Structural Insights into DENV-2 NS2B-NS3 Protease and Inhibition by Glutathione-Coated Gold Nanocluster

Structural Insights into DENV-2 NS2B-NS3 Protease and Inhibition by Glutathione-Coated Gold Nanocluster | 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 Structural Insights into DENV-2 NS2B-NS3 Protease and Inhibition by Glutathione-Coated Gold Nanocluster Haojia Zhu, Wenchao Niu, Yuancong Xu, Zhaoyang Li, Dongfang Xia, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8498727/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Dengue virus (DENV) continues to pose a significant global health threat, with increasing infection rates and limited treatment options. The viral NS2B-NS3 protease (NS2B-NS3pro), a highly conserved two-component enzyme essential for polyprotein processing and replication, is a key target for antiviral drug development. Here, we report the 2.25 Å-resolution crystal structure of DENV serotype 2 (DENV-2) NS2B-NS3pro, in which a PEG fragment is bound within a solvent-exposed pocket between β 10, β 11, β 14 and β 15 of NS3. This structure reveals a previously unidentified potential allosteric site, suggesting a novel pocket for inhibitor design. We also show that the glutathione-coated gold nanocluster (GSH-AuNC) directly inhibits DENV-2 NS2B-NS3pro. Bio-layer interferometry and fluorescence-based protease assays indicate that the nanocluster binds NS2B-NS3pro with a dissociation constant of 15.64 µM and inhibits its catalytic activity with an IC 50 of 16.04 µM, consistent with a direct inhibition mechanism. Molecular dynamics simulations further suggest that GSH-AuNC interacts with the catalytic triad of NS2B-NS3pro, forming stable electrostatic and van der Waals interactions that block substrate binding. Collectively, these findings provide a structural and mechanistic basis for the development of inhibitors targeting DENV-2 NS2B-NS3pro, offering new strategies for antiviral therapy using small molecules or nanomedicines. Dengue virus NS2B-NS3pro crystal structure glutathione-coated gold nanocluster Molecular dynamics simulations Molecular interactions Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Dengue, an infectious disease caused by DENV and transmitted by Aedes aegypti and Aedes albopictus mosquitoes, remains a major global health concern [ 1 ]. It is estimated that 2.5 billion people globally are at risk of dengue infection [ 2 ]. In 2024, around 14.1 million new cases and 9,508 deaths were reported, with both the in-cidence rate and the mortality rate increasing significantly compared to the previous year [ 3 ]. The clinical manifestations of DENV infection vary significantly in severity, ranging from mild dengue fever (DF) characterized by non-specific symptoms such as fever, headache, myalgia, and rash to life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) [ 4 , 5 ]. Unfortunately, there are currently no clini-cally approved drugs available for the treatment of dengue virus infections. Although licensed dengue vaccines have been developed, none of them can provide complete protection against infection, especially for those who have not been previously infected with dengue viruses [ 6 – 13 ]. DENVs belong to the genus Flavivirus within the family of Flaviviridae and consist of four serotypes (DENV-1 to -4). The virions are spherical, with a diameter of 40–50 nanometers and encapsulated by a lipopolysaccharide envelope. The positive-sense single-stranded RNA genome is approximately 11 kilobases in length and has a single open reading frame, encoding a polyprotein which is subsequently cleaved by either the host-encoded signal peptidase or the viral protease to generate three structural proteins (capsid (C), membrane (M)), and envelope (E)) and seven non-structural pro-teins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) [ 14 , 15 ]. The NS2B–NS3pro complex is a two-component serine protease, composed of the NS3 N-terminal protease domain and its essential catalytic cofactor NS2B, and plays a central role in polyprotein processing and RNA replication, making it a validated and druggable antiviral target. NS3 contains an N-terminal serine protease domain and a C-terminal helicase domain, while NS2B is indispensable for its proteolytic activity [ 16 – 20 ]. High-resolution crystal structures from multiple serotypes reveal that, although the catalytic triad is highly conserved, the arrangement of the NS2B cofactor, the substrate-recognition loops, and the overall conformational plasticity exhibit pronounced serotype-specific differences [ 21 – 23 ]. Notably, subtle variations in the position and conformation of the NS2B C-terminal β-hairpin can alter the accessibility of the active-site pocket. In addition, differences in the flexibility and orientation of NS3 substrate-binding loops and solvent-exposed pockets adjacent to the catalytic triad may further modulate substrate recognition and influence inhibitor specificity [ 20 , 21 ]. Over the past decade, most inhibitors of DENV NS2B-NS3pro have been broadly classified into two classes: substrate-mimetic peptides and macrocyclic compounds that occupy the catalytic cleft, and small-molecule inhibitors that bind to the hydrophobic allosteric pocket adjacent to the NS2B β -hairpin [ 20 , 24 ]. While these studies have established the validity of NS2B-NS3pro as an antiviral drug target, none of these inhibitors have advanced to clinical applications, partly due to the incomplete characterization of the protease’s druggable binding surfaces. Therefore, there is an urgent need for novel structural insights and mechanistic understanding to guide the development of new NS2B-NS3pro inhibitors. Gold nanoclusters (AuNCs) are special materials sized between individual gold atoms and plasmonic gold nanoparticles. Comprising several to hundreds of gold at-oms, they are smaller than 2 nm and have a typical core-shell structure. This structure comprises a metallic gold core, which is surrounded by stabilizing ligands that prevent aggregation and ensure structural stability [ 25 – 27 ]. Among them, GSH-AuNC represents a well-defined class of ultrasmall AuNCs coated and stabilized by glutathione, whose size-dependent properties, such as strong photoluminescence, high stability, and good biocompatibility, have enabled broad use in biomedical, catalytic and sensing applications. In recent years, a variety of nanomaterials, including AuNCs, have drawn increasing interest in antiviral research [ 28 – 30 ]. However, the specific targeting ability of GSH-AuNC toward NS2B-NS3pro has not been reported previously. This study determined the 2.25 Å crystal structure of DENV-2 NS2B-NS3pro, revealing a novel, solvent-exposed, positively charged groove, spatially distinct from the catalytic cleft and known allosteric site. Biochemical, biophysical, and molecular simulation approaches characterized the inhibitory effect and underlying mechanism by which GSH-AuNC inhibits NS2B-NS3pro. These findings provide a structural basis and target validation for allosteric modulation of flaviviral NS2B-NS3pro and the design of nanoscale/small-molecule inhibitors. Methods 2.1. Preparation of gold nanoclusters The preparation and characterization of glutathione-stabilized GSH-AuNC was exactly based on the method described in the previously published paper [31]. An aqueous solution of glutathione (GSH, 30 mM, 100 mL) was mixed with hydrogen tetrachloroaurate trihydrate (HAuCl 4 ·3H 2 O, 20 mM, 100 mL) and gently stirred at 25 °C for 10 min (500 rpm). The reaction mixture was then heated to 70 °C and maintained under stirring (500 rpm) for 12 h to obtain a fluorescent GSH-AuNC solution. For purification, ethanol was added to the reaction solution at a 3:1 volume ratio, followed by thorough mixing and centrifugation at 10,000 rpm for 15 min. The supernatant was discarded, and the precipitated clusters were washed three times with 75% ethanol to remove residual ligands and impurities. The purified clusters were dried and subsequently redispersed in ultrapure water with the assistance of NaOH. Finally, the clusters were further purified using an ultrafiltration tube with a molecular weight cutoff of 3 kDa to remove any remaining free ions, yielding high-purity GSH-AuNCs. 2.2. Expression and purification of NS2B-NS3pro The gene fragments encoding NS2B (residues 49-95) and NS3 (residues 1-185) of DENV-2 were cloned into the pET-28a(+) vector. The resulting construct expresses NS2B-NS3pro with an N-terminal His 6 tag and a Gly₄-Ser-Gly₄ linker. The recombinant plasmid was transformed into E. coli BL21(DE3) and screened on LB agar plates containing 50 μg/mL kanamycin. A single colony was inoculated into 10 mL LB medium containing kanamycin and grown overnight, then transferred into 1 L of LB medium and cultured at 37 °C and 180 rpm. Protein expression was induced with 0.2 mM IPTG when the OD 600 reached 0.6~0.8, followed by incubation at 16 °C, 150 rpm for 18 h. Cells were harvested by centrifugation at 6000 rpm for 10 min and resuspended in buffer containing 20 mM Tris-HCl (pH 8.0), 20 mM imidazole, and 500 mM NaCl. Cells were lysed using high-pressure homogenization in the presence of 1 mM PMSF, and the lysate was clarified by centrifugation at 15,000 rpm for 60 min. The supernatant was loaded onto a Ni 2+ -NTA affinity column and eluted with 20~300 mM imidazole gradient. The eluted protein was dialyzed overnight at 4 °C in buffer containing 50 mM Tris-HCl (pH 8.5), 50 mM NaCl, 1 mM TCEP, and 5% glycerol in the presence of Tobacco Etch Virus (TEV) protease (TEV: protein = 1:15, w/w). After dialysis, the sample was filtered through a 0.22 μm membrane and concentrated using a 10 kDa MWCO ultrafiltration tube to 20 mg/mL. The protein was further purified by an anion-exchange Q column using buffer A (50 mM Tris-HCl pH 8.5, 50 mM NaCl, 1 mM TCEP, 5% glycerol) and a 0~30% gradient of buffer B (50 mM Tris-HCl pH 8.5, 1 M NaCl, 1 mM TCEP, 5% glycerol). The collected components were concentrated using a 10 kDa MWCO centrifugal concentrator and further identified by size-exclusion chromatography (SEC) column preloaded with 20 mM Tris (pH 7.5) and 150 mM NaCl. Homogeneous fractions were concentrated to 20 mg/mL, flash-frozen in liquid nitrogen, and stored at -80 °C. 2.3. Crystallization of NS2B-NS3pro Crystals of NS2B-NS3pro were grown using the sitting-drop vapor diffusion method at 4 °C. Prior to crystallization, the protein was diluted to 4 mg/mL using buffer containing 20 mM Tris (pH 7.5) and 150 mM NaCl. Equal volumes (1 μL + 1 μL) of protein solution and reservoir solution were mixed and placed on 96-well crystallization plates with 100 μL reservoir solution in each well. Initial screening was performed using commercial sparse-matrix kits as well as in-house screens. Crystals suitable for X-ray diffraction were obtained from a reservoir solution containing 0.1 M sodium acetate (pH 5.5) and 44% (v/v) PEG-200. 2.4. Data Collection and Processing Diffraction data were collected at the beamline BL18U1 of the Shanghai Synchrotron Radiation Facility (SSRF). Data indexing and scaling were performed using XDS [32]. Molecular replacement was carried out using Phaser 2.8 within the CCP4 (v9.0) suite [33], with the DENV-2 NS2B-NS3pro structure (PDB code: 2FOM) as the search model. Manual model building was performed in Coot 0.9.8 [34], and structure refinement was completed using Phenix.refine 1.16 [35] and Refmac 5.8 [36]. The quality of the final structure was assessed using MolProbity ver. 4.5 [37]. Crystallographic data collection and refinement statistics are summarized in Table S1. 2.5. Bio-Layer Interferometry (BLI) analysis Avi tag (GLNDIFEAQKIEWHE) was added to the C-terminus of NS2B-NS3pro by PCR, using the pET-28a(+)-NS2B-NS3pro plasmid as the template [38]. The new construct (pET-28a(+)-NS2B-NS3pro-Avi tag) was co-expressed with biotin ligase (BirA) in E. coli . The cells were grown in 1 L of LB medium with kanamycin and ampicillin at 37 °C and 180 rpm. When OD₆₀₀ reached 0.6~0.8, 0.2 mM IPTG and 50 μM biotin were added to start protein expression, and the culture was continued at 16 °C and 150 rpm for 18 hours. The purification method was the same as the NS2B-NS3pro purification described earlier. Biotinylated protein was purified as described in Section 2.2. The binding experiment was carried out using the Octet® R8 instrument. Prior to the start of the experiment, biotinylated NS2B-NS3pro was diluted to 50 μg/mL in a buffer composed of 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.05% (v/v) Tween-20. After that, the sensors were placed in GSH-AuNC solutions at different concentrations (0, 21.25, 42.5, 85, 170, and 340 μM) to record association, followed by buffer for dissociation. Reference subtraction using unloaded biosensors was performed to correct for nonspecific signals. The data were analyzed using Octet software (v12, Sartorius), and the 1:1 model was used to obtain k on , k off , and K D values. 2.6. Protease activity assay The effect of GSH-AuNC on NS2B-NS3pro activity was measured using a continuous fluorescence assay. NS2B-NS3pro was used at 100 nM and mixed with GSH-AuNC at 1-276 μM (8-point two-fold dilution). GSH-AuNC and NS2B-NS3pro were mixed and pre-incubated at 37 °C for 30 min, followed by addition of the fluorogenic substrate Bz-nKRR-MCA to a final concentration of 50 mM [39]. Fluorescence was recorded immediately at Ex/Em = 360/450 nm at 1 min intervals for 10 min with brief shaking (5 s, 200 rpm) before each read. IC 50 values were determined by nonlinear regression using R software (version 4.5.1) [40]. 2.7. Molecular dynamics simulations The GSH-AuNCs structures were generated using GaussView 5.0, followed by geometry optimization via the Gaussian 09 suite employing density functional theory (DFT) [41]. For this optimization, the LANL2DZ basis set was applied to all gold atoms, while the 631G(d) basis set was utilized for carbon, hydrogen, oxygen, nitrogen, and sulfur atoms. The three-dimensional structure of NS2B-NS3pro was derived from X-ray crystallo-graphic data. Binding configurations were constructed using the Visual Molecular Dynamics (VMD) software [42], which also facilitated the generation of input files for subsequent MD simulations. All simulations were conducted under the NPT ensemble with peri-odic boundary conditions in all spatial dimensions. Temperature and pressure were regulated using the Langevin thermostat and the Nosé-Hoover Langevin piston method, ensuring full equilibration of the systems under physiological conditions. Electrostatic interactions were computed using the particle mesh Ewald (PME) tech-nique under conducting boundaries, while van der Waals forces were cutoff at 12 Å. The CHARMM22 and CHARMM36 force fields [43] were employed to describe the system, with explicit solvent molecules modeled using the TIP3P water model. Simula-tions were carried out using NAMD 2.13 [44], and trajectory analysis was performed with VMD 1.9.3. Prior to production runs, initial energy minimization involved 5,000 steps of steepest descent, followed by a 100 ns equilibration phase during which the system was gradually heated to 300 K. Thermal equilibration was achieved through coupling with a sodium ion solution. Results 3.1. Expression, purification and crystallization of DENV-2 NS2B-NS3pro fusion protein The recombinant DENV-2 NS2B-NS3pro construct carrying an N-terminal His₆ tag was expressed in E. coli BL21 (DE3). After induction, cells were harvested, resus-pended in lysis buffer, and clarified by centrifugation. The supernatant was applied to a Ni 2 +-NTA affinity chromatography, which removed most contaminating proteins and yielded His-tagged protein in the elution fractions ( Fig. 1A ). After dialysis, the His₆ tag was removed by TEV protease, and the sample was further purified on an anion-exchange Q column, which yielded three peaks ( Fig. 1B ). SDS-PAGE ( Fig . 1C ) revealed that peak 1 contained a ~28 kDa band corresponding to TEV protease, whereas peaks 2 and 3 both contained a band at approximately 37 kDa. This apparent molecular weight is higher than the calculated mass of NS2B-NS3pro (~26 kDa), consistent with the abnormal migration reported previously [45, 46]. Peaks 2 and 3 were analyzed by SEC and eluted as single, symmetric peaks at nearly identical volumes ( Fig. 1D ), indicating a homogeneous species with an apparent monomeric size in solution. These findings suggest the possible presence of minor conformational or charge variants in solution, and anion-exchange Q chromatography was necessary to obtain the target protein at higher purity. For crystallization, purified NS2B-NS3pro was screened using commercial kits and in-house conditions by sitting-drop vapor diffusion. Initial hits were obtained under several polyethylene glycol-based conditions. These were subsequently optimized by varying buffer composition. Well-diffracting crystals were finally obtained in 0.1 M sodium acetate (pH 5.5) and 44% (v/v) PEG 200. The crystals were uniform, with smooth, well-defined faces ( Fig. 1E ), and were suitable for high-resolution X-ray data collection. 3.2. NS2B-NS3pro structure revealing a PEG-binding pocket The crystal structure of DENV-2 NS2B-NS3pro reveals a functional enzyme complex composed of the NS2B cofactor and the NS3 protease domain, with an additional PEG molecule bound on the surface of NS3 ( Fig. 2A, B ). NS2B wraps around the N-terminal serine protease domain of NS3. NS3pro adopts a canonical chymotrypsin-like fold with a β-barrel core, while NS2B contributes four β-strands and one α-helix to stabilize the NS3pro structure. The substrate-binding pocket is formed by NS3 β-strands, with the catalytic triad residues His51, Asp75, and Ser135 located at the active site ( Fig. 2A ). The NS2B–NS3 interface is primarily stabilized by β -sheet-mediated interactions, consistent with previous structural reports ( Fig. S1, S2 ) [23]. A striking feature of the structure is a well-defined electron density corresponding to a PEG fragment bound on the NS3 surface within a shallow, solvent-exposed pocket formed by residues Phe116, Lys117, Thr118, Val154, Val155, Thr156, and Arg157. This pocket is surrounded by four β -strands ( β 10, β 11, β 14, and β 15), located adjacent to the C-terminal loop of NS3 ( Fig. 2A, C ). The analysis using parKVFinder [47] further revealed that the pocket has a surface area of ~ 69.4 Ų and a calculated volume of ~ 85.75 ų. The PEG molecule, derived from the PEG-200 used in crystallization, occupies a narrow groove next to this flexible C-terminal loop, making close contacts with the backbones of Val155 and Lys117 (~3.0 Å) and additional van der Waals interactions with Phe116 and the backbone of Thr156 ( Fig. 2C ). Electrostatic surface representation shows that this pocket has a positively charged entrance and a largely hydrophobic interior, creating a mixed hydrophobic and positively charged environment that can readily accommodate small, flexible ligands ( Fig. 2B ). To assess sequence and structural conservation, sequence and structural alignments of NS2B-NS3pro proteins from DENV serotypes 1-4, Yellow Fever virus (YFV), West Nile virus (WNV), and Zika virus (ZIKV) were conducted ( Fig. S3 ) [23, 39, 48-52]. Multiple sequence alignment showed 22.1% overall identity and 75.9% similarity. The catalytic triad (His51, Asp75, Ser135) and surrounding residues were strictly conserved, underscoring their central role in catalysis. Structural superposition yielded C α RMSD values of 0.189-0.829 Å, indicating a conserved serine-protease fold with the catalytic triad positioned almost identically. Despite this global similarity, DENV-2 NS2B-NS3pro features a previously unreported PEG-binding pocket on the NS3 surface, which is absent in the corresponding regions of other flaviviral proteases, suggesting that it may represent a DENV-2-specific potential allosteric binding site ( Fig. S3 ). Comparative analysis of complexes with both active‑site and allosteric inhibitors shows that the PEG‑binding crevice is spatially distant from the catalytic triad, the substrate‑binding cleft, and the previously described allosteric site ( Fig. 2D ) [22, 49, 53]. Thus, the PEG-bound groove represents a previously unrecognized solvent-exposed pocket, distinct from canonical regulatory sites, and may serve as a transient ligand-binding surface. Taken together, these observations suggest that this pocket may serve as a potential allosteric site on NS2B-NS3pro and could be explored in future fragment-based or structure-guided efforts to identify small-molecule modulators. 3.3. Binding and inhibition of NS2B-NS3pro by GSH-AuNC Previous studies have shown that the GSH-AuNC nanocluster can inhibit the main protease (Mpro) of SARS-CoV-2 and suppress viral replication, suggesting that AuNCs may act as direct protease modulators [31]. Motivated by these findings, we next examined whether the GSH-AuNC can also target the flaviviral NS2B-NS3pro. To characterize the interaction between GSH-AuNC and NS2B-NS3pro, we used bio-layer interferometry (BLI). NS2B-NS3pro was expressed with a C-terminal Avi tag (GLNDIFEAQKIEWHE), allowing in vivo biotinylation during E. coli expression. Then, the biotinylated NS2B-NS3pro was immobilized on streptavidin biosensors, and binding to GSH-AuNC was monitored by BLI. Exposure of the sensors to increasing concentrations of GSH-AuNC produced clear, concentration-dependent association and dissociation phases ( Fig. 3A ). Global fitting of the sensorgrams yielded an equilibrium dissociation constant ( K D ) of 15.64 μM ( R² = 0.98), indicating a direct interaction between the GSH-AuNC and NS2B-NS3pro in the micromolar affinity range. We then evaluated whether this interaction translated into functional inhibition using an in vitro fluorescence-based protease assay. NS2B-NS3pro was pre-incubated with increasing concentrations of GSH-AuNC before addition of a fluorogenic peptide substrate. Enzymatic activity decreased in a dose-dependent manner, and nonlinear fitting of the inhibition curve gave an IC 50 value of 16.04 μM ( Fig. 3B ). The close agreement between the binding affinity and the IC 50 strongly supports a direct inhibitory effect of the GSH-AuNC on NS2B-NS3pro, rather than nonspecific aggregation or indirect interference with the assay. 3.4. Molecular dynamics simulations of the NS2B-NS3pro-GSH-AuNC complex The structure of the GSH-AuNC nanocluster was constructed and optimized using quantum chemical calculations ( Fig. 4A ). GSH-AuNC consists of a well-defined gold core coordinated by glutathione ligands through Au-S bonds. The optimized GSH-AuNC model, together with our crystal structure of DENV-2 NS2B-NS3pro, was then used to set up molecular dynamics (MD) simulations of the NS2B-NS3pro-GSH-AuNC complex using the NAMD software package. A 100 ns MD trajectory was generated, and the root-mean-square deviation (RMSD) of the protein backbone was monitored to assess system stability ( Fig. 4B ). The complex reached equilibrium after ~10 ns and remained stable for the remainder of the simulation. Non-bonded interaction energies between NS2B-NS3pro and GSH-AuNC were decomposed into van der Waals and electrostatic components in MD simulations ( Fig. 4C ). Electrostatic interactions dominate the binding, with van der Waals contacts providing additional stabilization. Analysis of representative equilibrium conformations shows that GSH-AuNC is stably positioned adjacent to the catalytic triad (His51, Asp75, Ser135) ( Fig. 4D ). The cluster forms hydrogen bonds and salt bridges with Arg54 and Arg157, as well as hydrogen bonds with Ser135 and His51, thereby occupying the pocket adjacent to the active site. These interactions suggest that GSH-AuNC hampers substrate access and perturbs the catalytic environment by creating steric and electrostatic barriers at the entrance to the NS2B-NS3pro active center, providing a potential structural basis for the observed inhibition of protease activity. Notably, Arg157 also contributes to forming the PEG-binding pocket in our crystal structure ( Fig. 2C ), suggesting that the interaction of GSH-AuNC with this surface pocket may further stabilize an inhibitory conformation of NS2B-NS3pro and thereby impede substrate entry. Based on our combined structural, biophysical, and computational analyses, we propose a refined working model to illustrate how GSH-AuNC exerts its inhibitory effect on DENV-2 NS2B-NS3pro ( Fig. 4E ). Discussion NS2B-NS3pro is a key serine protease of DENV-2, predominantly localized on the cytoplasmic face of the host cell endoplasmic reticulum (ER) membrane [ 54 ]. It plays a critical role in viral polyprotein processing and viral particle maturation [ 55 ], making it an attractive target for antiviral drug development. Although significant progress has been made in developing NS2B-NS3pro inhibitors, the protease’s active site is relatively shallow and highly exposed, and its overall surface exhibits pronounced charge characteristics, which complicates the formation of stable, high-affinity interactions with small molecules [ 39 ]. Therefore, the exploration of inhibitors with novel chemical scaffolds and unique mechanisms of action remains essential to advance therapeutic strategies. In this study, we successfully resolved the high-resolution crystal structure of DENV-2 NS2B-NS3pro and identified a previously unreported PEG-binding pocket on the NS3 surface. The PEG molecule, originating from PEG-200 used in crystallization, occupies a shallow groove adjacent to the flexible C-terminal loop of NS3. Although this binding may be a crystallization artifact, its position highlights a solvent-exposed pocket capable of accommodating flexible ligands. Structural comparisons indicate that analogous binding sites have not been observed in other flaviviral NS3 proteases ( Fig. S3 ), suggesting that this pocket may serve as a potential DENV-2-specific allosteric binding site. Nanoparticles have attracted considerable attention in disease prevention and treatment owing to their exceptional tunability. Modifying their surface properties can enhance both selectivity and efficacy [ 56 , 57 ]. Here, we report for the first time that GSH-AuNCs can inhibit the dengue virus NS2B-NS3pro. Biochemical assays demonstrated that GSH-AuNCs bind the protease with micromolar affinity ( K D = 15.64 µM) and inhibit its catalytic activity with a comparable IC 50 value of 16.04 µM, supporting a direct inhibitory mechanism. MD simulations further elucidated the atomic-level basis of inhibition: GSH-AuNCs occupy regions adjacent to the catalytic triad (His51, Asp75, Ser135), forming persistent hydrogen bonds and electrostatic interactions with residues such as Arg157, Arg54, Ser135, and His51. This interaction sterically blocks the substrate-binding cleft and constrains the conformational changes required for substrate entry, thereby reducing catalytic efficiency ( Fig. S4 ). Notably, Arg157 also participates in the formation of the PEG-binding pocket observed in the crystal structure, suggesting that the interaction between GSH-AuNCs and this pocket may further enhance inhibition of NS2B-NS3pro. Given that efficient polyprotein cleavage by NS2B-NS3pro is essential for viral RNA replication [ 58 ], these inhibitory effects are expected to impair downstream viral replication processes. In summary, this study establishes a structural and mechanistic framework for NS2B-NS3pro inhibition and identifies a previously unrecognized solvent-exposed surface pocket on DENV-2 NS2B-NS3pro that has not been reported in other flaviviral proteases. This pocket may represent a DENV-2-specific inhibitory site with therapeutic potential. Moreover, GSH-AuNC is identified as a direct modulator of NS2B-NS3pro activity. Collectively, these findings expand the druggable landscape of DENV-2 NS2B-NS3pro and provide a structural basis for the rational development of small-molecule and nanomedicine-based antiviral strategies targeting flaviviral proteases. Declarations Declaration of competing interest The authors declare no known competing financial interests or personal relationships that could have influenced the work reported in this paper. Funding This research was funded by the R&D Program of Beijing Municipal Education Commission (KZ202210005001). Acknowledgements We thank the staff of beamlines BL18U1 and BL02U1 at the Shanghai Synchrotron Radiation Facility (SSRF) for assistance with data collection; Xuelin Zhao, Yangao Huo, Xiaoli Ma, Mei Li, and Tao Jiang (Institute of Biophysics, Chinese Academy of Sciences) for protein assays; Xiuqing Song, Yongwei Zhu, Wei Liu, and Huiqin Wang (Beijing University of Technology) for technical assistance; the Large-scale Instruments and Equipment Sharing Platform of Beijing University of Technology for support;and the R&D Program of the Beijing Municipal Education Commission for financial support (KZ202210005001). Data Availability Statement The atomic coordinate and structure factors have been deposited in the Protein Data Bank ( http://www.pdb.org ) under the accession code 9XVJ. References Mallapaty S (2024) The pathogens that could spark the next pandemic. 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(b) Q anion-exchange chromatography profile showing three elution peaks. (c) SDS-PAGE of fractions corresponding to the three peaks in (b). (d) SEC of proteins from peaks 2 and 3. (E) Crystals used for X-ray data collection.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8498727/v1/be1cc9eeb0340c687d920333.png"},{"id":100371231,"identity":"59f77d90-c37b-4ff2-8e4e-0f3006a396d7","added_by":"auto","created_at":"2026-01-16 08:09:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":9050324,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCrystal structure of DENV-2 NS2B-NS3pro bound to PEG.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Overall structure of NS2B-NS3pro (light-pink, NS2B; blue-white, NS3; orange, PEG fragment; cyan, the catalytic triad). (b) Electrostatic surface representation of NS2B-NS3pro. The Fo-Fc omit map of PEG fragment is contoured at 3\u003cem\u003eσ\u003c/em\u003e. (c) Close-up view of the PEG-binding site on NS3. Key interacting residues are shown as sticks. (d) Superposition of NS2B-NS3pro structures highlighting distinct ligand-binding sites, including the active-site region (blue dashed circle), a previously reported allosteric pocket (red dashed circle), and the newly identified PEG-binding site (green, PDB code: 5LC0; salmon, PDB code: 3U1I; light magenta, PDB code: 6MO0; slate, PDB code: 6MO1; white, PDB code: 6MO2).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8498727/v1/0ef08b2825f61fb9b5698878.png"},{"id":100237693,"identity":"6a29189f-60ea-419b-91a2-b5b8c7c4e56d","added_by":"auto","created_at":"2026-01-14 12:50:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":620951,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBinding and inhibition of GSH-AuNC on DENV-2 NS2B-NS3pro. \u003c/strong\u003e(a) BLI sensorgrams showing direct binding of GSH-AuNC to NS2B-NS3pro. (b) Fluorescence-based protease assay showing dose-dependent inhibition of NS2B-NS3pro by GSH-AuNC.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8498727/v1/ee145c51347065e184e949a6.png"},{"id":100370731,"identity":"bb6993a3-58a3-4d9f-8362-3944e1027d06","added_by":"auto","created_at":"2026-01-16 08:07:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":11790482,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMD simulation analysis of the NS2B-NS3pro-GSH-AuNC complex and proposed inhibition mechanism.\u003c/strong\u003e (a) Optimized structure of GSH-AuNC used in the simulations (yellow, Au atoms; pink, S atoms; blue, glutathione ligands). (b) Time evolution of the backbone RMSD of NS2B-NS3pro in complex with GSH-AuNC over a 100 ns MD trajectory. (c) Non-bonded interaction energy between NS2B-NS3pro and GSH-AuNC. (d) Representative binding mode of GSH-AuNC on the NS2B-NS3pro surface. (e) Schematic model of the proposed mechanism by which GSH-AuNC targets NS2B-NS3pro and interferes with viral polyprotein processing.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8498727/v1/6854beca64aba3845cd0191b.png"},{"id":100546016,"identity":"98a5b71f-6ee8-42da-93ef-5e3ff595d514","added_by":"auto","created_at":"2026-01-19 07:33:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":37304417,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8498727/v1/6ebd364b-4680-43c3-bbcd-ead3d4184a53.pdf"},{"id":100237697,"identity":"ab4c5eae-6fe4-458e-b2af-1413d5482568","added_by":"auto","created_at":"2026-01-14 12:50:06","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":1767885,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarymaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8498727/v1/97dc5a04bb350608a8f3a20b.pdf"}],"financialInterests":"","formattedTitle":"Structural Insights into DENV-2 NS2B-NS3 Protease and Inhibition by Glutathione-Coated Gold Nanocluster","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDengue, an infectious disease caused by DENV and transmitted by \u003cem\u003eAedes aegypti\u003c/em\u003e and \u003cem\u003eAedes albopictus\u003c/em\u003e mosquitoes, remains a major global health concern [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It is estimated that 2.5\u0026nbsp;billion people globally are at risk of dengue infection [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In 2024, around 14.1\u0026nbsp;million new cases and 9,508 deaths were reported, with both the in-cidence rate and the mortality rate increasing significantly compared to the previous year [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The clinical manifestations of DENV infection vary significantly in severity, ranging from mild dengue fever (DF) characterized by non-specific symptoms such as fever, headache, myalgia, and rash to life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Unfortunately, there are currently no clini-cally approved drugs available for the treatment of dengue virus infections. Although licensed dengue vaccines have been developed, none of them can provide complete protection against infection, especially for those who have not been previously infected with dengue viruses [\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10 CR11 CR12\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDENVs belong to the genus Flavivirus within the family of Flaviviridae and consist of four serotypes (DENV-1 to -4). The virions are spherical, with a diameter of 40\u0026ndash;50 nanometers and encapsulated by a lipopolysaccharide envelope. The positive-sense single-stranded RNA genome is approximately 11 kilobases in length and has a single open reading frame, encoding a polyprotein which is subsequently cleaved by either the host-encoded signal peptidase or the viral protease to generate three structural proteins (capsid (C), membrane (M)), and envelope (E)) and seven non-structural pro-teins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The NS2B\u0026ndash;NS3pro complex is a two-component serine protease, composed of the NS3 N-terminal protease domain and its essential catalytic cofactor NS2B, and plays a central role in polyprotein processing and RNA replication, making it a validated and druggable antiviral target. NS3 contains an N-terminal serine protease domain and a C-terminal helicase domain, while NS2B is indispensable for its proteolytic activity [\u003cspan additionalcitationids=\"CR17 CR18 CR19\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. High-resolution crystal structures from multiple serotypes reveal that, although the catalytic triad is highly conserved, the arrangement of the NS2B cofactor, the substrate-recognition loops, and the overall conformational plasticity exhibit pronounced serotype-specific differences [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Notably, subtle variations in the position and conformation of the NS2B C-terminal β-hairpin can alter the accessibility of the active-site pocket. In addition, differences in the flexibility and orientation of NS3 substrate-binding loops and solvent-exposed pockets adjacent to the catalytic triad may further modulate substrate recognition and influence inhibitor specificity [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Over the past decade, most inhibitors of DENV NS2B-NS3pro have been broadly classified into two classes: substrate-mimetic peptides and macrocyclic compounds that occupy the catalytic cleft, and small-molecule inhibitors that bind to the hydrophobic allosteric pocket adjacent to the NS2B \u003cem\u003eβ\u003c/em\u003e-hairpin [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. While these studies have established the validity of NS2B-NS3pro as an antiviral drug target, none of these inhibitors have advanced to clinical applications, partly due to the incomplete characterization of the protease\u0026rsquo;s druggable binding surfaces. Therefore, there is an urgent need for novel structural insights and mechanistic understanding to guide the development of new NS2B-NS3pro inhibitors.\u003c/p\u003e \u003cp\u003eGold nanoclusters (AuNCs) are special materials sized between individual gold atoms and plasmonic gold nanoparticles. Comprising several to hundreds of gold at-oms, they are smaller than 2 nm and have a typical core-shell structure. This structure comprises a metallic gold core, which is surrounded by stabilizing ligands that prevent aggregation and ensure structural stability [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Among them, GSH-AuNC represents a well-defined class of ultrasmall AuNCs coated and stabilized by glutathione, whose size-dependent properties, such as strong photoluminescence, high stability, and good biocompatibility, have enabled broad use in biomedical, catalytic and sensing applications. In recent years, a variety of nanomaterials, including AuNCs, have drawn increasing interest in antiviral research [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. However, the specific targeting ability of GSH-AuNC toward NS2B-NS3pro has not been reported previously.\u003c/p\u003e \u003cp\u003eThis study determined the 2.25 \u0026Aring; crystal structure of DENV-2 NS2B-NS3pro, revealing a novel, solvent-exposed, positively charged groove, spatially distinct from the catalytic cleft and known allosteric site. Biochemical, biophysical, and molecular simulation approaches characterized the inhibitory effect and underlying mechanism by which GSH-AuNC inhibits NS2B-NS3pro. These findings provide a structural basis and target validation for allosteric modulation of flaviviral NS2B-NS3pro and the design of nanoscale/small-molecule inhibitors.\u003c/p\u003e"},{"header":"Methods","content":"\u003ch2\u003e2.1. Preparation of gold nanoclusters\u003c/h2\u003e\n\u003cp\u003eThe preparation and characterization of glutathione-stabilized GSH-AuNC was exactly based on the method described in the previously published paper [31]. An aqueous solution of glutathione (GSH, 30 mM, 100 mL) was mixed with hydrogen tetrachloroaurate trihydrate (HAuCl\u003csub\u003e4\u003c/sub\u003e·3H\u003csub\u003e2\u003c/sub\u003eO, 20 mM, 100 mL) and gently stirred at 25 °C for 10 min (500 rpm). The reaction mixture was then heated to 70 °C and maintained under stirring (500 rpm) for 12 h to obtain a fluorescent GSH-AuNC solution. For purification, ethanol was added to the reaction solution at a 3:1 volume ratio, followed by thorough mixing and centrifugation at 10,000 rpm for 15 min. The supernatant was discarded, and the precipitated clusters were washed three times with 75% ethanol to remove residual ligands and impurities. The purified clusters were dried and subsequently redispersed in ultrapure water with the assistance of NaOH. Finally, the clusters were further purified using an ultrafiltration tube with a molecular weight cutoff of 3 kDa to remove any remaining free ions, yielding high-purity GSH-AuNCs.\u003c/p\u003e\n\u003ch2\u003e2.2. Expression and purification of NS2B-NS3pro\u003c/h2\u003e\n\u003cp\u003eThe gene fragments encoding NS2B (residues 49-95) and NS3 (residues 1-185) of DENV-2 were cloned into the pET-28a(+) vector. The resulting construct expresses NS2B-NS3pro with an N-terminal His\u003csub\u003e6\u003c/sub\u003e tag and a Gly₄-Ser-Gly₄ linker. The recombinant plasmid was transformed into\u003cem\u003e E. coli \u003c/em\u003eBL21(DE3) and screened on LB agar plates containing 50 μg/mL kanamycin. A single colony was inoculated into 10 mL LB medium containing kanamycin and grown overnight, then transferred into 1 L of LB medium and cultured at 37 °C and 180 rpm. Protein expression was induced with 0.2 mM IPTG when the OD\u003csub\u003e600\u003c/sub\u003e reached 0.6~0.8, followed by incubation at 16 °C, 150 rpm for 18 h. Cells were harvested by centrifugation at 6000 rpm for 10 min and resuspended in buffer containing 20 mM Tris-HCl (pH 8.0), 20 mM imidazole, and 500 mM NaCl. Cells were lysed using high-pressure homogenization in the presence of 1 mM PMSF, and the lysate was clarified by centrifugation at 15,000 rpm for 60 min. The supernatant was loaded onto a Ni\u003csup\u003e2+\u003c/sup\u003e-NTA affinity column and eluted with 20~300 mM imidazole gradient. The eluted protein was dialyzed overnight at 4 °C in buffer containing 50 mM Tris-HCl (pH 8.5), 50 mM NaCl, 1 mM TCEP, and 5% glycerol in the presence of Tobacco Etch Virus (TEV) protease (TEV: protein = 1:15, w/w). After dialysis, the sample was filtered through a 0.22 μm membrane and concentrated using a 10 kDa MWCO ultrafiltration tube to 20 mg/mL. \u003c/p\u003e\n\u003cp\u003eThe protein was further purified by an anion-exchange Q column using buffer A (50 mM Tris-HCl pH 8.5, 50 mM NaCl, 1 mM TCEP, 5% glycerol) and a 0~30% gradient of buffer B (50 mM Tris-HCl pH 8.5, 1 M NaCl, 1 mM TCEP, 5% glycerol). The collected components were concentrated using a 10 kDa MWCO centrifugal concentrator and further identified by size-exclusion chromatography (SEC) column preloaded with 20 mM Tris (pH 7.5) and 150 mM NaCl. Homogeneous fractions were concentrated to 20 mg/mL, flash-frozen in liquid nitrogen, and stored at -80 °C.\u003c/p\u003e\n\u003ch2\u003e2.3. Crystallization of NS2B-NS3pro\u003c/h2\u003e\n\u003cp\u003eCrystals of NS2B-NS3pro were grown using the sitting-drop vapor diffusion method at 4 °C. Prior to crystallization, the protein was diluted to 4 mg/mL using buffer containing 20 mM Tris (pH 7.5) and 150 mM NaCl. Equal volumes (1 μL + 1 μL) of protein solution and reservoir solution were mixed and placed on 96-well crystallization plates with 100 μL reservoir solution in each well. Initial screening was performed using commercial sparse-matrix kits as well as in-house screens. Crystals suitable for X-ray diffraction were obtained from a reservoir solution containing 0.1 M sodium acetate (pH 5.5) and 44% (v/v) PEG-200.\u003c/p\u003e\n\u003ch2\u003e2.4. Data Collection and Processing\u003c/h2\u003e\n\u003cp\u003eDiffraction data were collected at the beamline BL18U1 of the Shanghai Synchrotron Radiation Facility (SSRF). Data indexing and scaling were performed using XDS [32]. Molecular replacement was carried out using Phaser 2.8 within the CCP4 (v9.0) suite [33], with the DENV-2 NS2B-NS3pro structure (PDB code: 2FOM) as the search model. Manual model building was performed in Coot 0.9.8 [34], and structure refinement was completed using Phenix.refine 1.16 [35] and Refmac 5.8 [36]. The quality of the final structure was assessed using MolProbity ver. 4.5 [37]. Crystallographic data collection and refinement statistics are summarized in Table S1.\u003c/p\u003e\n\u003ch2\u003e2.5. Bio-Layer Interferometry (BLI) analysis\u003c/h2\u003e\n\u003cp\u003eAvi tag (GLNDIFEAQKIEWHE) was added to the C-terminus of NS2B-NS3pro by PCR, using the pET-28a(+)-NS2B-NS3pro plasmid as the template [38]. The new construct (pET-28a(+)-NS2B-NS3pro-Avi tag) was co-expressed with biotin ligase (BirA) in \u003cem\u003eE. coli\u003c/em\u003e. The cells were grown in 1 L of LB medium with kanamycin and ampicillin at 37 °C and 180 rpm. When OD₆₀₀ reached 0.6~0.8, 0.2 mM IPTG and 50 μM biotin were added to start protein expression, and the culture was continued at 16 °C and 150 rpm for 18 hours. The purification method was the same as the NS2B-NS3pro purification described earlier. Biotinylated protein was purified as described in Section 2.2.\u003c/p\u003e\n\u003cp\u003eThe binding experiment was carried out using the Octet® R8 instrument. Prior to the start of the experiment, biotinylated NS2B-NS3pro was diluted to 50 μg/mL in a buffer composed of 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.05% (v/v) Tween-20. After that, the sensors were placed in GSH-AuNC solutions at different concentrations (0, 21.25, 42.5, 85, 170, and 340 μM) to record association, followed by buffer for dissociation. Reference subtraction using unloaded biosensors was performed to correct for nonspecific signals. The data were analyzed using Octet software (v12, Sartorius), and the 1:1 model was used to obtain \u003cem\u003ek\u003c/em\u003e\u003csub\u003eon\u003c/sub\u003e, \u003cem\u003ek\u003c/em\u003e\u003csub\u003eoff\u003c/sub\u003e, and \u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e values.\u003c/p\u003e\n\u003ch2\u003e2.6. Protease activity assay\u003c/h2\u003e\n\u003cp\u003eThe effect of GSH-AuNC on NS2B-NS3pro activity was measured using a continuous fluorescence assay. NS2B-NS3pro was used at 100 nM and mixed with GSH-AuNC at 1-276 μM (8-point two-fold dilution). GSH-AuNC and NS2B-NS3pro were mixed and pre-incubated at 37 °C for 30 min, followed by addition of the fluorogenic substrate Bz-nKRR-MCA to a final concentration of 50 mM [39]. Fluorescence was recorded immediately at Ex/Em = 360/450 nm at 1 min intervals for 10 min with brief shaking (5 s, 200 rpm) before each read. IC\u003csub\u003e50\u003c/sub\u003e values were determined by nonlinear regression using R software (version 4.5.1) [40].\u003c/p\u003e\n\u003ch2\u003e2.7. Molecular dynamics simulations\u003c/h2\u003e\n\u003cp\u003eThe GSH-AuNCs structures were generated using GaussView 5.0, followed by geometry optimization via the Gaussian 09 suite employing density functional theory (DFT) [41]. For this optimization, the LANL2DZ basis set was applied to all gold atoms, while the 631G(d) basis set was utilized for carbon, hydrogen, oxygen, nitrogen, and sulfur atoms. The three-dimensional structure of NS2B-NS3pro was derived from X-ray crystallo-graphic data.\u003c/p\u003e\n\u003cp\u003eBinding configurations were constructed using the Visual Molecular Dynamics (VMD) software [42], which also facilitated the generation of input files for subsequent MD simulations. All simulations were conducted under the NPT ensemble with peri-odic boundary conditions in all spatial dimensions. Temperature and pressure were regulated using the Langevin thermostat and the Nosé-Hoover Langevin piston method, ensuring full equilibration of the systems under physiological conditions. Electrostatic interactions were computed using the particle mesh Ewald (PME) tech-nique under conducting boundaries, while van der Waals forces were cutoff at 12 Å. The CHARMM22 and CHARMM36 force fields [43] were employed to describe the system, with explicit solvent molecules modeled using the TIP3P water model. Simula-tions were carried out using NAMD 2.13 [44], and trajectory analysis was performed with VMD 1.9.3. Prior to production runs, initial energy minimization involved 5,000 steps of steepest descent, followed by a 100 ns equilibration phase during which the system was gradually heated to 300 K. Thermal equilibration was achieved through coupling with a sodium ion solution.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003e3.1. Expression, purification and crystallization of DENV-2 NS2B-NS3pro fusion protein\u003c/h2\u003e\n\u003cp\u003eThe recombinant DENV-2 NS2B-NS3pro construct carrying an N-terminal His₆ tag was expressed in\u003cem\u003e E. coli\u003c/em\u003e BL21 (DE3). After induction, cells were harvested, resus-pended in lysis buffer, and clarified by centrifugation. The supernatant was applied to a Ni\u003csup\u003e2\u003c/sup\u003e+-NTA affinity chromatography, which removed most contaminating proteins and yielded His-tagged protein in the elution fractions (\u003cstrong\u003eFig. 1A\u003c/strong\u003e). After dialysis, the His₆ tag was removed by TEV protease, and the sample was further purified on an anion-exchange Q column, which yielded three peaks (\u003cstrong\u003eFig. 1B\u003c/strong\u003e). SDS-PAGE (\u003cstrong\u003eFig\u003c/strong\u003e\u003cstrong\u003e. \u003c/strong\u003e\u003cstrong\u003e1C\u003c/strong\u003e) revealed that peak 1 contained a ~28 kDa band corresponding to TEV protease, whereas peaks 2 and 3 both contained a band at approximately 37 kDa. This apparent molecular weight is higher than the calculated mass of NS2B-NS3pro (~26 kDa), consistent with the abnormal migration reported previously [45, 46]. Peaks 2 and 3 were analyzed by SEC and eluted as single, symmetric peaks at nearly identical volumes (\u003cstrong\u003eFig. 1D\u003c/strong\u003e), indicating a homogeneous species with an apparent monomeric size in solution. These findings suggest the possible presence of minor conformational or charge variants in solution, and anion-exchange Q chromatography was necessary to obtain the target protein at higher purity.\u003c/p\u003e\n\u003cp\u003eFor crystallization, purified NS2B-NS3pro was screened using commercial kits and in-house conditions by sitting-drop vapor diffusion. Initial hits were obtained under several polyethylene glycol-based conditions. These were subsequently optimized by varying buffer composition. Well-diffracting crystals were finally obtained in 0.1 M sodium acetate (pH 5.5) and 44% (v/v) PEG 200. The crystals were uniform, with smooth, well-defined faces (\u003cstrong\u003eFig. 1E\u003c/strong\u003e), and were suitable for high-resolution X-ray data collection.\u003c/p\u003e\n\u003ch2\u003e3.2. NS2B-NS3pro structure revealing a PEG-binding pocket\u003c/h2\u003e\n\u003cp\u003eThe crystal structure of DENV-2 NS2B-NS3pro reveals a functional enzyme complex composed of the NS2B cofactor and the NS3 protease domain, with an additional PEG molecule bound on the surface of NS3 (\u003cstrong\u003eFig. 2A, B\u003c/strong\u003e). NS2B wraps around the N-terminal serine protease domain of NS3. NS3pro adopts a canonical chymotrypsin-like fold with a \u0026beta;-barrel core, while NS2B contributes four \u0026beta;-strands and one \u0026alpha;-helix to stabilize the NS3pro structure. The substrate-binding pocket is formed by NS3 \u0026beta;-strands, with the catalytic triad residues His51, Asp75, and Ser135 located at the active site (\u003cstrong\u003eFig. 2A\u003c/strong\u003e). The NS2B\u0026ndash;NS3 interface is primarily stabilized by \u003cem\u003e\u0026beta;\u003c/em\u003e-sheet-mediated interactions, consistent with previous structural reports (\u003cstrong\u003eFig. S1, S2\u003c/strong\u003e) [23].\u003c/p\u003e\n\u003cp\u003eA striking feature of the structure is a well-defined electron density corresponding to a PEG fragment bound on the NS3 surface within a shallow, solvent-exposed pocket formed by residues Phe116, Lys117, Thr118, Val154, Val155, Thr156, and Arg157. This pocket is surrounded by four \u003cem\u003e\u0026beta;\u003c/em\u003e-strands (\u003cem\u003e\u0026beta;\u003c/em\u003e10, \u003cem\u003e\u0026beta;\u003c/em\u003e11, \u003cem\u003e\u0026beta;\u003c/em\u003e14, and \u003cem\u003e\u0026beta;\u003c/em\u003e15), located adjacent to the C-terminal loop of NS3 (\u003cstrong\u003eFig. 2A, C\u003c/strong\u003e). The analysis using parKVFinder [47] further revealed that the pocket has a surface area of ~ 69.4 \u0026Aring;\u0026sup2; and a calculated volume of ~ 85.75 \u0026Aring;\u0026sup3;. The PEG molecule, derived from the PEG-200 used in crystallization, occupies a narrow groove next to this flexible C-terminal loop, making close contacts with the backbones of Val155 and Lys117 (~3.0 \u0026Aring;) and additional van der Waals interactions with Phe116 and the backbone of Thr156 (\u003cstrong\u003eFig. 2C\u003c/strong\u003e). Electrostatic surface representation shows that this pocket has a positively charged entrance and a largely hydrophobic interior, creating a mixed hydrophobic and positively charged environment that can readily accommodate small, flexible ligands (\u003cstrong\u003eFig. 2B\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eTo assess sequence and structural conservation, sequence and structural alignments of NS2B-NS3pro proteins from DENV serotypes 1-4, Yellow Fever virus (YFV), West Nile virus (WNV), and Zika virus (ZIKV) were conducted (\u003cstrong\u003eFig. S3\u003c/strong\u003e) [23, 39, 48-52]. Multiple sequence alignment showed 22.1% overall identity and 75.9% similarity. The catalytic triad (His51, Asp75, Ser135) and surrounding residues were strictly conserved, underscoring their central role in catalysis. Structural superposition yielded C\u003csub\u003e\u0026alpha;\u003c/sub\u003e RMSD values of 0.189-0.829 \u0026Aring;, indicating a conserved serine-protease fold with the catalytic triad positioned almost identically. Despite this global similarity, DENV-2 NS2B-NS3pro features a previously unreported PEG-binding pocket on the NS3 surface, which is absent in the corresponding regions of other flaviviral proteases, suggesting that it may represent a DENV-2-specific potential allosteric binding site (\u003cstrong\u003eFig. S3\u003c/strong\u003e). Comparative analysis of complexes with both active‑site and allosteric inhibitors shows that the PEG‑binding crevice is spatially distant from the catalytic triad, the substrate‑binding cleft, and the previously described allosteric site (\u003cstrong\u003eFig. 2D\u003c/strong\u003e) [22, 49, 53]. Thus, the PEG-bound groove represents a previously unrecognized solvent-exposed pocket, distinct from canonical regulatory sites, and may serve as a transient ligand-binding surface. Taken together, these observations suggest that this pocket may serve as a potential allosteric site on NS2B-NS3pro and could be explored in future fragment-based or structure-guided efforts to identify small-molecule modulators.\u003c/p\u003e\n\u003ch2\u003e3.3. Binding and inhibition of NS2B-NS3pro by GSH-AuNC\u003c/h2\u003e\n\u003cp\u003ePrevious studies have shown that the GSH-AuNC nanocluster can inhibit the main protease (Mpro) of SARS-CoV-2 and suppress viral replication, suggesting that AuNCs may act as direct protease modulators [31]. Motivated by these findings, we next examined whether the GSH-AuNC can also target the flaviviral NS2B-NS3pro.\u003c/p\u003e\n\u003cp\u003eTo characterize the interaction between GSH-AuNC and NS2B-NS3pro, we used bio-layer interferometry (BLI). NS2B-NS3pro was expressed with a C-terminal Avi tag (GLNDIFEAQKIEWHE), allowing in vivo biotinylation during \u003cem\u003eE. coli\u003c/em\u003e expression. Then, the biotinylated NS2B-NS3pro was immobilized on streptavidin biosensors, and binding to GSH-AuNC was monitored by BLI. Exposure of the sensors to increasing concentrations of GSH-AuNC produced clear, concentration-dependent association and dissociation phases (\u003cstrong\u003eFig. 3A\u003c/strong\u003e). Global fitting of the sensorgrams yielded an equilibrium dissociation constant (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e) of 15.64 \u0026mu;M (\u003cem\u003eR\u0026sup2;\u003c/em\u003e = 0.98), indicating a direct interaction between the GSH-AuNC and NS2B-NS3pro in the micromolar affinity range.\u003c/p\u003e\n\u003cp\u003eWe then evaluated whether this interaction translated into functional inhibition using an in vitro fluorescence-based protease assay. NS2B-NS3pro was pre-incubated with increasing concentrations of GSH-AuNC before addition of a fluorogenic peptide substrate. Enzymatic activity decreased in a dose-dependent manner, and nonlinear fitting of the inhibition curve gave an IC\u003csub\u003e50\u003c/sub\u003e value of 16.04 \u0026mu;M (\u003cstrong\u003eFig. 3B\u003c/strong\u003e). The close agreement between the binding affinity and the IC\u003csub\u003e50\u003c/sub\u003e strongly supports a direct inhibitory effect of the GSH-AuNC on NS2B-NS3pro, rather than nonspecific aggregation or indirect interference with the assay.\u003c/p\u003e\n\u003ch2\u003e3.4. Molecular dynamics simulations of the NS2B-NS3pro-GSH-AuNC complex\u003c/h2\u003e\n\u003cp\u003eThe structure of the GSH-AuNC nanocluster was constructed and optimized using quantum chemical calculations (\u003cstrong\u003eFig. 4A\u003c/strong\u003e). GSH-AuNC consists of a well-defined gold core coordinated by glutathione ligands through Au-S bonds. The optimized GSH-AuNC model, together with our crystal structure of DENV-2 NS2B-NS3pro, was then used to set up molecular dynamics (MD) simulations of the NS2B-NS3pro-GSH-AuNC complex using the NAMD software package. A 100 ns MD trajectory was generated, and the root-mean-square deviation (RMSD) of the protein backbone was monitored to assess system stability (\u003cstrong\u003eFig. 4B\u003c/strong\u003e). The complex reached equilibrium after ~10 ns and remained stable for the remainder of the simulation.\u003c/p\u003e\n\u003cp\u003eNon-bonded interaction energies between NS2B-NS3pro and GSH-AuNC were decomposed into van der Waals and electrostatic components in MD simulations (\u003cstrong\u003eFig. 4C\u003c/strong\u003e). Electrostatic interactions dominate the binding, with van der Waals contacts providing additional stabilization. Analysis of representative equilibrium conformations shows that GSH-AuNC is stably positioned adjacent to the catalytic triad (His51, Asp75, Ser135) (\u003cstrong\u003eFig. 4D\u003c/strong\u003e). The cluster forms hydrogen bonds and salt bridges with Arg54 and Arg157, as well as hydrogen bonds with Ser135 and His51, thereby occupying the pocket adjacent to the active site. These interactions suggest that GSH-AuNC hampers substrate access and perturbs the catalytic environment by creating steric and electrostatic barriers at the entrance to the NS2B-NS3pro active center, providing a potential structural basis for the observed inhibition of protease activity. Notably, Arg157 also contributes to forming the PEG-binding pocket in our crystal structure (\u003cstrong\u003eFig. 2C\u003c/strong\u003e), suggesting that the interaction of GSH-AuNC with this surface pocket may further stabilize an inhibitory conformation of NS2B-NS3pro and thereby impede substrate entry. Based on our combined structural, biophysical, and computational analyses, we propose a refined working model to illustrate how GSH-AuNC exerts its inhibitory effect on DENV-2 NS2B-NS3pro (\u003cstrong\u003eFig. 4E\u003c/strong\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eNS2B-NS3pro is a key serine protease of DENV-2, predominantly localized on the cytoplasmic face of the host cell endoplasmic reticulum (ER) membrane [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. It plays a critical role in viral polyprotein processing and viral particle maturation [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], making it an attractive target for antiviral drug development. Although significant progress has been made in developing NS2B-NS3pro inhibitors, the protease\u0026rsquo;s active site is relatively shallow and highly exposed, and its overall surface exhibits pronounced charge characteristics, which complicates the formation of stable, high-affinity interactions with small molecules [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Therefore, the exploration of inhibitors with novel chemical scaffolds and unique mechanisms of action remains essential to advance therapeutic strategies.\u003c/p\u003e \u003cp\u003eIn this study, we successfully resolved the high-resolution crystal structure of DENV-2 NS2B-NS3pro and identified a previously unreported PEG-binding pocket on the NS3 surface. The PEG molecule, originating from PEG-200 used in crystallization, occupies a shallow groove adjacent to the flexible C-terminal loop of NS3. Although this binding may be a crystallization artifact, its position highlights a solvent-exposed pocket capable of accommodating flexible ligands. Structural comparisons indicate that analogous binding sites have not been observed in other flaviviral NS3 proteases (\u003cb\u003eFig. S3\u003c/b\u003e), suggesting that this pocket may serve as a potential DENV-2-specific allosteric binding site.\u003c/p\u003e \u003cp\u003eNanoparticles have attracted considerable attention in disease prevention and treatment owing to their exceptional tunability. Modifying their surface properties can enhance both selectivity and efficacy [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Here, we report for the first time that GSH-AuNCs can inhibit the dengue virus NS2B-NS3pro. Biochemical assays demonstrated that GSH-AuNCs bind the protease with micromolar affinity (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e = 15.64 \u0026micro;M) and inhibit its catalytic activity with a comparable IC\u003csub\u003e50\u003c/sub\u003e value of 16.04 \u0026micro;M, supporting a direct inhibitory mechanism.\u003c/p\u003e \u003cp\u003eMD simulations further elucidated the atomic-level basis of inhibition: GSH-AuNCs occupy regions adjacent to the catalytic triad (His51, Asp75, Ser135), forming persistent hydrogen bonds and electrostatic interactions with residues such as Arg157, Arg54, Ser135, and His51. This interaction sterically blocks the substrate-binding cleft and constrains the conformational changes required for substrate entry, thereby reducing catalytic efficiency (\u003cb\u003eFig. S4\u003c/b\u003e). Notably, Arg157 also participates in the formation of the PEG-binding pocket observed in the crystal structure, suggesting that the interaction between GSH-AuNCs and this pocket may further enhance inhibition of NS2B-NS3pro. Given that efficient polyprotein cleavage by NS2B-NS3pro is essential for viral RNA replication [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], these inhibitory effects are expected to impair downstream viral replication processes.\u003c/p\u003e \u003cp\u003eIn summary, this study establishes a structural and mechanistic framework for NS2B-NS3pro inhibition and identifies a previously unrecognized solvent-exposed surface pocket on DENV-2 NS2B-NS3pro that has not been reported in other flaviviral proteases. This pocket may represent a DENV-2-specific inhibitory site with therapeutic potential. Moreover, GSH-AuNC is identified as a direct modulator of NS2B-NS3pro activity. Collectively, these findings expand the druggable landscape of DENV-2 NS2B-NS3pro and provide a structural basis for the rational development of small-molecule and nanomedicine-based antiviral strategies targeting flaviviral proteases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e \u003cp\u003eThe authors declare no known competing financial interests or personal relationships that could have influenced the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was funded by the R\u0026amp;D Program of Beijing Municipal Education Commission (KZ202210005001).\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe thank the staff of beamlines BL18U1 and BL02U1 at the Shanghai Synchrotron Radiation Facility (SSRF) for assistance with data collection; Xuelin Zhao, Yangao Huo, Xiaoli Ma, Mei Li, and Tao Jiang (Institute of Biophysics, Chinese Academy of Sciences) for protein assays; Xiuqing Song, Yongwei Zhu, Wei Liu, and Huiqin Wang (Beijing University of Technology) for technical assistance; the Large-scale Instruments and Equipment Sharing Platform of Beijing University of Technology for support;and the R\u0026amp;D Program of the Beijing Municipal Education Commission for financial support (KZ202210005001).\u003c/p\u003e\u003ch2\u003eData Availability Statement\u003c/h2\u003e \u003cp\u003eThe atomic coordinate and structure factors have been deposited in the Protein Data Bank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.pdb.org\u003c/span\u003e\u003cspan address=\"http://www.pdb.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) under the accession code 9XVJ.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMallapaty S (2024) The pathogens that could spark the next pandemic. 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Virol J 14:95. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1186/s12985-017-0761-1\u003c/span\u003e\u003cspan address=\"10.1186/s12985-017-0761-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Dengue virus, NS2B-NS3pro, crystal structure, glutathione-coated gold nanocluster, Molecular dynamics simulations, Molecular interactions","lastPublishedDoi":"10.21203/rs.3.rs-8498727/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8498727/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDengue virus (DENV) continues to pose a significant global health threat, with increasing infection rates and limited treatment options. The viral NS2B-NS3 protease (NS2B-NS3pro), a highly conserved two-component enzyme essential for polyprotein processing and replication, is a key target for antiviral drug development. Here, we report the 2.25 \u0026Aring;-resolution crystal structure of DENV serotype 2 (DENV-2) NS2B-NS3pro, in which a PEG fragment is bound within a solvent-exposed pocket between \u003cem\u003eβ\u003c/em\u003e10, \u003cem\u003eβ\u003c/em\u003e11, \u003cem\u003eβ\u003c/em\u003e14 and \u003cem\u003eβ\u003c/em\u003e15 of NS3. This structure reveals a previously unidentified potential allosteric site, suggesting a novel pocket for inhibitor design. We also show that the glutathione-coated gold nanocluster (GSH-AuNC) directly inhibits DENV-2 NS2B-NS3pro. Bio-layer interferometry and fluorescence-based protease assays indicate that the nanocluster binds NS2B-NS3pro with a dissociation constant of 15.64 \u0026micro;M and inhibits its catalytic activity with an IC\u003csub\u003e50\u003c/sub\u003e of 16.04 \u0026micro;M, consistent with a direct inhibition mechanism. Molecular dynamics simulations further suggest that GSH-AuNC interacts with the catalytic triad of NS2B-NS3pro, forming stable electrostatic and van der Waals interactions that block substrate binding. Collectively, these findings provide a structural and mechanistic basis for the development of inhibitors targeting DENV-2 NS2B-NS3pro, offering new strategies for antiviral therapy using small molecules or nanomedicines.\u003c/p\u003e","manuscriptTitle":"Structural Insights into DENV-2 NS2B-NS3 Protease and Inhibition by Glutathione-Coated Gold Nanocluster","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-14 12:50:01","doi":"10.21203/rs.3.rs-8498727/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-02-20T08:08:10+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-13T08:34:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-03T02:17:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Virology","date":"2026-01-02T01:57:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a3cd0832-5210-4b16-9ba0-14b7b4c0a45f","owner":[],"postedDate":"January 14th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-19T06:56:49+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-14 12:50:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8498727","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8498727","identity":"rs-8498727","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}