u0026middot;\u0026middot;\u0026middot;H/H\u0026middot;\u0026middot;\u0026middot;O and H\u0026middot;\u0026middot;\u0026middot;H interactions. The shape index shows complementary red/blue triangular motifs on the flavonoid faces, diagnostic of \u0026pi;-\u0026pi; stacking, while the predominantly green curvedness map with limited blue ridges indicates extended flat regions separated by edges. Together, the six maps depict a surface that blends directional hydrogen bonding with aromatic stacking and moderate hydrophobic contacts, consistent with the packing features inferred from the quantitative metrics.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e presents Hirshfeld fingerprint plots resolved by atom identity (inside vs. outside). Inside the surface, H atoms dominate the contacts (58.2%), followed by O (25.9%) and C (16.0%); for outside atoms the shares are H 52.0%, O 33.3%, and C 14.7%. The sharp spikes at low d\u003csub\u003ei\u003c/sub\u003e/d\u003csub\u003ee\u003c/sub\u003e in the O(in)/H(out) and H(in)/O(out) maps diagnose directional O\u0026middot;\u0026middot;\u0026middot;H/H\u0026middot;\u0026middot;\u0026middot;O hydrogen bonds as the principal motif, in line with Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and the red hotspots on d\u003csub\u003enorm\u003c/sub\u003e. The broader wings in the H-resolved maps indicate numerous H\u0026middot;\u0026middot;\u0026middot;H contacts, whereas the smaller carbon fractions point to a secondary hydrophobic component (C\u0026middot;\u0026middot;\u0026middot;H/H\u0026middot;\u0026middot;\u0026middot;C) rather than dominant face-to-face \u0026pi;-\u0026pi; stacking. Overall, Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e shows a hydrogen-bond-led packing network complemented by moderate hydrophobic interactions.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. Molecular docking\u003c/h2\u003e\n \u003cp\u003eMolecular docking offers a mechanistic bridge between chemical structure and target modulation by predicting low-energy poses and the noncovalent interaction network within a protein active site. For Isorhamnetin-3-O-glucoside (I3OG) a polyphenolic glycoside with multiple H-bond donors/acceptors and an extended \u0026pi; system docking is particularly informative because it tests whether the flavonoid core and sugar hydroxyls can cooperatively engage the polar access channel and the heme-proximal pocket of inducible nitric-oxide synthase (iNOS). We selected the oxygenase domain from mouse (PDB 3E6T, 2.50 \u0026Aring;) and human (PDB 3E7G, 2.20 \u0026Aring;) to capture species-conserved recognition features and ensure translational relevance\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e; both entries contain the catalytic heme prosthetic group and co-bound reference inhibitors, which permits grid definition around the native ligand and preservation of the correct Fe-heme environment. Pharmacologically, iNOS inhibition is a validated anti-inflammatory strategy because pathological NO overproduction drives oxidative stress and tissue injury; thus, evaluating I3OG against these two high-quality structures tests whether a natural antioxidant scaffold can sterically and electronically complement the iNOS active site.\u003c/p\u003e\n \u003cp\u003eAs shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, panel A (3E6T) and panel B (3E7G) depict Isorhamnetin-3-O-glucoside (red) occupying a well-defined binding pocket (grey) on the protein surface (blue). In both structures, the flavonoid core sits deeper along a hydrophobic corridor, while the glucose moiety projects toward the pocket rim where polar donors/acceptors enable plausible hydrogen bonds, yielding minor pose differences but a conserved recognition pattern across mouse and human iNOS oxygenase domains.\u003c/p\u003e\n \u003cp\u003eTo account for the docking scores (-10.1 kcal/mol with 3E6T and \u0026minus;\u0026thinsp;9.7 kcal/mol with 3E7G), Figs. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e reveal a coherent interaction network that explains pose stability within the iNOS oxygenase pocket. In Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e (3E6T), three conventional hydrogen bonds with Gly365, Asn364, and Thr184 anchor the ligand\u0026rsquo;s orientation in the polar channel and cooperate with four \u0026pi;-\u0026pi; stacking contacts two with Trp188 and two with Phe363 to bury the aromatic core and reinforce shape complementarity\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e; these are complemented by five alkyl/\u0026pi;-alkyl contacts, providing hydrophobic support that lowers the desolvation cost. In Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e (3E7G), the ligand retains three conventional hydrogen bonds (two with Trp372 and one with Cys200), together with three \u0026pi;-\u0026pi; stacking interactions involving Phe369, Asn370, and Trp194, two \u0026pi;-\u0026sigma; contacts with Gly202 and Gly371, and a \u0026pi;-sulfur contact with Met434; six additional alkyl/\u0026pi;-alkyl contacts further tighten hydrophobic burial\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. This balance of hydrogen bonding, aromatic stacking, and hydrophobic contacts across both structures explains the slight advantage for 3E6T and supports realistic shape-electrostatic complementarity for the ligand, motivating follow-up explicit-solvent MD and MM/GBSA to verify pose stability and estimate binding free energy.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. Molecular Dynamics Simulation\u003c/h2\u003e\n \u003cp\u003eMolecular dynamics (MD) is the critical stress-test that turns a static docking pose into a time-resolved, solvent-aware assessment of binding stability. Across a 100-ns trajectory, four standard readouts provide complementary evidence: the ligand RMSD (pose persistence relative to the protein frame), the protein backbone RMSF (local flexibility of residues surrounding the pocket), the time-series of hydrogen bonds (polar anchoring and persistence), and the protein\u0026rsquo;s radius of gyration, Rg (global compactness). Together they verify whether the docked pose for I3OG, is both kinetically stable and structurally sensible before proceeding to MM/GBSA\u003csup\u003e36\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e (ligand RMSD after least-squares fit to the protein) shows that I3OG remains well confined in both complexes. The 3E6T system (red) stabilizes rapidly within a narrow band of ~\u0026thinsp;0.17\u0026ndash;0.22 nm, whereas 3E7G (black) plateaus around ~\u0026thinsp;0.07\u0026ndash;0.17 nm but with a broader spread and occasional short-lived excursions toward ~\u0026thinsp;0.20 nm\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. The absence of long-term drift in either trace indicates stable poses; however, the tighter RMSD fluctuations in 3E6T despite its slightly higher mean RMSD than 3E7G point to a more consistently anchored ligand. In short, lower RMSD variability (not just a lower mean) signals greater target stability, so I3OG appears more stable with 3E6T. Figure \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e (backbone RMSF) shows low baseline fluctuations (~\u0026thinsp;0.05\u0026ndash;0.15 nm) across most residues for both proteins, consistent with a rigid catalytic core; 3E6T exhibits an isolated spike at the N-terminus and a modest rise near the C-terminus typical for solvent-exposed termini whereas 3E7G displays higher and more frequent loop mobilities (several peaks\u0026thinsp;~\u0026thinsp;0.25\u0026ndash;0.35 nm), suggesting a slightly softer pocket environment around the ligand in the human isoform\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eIn \u003cstrong\u003eFig.\u0026nbsp;10\u003c/strong\u003e, the hydrogen-bond trace reveals a clear difference in polar retention: I3OG forms a persistent network of ~\u0026thinsp;4\u0026ndash;6 H-bonds in 3E6T (frequent bursts to 7\u0026ndash;8), versus a thinner, mostly 1\u0026ndash;3 H-bond pattern in 3E7G; in parallel, Rg remains flat in both trajectories (\u0026asymp;\u0026thinsp;2.23\u0026ndash;2.29 nm) with an early, gentle compaction in 3E6T, confirming the complexes maintain global structural integrity without unfolding. Overall, MD corroborates the docking results for I3OG in both systems and favors 3E6T as the more stable complex, supported by lower ligand RMSD, damped backbone fluctuations near the binding site, a denser H-bonding network, and slightly greater compactness features that foreshadow a more favorable MM/GBSA binding free energy for the mouse iNOS oxygenase domain.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4. MM/GBSA Free-Energy Analysis\u003c/h2\u003e\n \u003cp\u003eMM/GBSA complements docking and MD by estimating the binding free energy from equilibrated MD frames while decomposing the driving forces into van der Waals and electrostatics in the gas phase (\u0026Delta;G\u003csub\u003egas\u003c/sub\u003e) and the polar/non-polar solvation response (\u0026Delta;G\u003csub\u003esolv\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;\u0026Delta;E\u003csub\u003eGB\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;\u0026Delta;E\u003csub\u003esurf\u003c/sub\u003e). Using 100 snapshots extracted from the 60\u0026ndash;100 ns window to ensure well-equilibrated sampling, Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e shows that both complexes are favorable in water (negative \u0026Delta;G\u003csub\u003ebind\u003c/sub\u003e).\u003c/p\u003e\n \u003cdiv id=\"Equa\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003eFor I3OG-3E6T, the interaction is strongly driven by gas-phase terms (\u0026Delta;E\u003csub\u003evdW\u003c/sub\u003e \u0026asymp; -50.5 kcal/mol and \u0026Delta;E\u003csub\u003eelec\u003c/sub\u003e \u0026asymp; -62.2 kcal/mol; \u0026Delta;G\u003csub\u003egas\u003c/sub\u003e \u0026asymp; -112.7), partly offset by a polar desolvation penalty (\u0026Delta;E\u003csub\u003eGB\u003c/sub\u003e\u0026thinsp;\u0026asymp;\u0026thinsp;+\u0026thinsp;74.6) with a small non-polar gain (\u0026Delta;E\u003csub\u003esurf\u003c/sub\u003e \u0026asymp; -6.8), yielding \u0026Delta;G\u003csub\u003ebind\u003c/sub\u003e \u0026asymp; -44.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9 kcal/mol (SEM\u0026thinsp;\u0026asymp;\u0026thinsp;0.44)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. For I3OG-3E7G, the pattern is similar but weaker electrostatics (\u0026Delta;E\u003csub\u003evdW\u003c/sub\u003e \u0026asymp; -54.2; \u0026Delta;E\u003csub\u003eelec\u003c/sub\u003e \u0026asymp; -19.7; \u0026Delta;G\u003csub\u003egas\u003c/sub\u003e \u0026asymp; -73.9) and a smaller polar penalty (\u0026Delta;E\u003csub\u003eGB\u003c/sub\u003e\u0026thinsp;\u0026asymp;\u0026thinsp;+\u0026thinsp;44.2; \u0026Delta;E\u003csub\u003esurf\u003c/sub\u003e \u0026asymp; -6.4) lead to \u0026Delta;G\u003csub\u003ebind\u003c/sub\u003e \u0026asymp; -36.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7 kcal/mol (SEM\u0026thinsp;\u0026asymp;\u0026thinsp;0.31). Thus, both proteins stabilize I3OG, but 3E6T is favored by ~\u0026thinsp;9 kcal/mol because its stronger gas-phase attraction especially electrostatics more than compensates its larger desolvation cost. This energetic profile is fully consistent with the MD readouts (lower ligand RMSD and denser H-bonding in 3E6T), reinforcing the conclusion that I3OG binds more stably to 3E6T.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003e presents the residue-wise MM/GBSA decomposition from 100 snapshots taken between 60\u0026ndash;100 ns, mapping the \u0026ldquo;hot spots\u0026rdquo; that anchor I3OG in the active site; residues with per-residue contributions \u0026lt; -1 kcal/mol are considered stabilizing. In I3OG-3E6T, Trp188, Cys194, Trp366, and Phe363 show \u0026lt; -2 kcal/mol and thus act as the primary anchors fully consistent with the docking contacts and additional pocket residues just below \u0026minus;\u0026thinsp;1 kcal/mol form a hydrophobic belt that helps keep the ligand seated. In I3OG-3E7G, three key residues dominate (Trp194, Phe369, Trp372), each \u0026lt; -2 kcal/mol; notably, Trp372 was flagged in docking for two conventional hydrogen bonds, which agrees with the MD hydrogen-bond trace (Fig. 10) where two H-bonds persist from ~\u0026thinsp;60 ns to the end of the run. Overall, Fig. \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003e confirms that the ligand is well stabilized in the active site of both proteins, with deeper hot spots in 3E6T, in line with its more favorable \u0026Delta;G\u003csub\u003ebind\u003c/sub\u003e and MD stability readouts.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e13\u003c/span\u003e is a time-resolved heat map of the per-residue MM/GBSA interaction energies over the 60-100 ns window (blue = favorable/negative; pale = weak or transient). In I3OG\u0026ndash;3E6T, the darkest, most continuous bands sit on Phe363 (dominant hotspot) and Trp366, with persistent contributions from Trp188 and Cys194 exactly the anchors highlighted by Figure \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003e while loop residues show intermittent, lighter stripes indicative of fleeting contacts. In I3OG-3E7G, Trp372 is the chief hotspot, followed by Phe369 and Trp194; the sustained dark-blue signal on Trp372 agrees with docking (two conventional H-bonds) and with the MD H-bond trace in \u003cstrong\u003eFigure 10\u003c/strong\u003e, where two hydrogen bonds persist from ~60 ns to the end. The bottom LIG (I3OG) row remains strongly negative throughout in both panels, confirming a stable net attraction. Overall, Figure \u003cspan class=\"InternalRef\"\u003e13\u003c/span\u003e corroborates Figure \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003e: the key hotspots stay engaged across the trajectory, with more continuous strong contacts in 3E6T, consistent with its more favorable \u0026Delta;G\u003csub\u003ebind\u003c/sub\u003e.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis work identifies isorhamnetin-3-O-glucoside (I3OG) as a credible, mechanism-aligned scaffold for attenuating iNOS activity. Consistent docking, restrained ligand dynamics, and favorable MM/GBSA estimates converge to support stable engagement of the iNOS oxygenase domain, with stronger stabilization in the murine (3E6T) than the human (3E7G) construct and clear residue-level hot spots that rationalize this species dependence. These results argue that I3OG and closely related chromone/flavonol chemotypes merit progression beyond in silico screening. Immediate priorities are orthogonal biophysical validation (SPR/ITC), enzyme inhibition and NO-suppression assays in relevant macrophage models, and selectivity profiling against eNOS/nNOS. Given the permeability liabilities of glycosides, medicinal chemistry should explore aglycone analogs, sugar bioisosteres, and prodrug strategies while preserving the π-stacking and hydrogen-bonding motifs highlighted here. Finally, longer simulations and models incorporating full cofactor/dimer contexts will refine translatability to the human enzyme. Collectively, our data provide a quantitative blueprint for structure-guided iNOS inhibitor design and a tractable starting point for anti-inflammatory lead optimization.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"524\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eRMSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;Root Mean Square Deviation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eRMSF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;Root Mean Square Fluctuation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eiNOS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u0026nbsp;Inducible nitric oxide synthase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eADME\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;Absorption, Distribution, Metabolism, and Excretion\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eI3OG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;Isorhamnetin-3-O-Glucoside\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003eAuthor Contributions:\u0026nbsp;O, K; H, B; Y, H; M, B; M, M Methodology and Writing original draft. S, A; B,B ;A, C Supervision and Project administration. O, K Docking and dynamic simulation.\u0026nbsp;O, K; H,B; Y,H; M, B; M,M; S,A; M, AL z; F A.N; A, A.Q; B,B; A, C Writing review and editing.\u003c/p\u003e\n\u003cp\u003eFunding:\u0026nbsp;Biology Department, College of Science, Imam Mohammad Ibn Saud Islamic University (IMSIU) (grant number IMSIU-DDRSP2501), Riyadh, 11623, Saudi Arabia. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInstitutional Review Board Statement: Not applicable.\u003c/p\u003e\n\u003cp\u003eInformed Consent Statement: Not applicable.\u003c/p\u003e\n\u003cp\u003eData Availability Statement: All datasets generated and/or analyzed in this study are publicly available at the open-access repository https://github.com/khibech/I3OG-to-iNOS. The repository includes: the crystallographic .CIF file of the compound together with the CrystalExplorer project (.cxp) and output files; AutoDock Vina input/output files for both targets (3E6T and 3E7G); GROMACS system/parameter files (.mdp, .itp, .gro); and MM/GBSA input and result files. No additional data are required to support the findings of this work.\u003c/p\u003e\n\u003cp\u003eAcknowledgements:\u0026nbsp;This work was supported and funded by the Deanship of Scientific Research at Imam Mohammad Ibn Saud Islamic University (IMSIU) (grant number IMSIU-DDRSP2501). We gratefully acknowledge the HPC Marwan team for granting us privileged access to their high-performance computing resources. The exceptional computational power and responsive technical support they provided were instrumental in carrying out the molecular-dynamics simulations reported in this study. Their contribution greatly enhanced the quality and robustness of our results, and we are sincerely thankful for their assistance.\u003c/p\u003e\n\u003cp\u003eConflicts of Interest: The authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eF\u0026ouml;rstermann, U. \u0026amp; Sessa, W. C. Nitric oxide synthases: Regulation and function. \u003cem\u003eEur. Heart J.\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 1\u0026ndash;13 (2012).\u003c/li\u003e\n \u003cli\u003eBogdan, C. Nitric oxide and the immune response - Nature Immunology. \u003cem\u003eNat. Immunol.\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 907\u0026ndash;916 (2001).\u003c/li\u003e\n \u003cli\u003eMacMicking, J., Xie, Q. W. \u0026amp; Nathan, C. Nitric oxide and macrophage function. \u003cem\u003eAnnu. Rev. 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Rev.\u003c/em\u003e \u003cstrong\u003e119\u003c/strong\u003e, 9478\u0026ndash;9508 (2019).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"Hirshfeld surface analysis, iNOS, I3OG, MM/GBSA, MD","lastPublishedDoi":"10.21203/rs.3.rs-7400020/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7400020/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eInducible nitric oxide synthase (iNOS) overproduction drives chronic inflammation and oncogenic signaling, yet selective small-molecule modulation remains elusive. We interrogated isorhamnetin-3-O-glucoside (I3OG), a dietary flavonol glycoside, against murine (3E6T) and human (3E7G) iNOS oxygenase domains using a validated in silico pipeline spanning redocking, explicit-solvent molecular dynamics (100 ns), and MM/GBSA free-energy analysis. Redocking reproduced co-crystal poses (RMSD 1.21/1.25 Å), and Vina ranked I3OG favorably (-10.1/-9.7 kcal·mol⁻¹). MD revealed confined ligand motions and intact protein compaction; 3E6T displayed tighter RMSD variability and a denser hydrogen-bond network (≈4-6 persistent bonds) than 3E7G (≈1-3), with damped local flexibility around the pocket. MM/GBSA from equilibrated frames yielded ΔGbind = -44.9 ± 3.9 kcal·mol⁻¹ (3E6T) versus -36.1 ± 3.7 kcal·mol⁻¹ (3E7G), driven by favorable gas-phase van der Waals/electrostatics that outweigh polar desolvation. Hot-spot residues (3E6T: Trp188, Cys194, Trp366, Phe363; 3E7G: Trp194, Phe369, Trp372) rationalize species-dependent stabilization. Complementary Hirshfeld analysis of I3OG crystals highlights dominant O···H/H···O contacts and ancillary π-stacking, mirroring solution-phase recognition. Collectively, I3OG emerges as a mechanism-aware, tractable scaffold for iNOS attenuation, with the murine domain offering a more avid binding environment and a quantitative benchmark for future selective inhibitor design.\u003c/p\u003e","manuscriptTitle":"Elucidating the Isorhamnetin-3-O-glucoside-iNOS Interaction via Molecular Dynamics and Hirshfeld Surface Analyses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-09 17:42:52","doi":"10.21203/rs.3.rs-7400020/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":"2c064ffa-51a4-4a02-86fa-e5a50b5a1504","owner":[],"postedDate":"September 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54095727,"name":"Biological sciences/Biochemistry"},{"id":54095728,"name":"Biological sciences/Biophysics"},{"id":54095729,"name":"Biological sciences/Chemical biology"},{"id":54095730,"name":"Physical sciences/Chemistry"},{"id":54095731,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":54095732,"name":"Biological sciences/Drug discovery"}],"tags":[],"updatedAt":"2025-09-25T15:38:47+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-09 17:42:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7400020","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7400020","identity":"rs-7400020","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}