Lupeol as a Modulator of Bacterial Resistance Mediated by the MepA Efflux Pump in Staphylococcus aureus

Lupeol as a Modulator of Bacterial Resistance Mediated by the MepA Efflux Pump in Staphylococcus aureus | 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 Lupeol as a Modulator of Bacterial Resistance Mediated by the MepA Efflux Pump in Staphylococcus aureus Nara Juliana Santos Araújo, Camila Aparecida Pereira Silva, Cicera Datiane Morais Oliveira-Tintino, and 16 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8939866/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Increasing bacterial resistance has stimulated the search for alternative therapeutic strategies, particularly efflux pump inhibitors, which enhance antimicrobial activity by increasing intracellular drug concentration. Natural compounds such as Lupeol have emerged as promising resistance modulators. This study investigated the ability of Lupeol to inhibit the MepA efflux pump in the K2068 strain of Staphylococcus aureus . Minimum inhibitory concentration, efflux inhibition by microdilution, membrane permeability (Sytox Green), ethidium bromide fluorescence, gene expression by RT-qPCR, and molecular docking analyses were performed. Lupeol showed no intrinsic antibacterial or cell wall activity but effectively inhibited the MepA efflux pump. Its effect on ethidium bromide fluorescence was dose-dependent, and RT-qPCR confirmed significant suppression of mepA gene expression. Molecular docking revealed strong binding affinity (− 9.010 kcal/mol) involving van der Waals, hydrophobic, and hydrogen interactions. These findings indicate that Lupeol is a promising efflux pump inhibitor with potential to combat bacterial resistance. Triterpenes molecular docking RT-qPCR membrane permeability efflux inhibitor Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1.INTRODUCTION Bacterial resistance to antibiotics has become a growing challenge for healthcare institutions globally, representing a direct threat to human life [ 1 ]. This phenomenon is intensified by factors such as the indiscriminate use of antimicrobials—widely observed during the COVID-19 pandemic—and by genetic mechanisms that favor the maintenance and spread of resistance among bacterial strains [ 2 ]. Given this alarming scenario, investment in research aimed at identifying compounds capable of effectively and safely combating multidrug-resistant organisms becomes urgent [ 3 ]. Among emerging strategies, the development of efflux pump inhibitors stands out, whose action consists of blocking bacterial systems that reduce the intracellular concentration of drugs, compromising their effectiveness. Efflux pumps are transmembrane proteins that act in the active extrusion of toxic substances and antimicrobials into the extracellular environment. The MepA pump, belonging to the MATE (Multidrug and Toxic Compound Extrusion) family, is a relevant example due to its ability to expel various compounds from the cell interior [ 1 , 2 , 3 , 4 ]. In this context, secondary metabolites have gained prominence due to their diverse chemical structures and promising mechanisms of action for the development of new therapeutic agents [ 5 ]. Among these compounds, terpenoids have attracted interest for their antinociceptive, antiviral, anti-inflammatory, and antibacterial properties [ 6 ]. Lupeol, a triterpene widely distributed in nature, stands out for its broad range of pharmacological activities, including antibacterial, anti-inflammatory, and wound-healing effects [ 7 , 8 ]. Therefore, the present study aimed to evaluate the inhibitory capacity of the triterpene Lupeol on the MepA efflux pump, seeking to explore its potential as a modulator of bacterial resistance. The K2068 strain of Staphylococcus aureus , used in this study, is a clinical strain characterized by exhibiting resistance to multiple antimicrobials and expressing the MepA efflux pump. 2. MATERIALS AND METHODS 2.1- Lupeol and bacterial strain The triterpene Lupeol (C 30 H 50 O), used in this study, was acquired from Sigma-Aldrich (St. Louis, MO, USA). The bacterial strain employed was the K2068 strain of Staphylococcus aureus , known to express the MepA efflux pump, acquired from the microorganism collection of the Microbiology and Molecular Biology Laboratory of the Regional University of Cariri (LMBM/URCA). 2.2-Determination of minimum inhibitory concentration and efflux pump inhibitory potential The minimum inhibitory concentration (MIC) was determined by the microdilution method in 96-well plates, according to the guidelines established by CLSI [ 9 ]. The experiments were performed in triplicate, using resazurin as an indicator of bacterial viability. Lupeol dilutions and standard inhibitors were prepared in BHI medium and the plates were incubated at 35°C for 24 hours. The MIC reading was performed by observing the change in resazurin color, which indicated the presence or absence of bacterial growth. The evaluation of efflux pump inhibition was conducted using a subinhibitory concentration (MIC/8) of standard inhibitors and Lupeol. A solution containing 10% bacterial inoculum, the inhibitors, and Lupeol was prepared and completed with BHI medium, which was transferred to 96-well microdilution plates. Then, 100 µL of ciprofloxacin or ethidium bromide (EtBr) were added in serial dilutions, ranging from 0.5 to 512 µg/mL. The plates were incubated at 35°C for 24 hours, and resazurin was again used to detect bacterial viability [ 9 , 10 ]. 2.3-Membrane permeability assessment Membrane permeability was assessed using SYTOX Green dye. Inocula of S. aureus K2068, adjusted to the 0.5 standard of the McFarland scale, were distributed in 96-well black plates. Lupeol was added at final concentrations of 200, 100, and 50 µg/mL, while polymyxin B was used as a positive control at concentrations of 100, 50, and 25 µg/mL, and PBS as a negative control. After 1 hour of incubation, 100 µL of SYTOX Green (1 µM) were added, followed by another 30 minutes of incubation. Fluorescence was then measured using the Cytation 1 reader (BioTek ® ) with Gen5™ 3.11 software, using 485 nm excitation and 528 nm emission filters. The tests were performed in triplicate, as described by Yuen et al . [ 11 ]. 2.4-Fluorescence assessment of ethidium bromide S. aureus K2068 strains were cultured and incubated at 37°C. The bacterial inoculum was adjusted to the 0.5 standard of the McFarland scale in PBS and combined with Lupeol at concentrations of 10, 20, and 50 µg/mL. CCCP (50 µg/mL) was used as a positive control, and PBS containing only the inoculum was the negative control. After incubation for 1 hour and 30 minutes, EtBr (100 µg/mL) was added, followed by a new incubation. The samples were then centrifuged, washed with PBS, and resuspended. The resulting pellet was transferred to 96-well black plates, and fluorescence was measured using the Cytation 1 reader (BioTek®) with Gen5™ 3.22 software, using excitation filters at 530 nm and emission filters at 590 nm. The tests were performed in triplicate, as described by Oliveira-Tintino and colleagues [ 12 ]. 2.5-Quantification of MepA gene expression by RT-Qpcr The Staphylococcus aureus K2068 strain was cultured in BHI medium at 37°C for 24 h. Subsequently, the bacterial suspension was incubated in liquid BHI medium under two experimental conditions: control, containing ciprofloxacin at a sub-inhibitory concentration (MIC/8), and test, containing ciprofloxacin (MIC/8) associated with Lupeol (MIC/8). All conditions were prepared in triplicate, with a final volume of up to 5 mL, and incubated at 37°C for 24 h. After incubation, total RNA was extracted using TRIzol™ (Thermo Fisher Scientific), preceded by cell lysis with lysozyme and Tris-EDTA buffer. The RNA obtained was quantified and used for cDNA synthesis using the SuperScript™ VILO™ Master Mix kit (Thermo Fisher Scientific), according to the manufacturer's instructions. The expression of the mepA gene was evaluated by RT-qPCR using the SYBR™ Green PCR Master Mix (Thermo Fisher Scientific) on the qTowerG3 instrument (Analytik Jena). The 23S gene was used as an endogenous control. Reactions were performed with an annealing temperature of 61°C, and relative gene expression was determined by the ΔΔCt method. 2.6-Molecular docking The structures of lupeol, ethidium bromide, ciprofloxacin, and CCCP were generated in MarvInSketch software (Chemaxon Ltd., version 23.14) and optimized with Open Babel (version 2.4.1) using the MMFF94 force field. The three-dimensional structure of the MepA protein was modeled by homology in SWISS-MODEL, with the UniProt Q2G140 sequence from Staphylococcus aureus (NCTC 8325/PS 47) and the 3W4T model from PDB [ 13 ]. Molecular docking was performed in AutoDock Vina (version 1.2.5) using AutoDock Tools (MGLTools, version 1.5.7), configuring a 50 Å grid box centered on the conveyor cavity (coordinates: x = -32.554; y = 0.012; z = 11.605). The fit validation followed the methodology of Martin et al. (2024), with a limit of 2.0 Å for the root mean square deviation (RMSD) [ 14 , 15 ]. 2.7-Statistical Analysis Statistical analysis was performed using GraphPad Prism 8.01 software. Comparisons between multiple groups were made using two-way ANOVA, followed by the Bonferroni post-hoc test. For comparisons between independent groups with a single variable, one-way ANOVA was used, followed by Tukey's test. The significance level adopted was p < 0.01. Results are expressed as mean ± standard error of the mean. RT-qPCR results were expressed as geometric mean + standard deviation, statistically evaluated using the T-test, using GraphPad Prism 9.2.0 software. Differences were considered significant when p < 0.05. 3. RESULTS 3.1-Determination of the minimum inhibitory concentration and evaluation of efflux pump inhibition After the minimum inhibitory concentration (MIC) assays and evaluation of the inhibitory potential of the MepA efflux pump, it was observed that Lupeol showed isolated antibacterial activity, but without clinical relevance, with an MIC ≥ 1024 µg/mL. In the efflux pump inhibition tests, Lupeol promoted a significant reduction in the MIC of ciprofloxacin (from 64 µg/mL to 20.1 µg/mL) and ethidium bromide (from 256 µg/mL to 64 µg/mL), compared to the results obtained with these compounds in isolation. This reduction was more significant than that observed with the positive control CCCP, which showed an MIC of 32 µg/mL for ciprofloxacin and 101.6 µg/mL for ethidium bromide (Fig. 1 ). These results suggest that Lupeol exhibits more efficient inhibition of the MepA efflux pump compared to the standard CCCP inhibitor. 3.2-Membrane permeability assessment and fluorescence assessment of ethidium bromide In the membrane permeability assessment, it was observed that, in the presence of the K2068 strain, none of the Lupeol concentrations used in the assay significantly altered the fluorescence emission of Sytox green, indicating that the metabolite does not exhibit cell wall activity. The positive control of the test showed a change in the fluorescence level, validating the assay performed (Fig. 2 ). In the fluorescence assessment of ethidium bromide, it was possible to perceive that, in the presence of K2068, a dose-dependent effect occurred, in which it is possible to see that the higher the concentration of Lupeol used, the greater its effect on increasing the fluorescence intensity of bromide, indicating inhibition of the MepA efflux pump. The CCCP control considerably increased fluorescence emission compared to the negative control, indicating the reproducibility of the assay (Fig. 3 ). 3.3- Quantification of mepA gene expression by RT-qPCR RT-qPCR analysis revealed that the combination of ciprofloxacin with Lupeol significantly reduced mepA gene expression compared to the control, showing ΔCt ≈ − 2.5, which corresponds to a decrease of approximately 5.7 times in transcription levels, suggesting a modulating effect of Lupeol on the bacterial efflux system (Fig. 4 ). 3.4-Molecular docking Molecular docking of Lupeol with the MepA model resulted in a binding energy equivalent to -9,010 Kcal/mol. The triterpenoid conformation corresponding to this result ensures the establishment of a hydrogen bond with Glu156 (1.90 Å distance) and three hydrophobic (alkyl) interactions with Ala247 (3.47 Å), Val74 (4.17 Å) and Met250 (4.86 Å). Van der Waals interactions (with a maximum distance of 5.00 Å) were also identified, with a predominance of methionine, phenylalanine and serine residues (Fig. 5 ). Based on the data mentioned above and those described in Table 1 , it is clear that, among the ligands analyzed, Lupeol has the highest affinity for the multidrug transporter. Furthermore, the residues with which these compounds interacted also indicate that the respective conformations occur in the same portion of MepA, revealing its importance for the efflux pump and corroborating the potential of the triterpenoid in question as an efflux inhibitor. Table 1 Docking results with ciprofloxacin, ethidium bromide and CCCP Results Ciprofloxacin Ethidium Bromide CCCP Binding Energy -8.236 Kcal/mol -8.596 Kcal/mol -7.493 Kcal/mol Conventional Hydrogen Bond Ala247 (1.70) Gln380 (2.32) Glu156 (2.12) Ala247 (2.51) Gln157 (2.71) Glu295 (2.50) Ser251 (2.78) Arg160 (2.33) Asn70 (3.00) Gln157 (2.49) Carbon-Hydrogen Bond Glu295 (3.28) ---- ---- Pi-Anion Interaction Glu295 (4.01) Arg160 (3.76) Glu295 (3.89) Glu295 (4.24) Alkyl Interaction Val74 (4.21) Val298 (5.21) Val74 (4.47) Val298 (3.93) Pi-Alkyl Interaction Ala247 (5.05) Val74 (5.11) Val74 (5.40) Ala247 (5.11) Pi-Sigma Interaction ---- ---- Val74 (3.44) Pi-Sulfur Interaction ---- Met250 (5.02) ---- *Distances (in parentheses) are expressed in Angstrom (Å). 4.DISCUSSION According to Rosandy et al . [ 16 ], Lupeol, a natural pentacyclic triterpene, exhibits antibacterial activity against Gram-positive and Gram-negative strains. The variation in susceptibility between these groups can be attributed to structural differences in their cell walls: Gram-positive bacteria have a thick peptidoglycan layer, while Gram-negative bacteria have an outer membrane rich in lipopolysaccharides (LPS), which acts as a selective barrier, restricting the entry of many antimicrobial agents [ 17 ]. However, the antimicrobial activity of triterpenoids is not universal. Al-Ansi et al. [ 18 ] observed that compounds of this class, extracted from the latex of Euphorbia arbuscula , showed weak or absent antibacterial activity. These discrepancies may be related to structural variations among triterpenes, their bioavailability, affinity for specific molecular targets, and the presence of bacterial resistance mechanisms, such as efflux pumps and modifying enzymes [ 19 , 17 ]. Lupeol activity appears to be concentration-dependent, potentially exerting bactericidal or bacteriostatic effects, as highlighted by Adnan et al . [ 20 ]. Kwon et al . [ 21 ] demonstrated that this triterpene is capable of inhibiting bacterial protein synthesis, which may represent an additional mechanism to its effect on efflux pumps. This inhibition, by compromising translation and the functional integrity of ribosomes, is in agreement with the findings of Adnan et al . [ 20 ], who reported the negative regulation of ribosomal genes of the 30S and 50S subunits and RNA polymerase, directly interfering with bacterial growth. The absence of significant changes in cell membrane permeability, observed in the experiments of the present study, is in agreement with the data of Nonato and colleagues [ 22 ], who describe Lupeol as a membrane stabilizer, limiting the permeabilization of lipid bilayers. Furthermore, Souza [ 23 ] suggests that its structural similarity to cholesterol may favor interactions with specific lipid domains, affecting the fluidity and functional activity of transmembrane proteins, including active transporters such as MATE pumps. At the molecular level, Labbozzetta, Pomo and Notarbartolo [ 24 ] showed that Lupeol can inhibit the transcription of P-gp protein mRNA in acute myeloid leukemia cell lines, also reducing the expression of other oncoproteins. These findings support the hypothesis that Lupeol exerts modulatory effects on high functional weight membrane proteins, including bacterial efflux pumps. Docking assays performed with abietic acid, a diterpene structurally distinct from Lupeol, against the MepA pump, revealed predominantly hydrophobic interactions with nonpolar residues, such as methionine and phenylalanine, in addition to hydrogen bonding interactions with polar residues such as asparagine, glutamine, serine, threonine, and glutamic acid [ 25 ]. Similar patterns were described by Almeida et al . [ 26 ] with safrole, reinforcing the relevance of hydrophobic domains in the structure of MATE family pumps. This interaction pattern is consistent with the functional model of MepA, whose architecture includes binding sites rich in nonpolar residues, fundamental for the recognition and translocation of lipophilic compounds [ 27 ]. In particular, the observed interaction between Lupeol and the Glu156 residue—previously described as essential for MepA function—may be crucial for both blocking the active site and modulating the pump's conformation, directly impacting its ability to expel antimicrobial compounds [28, 29]. 5.CONCLUSIONS This research proposes an innovative approach by exploring isolated Lupeol as a promising alternative in combating bacterial resistance. The results obtained highlight its ability to inhibit the MepA efflux pump, a central mechanism in bacterial resistance. Although the minimum inhibitory concentration of Lupeol did not show clinical relevance, the data regarding membrane permeability and fluorescence with ethidium bromide confirm its specific action, independent of interactions in the cell wall. Furthermore, molecular docking results revealed a robust interaction between Lupeol and the target protein, with a binding energy of -9,010 Kcal/mol. The conformation of Lupeol, with key interactions such as hydrogen bonds and hydrophobic interactions with specific residues, reinforces its potential as a valuable tool in the modulation of efflux pumps. These findings highlight the need for further investigations, both to optimize the structure of Lupeol in search of greater efficacy and to expand the understanding of its possible therapeutic implications. The role of natural products, such as Lupeol, is crucial in tackling bacterial resistance, offering new perspectives for the development of innovative and sustainable drugs capable of overcoming the challenges posed by this global phenomenon. Declarations CONFLICT OF INTEREST The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. FUNDING Not applicable. Author Contribution Conceptualization: JCA-P, HDMC, JMB-F, NJSA. Method: NJSA, CAPS, VLB, CDMO-T, GGA, MSC, ARPS, JBA-N, JBS, VLCR, JMB-F, JER, ACFA, JAOB, GAA. Writing: NJSA, VLB, JBA-N, JCA-P, JTCS.Review: JCA-P, HDMC, SRT.Supervision: JCA-P, JER, HDMC. ACKNOWLEDGMENTS To the Laboratory of Microbiology and Molecular Biology - LMBM, Regional University of Cariri (URCA) Brazil; Laboratory of Applied Microbiology - LAMAP, Federal University of Cariri - UFCA, to the National Council for Scientific and Technological Development - CNPq and to the Ceará Foundation for Support of Scientific and Technological Development - FUNCAP, for financing the project “Pentacyclic Triterpenes: In Vitro and In Silico Evaluation in Single-Species and Multi-Species Bacterial Biofilms (BP6-0241-00330.01.00/25)”. DATA AVAILABILITY STATEMENT The data that suport the findings of this study are avaliable from the corresponding author upon reasonable request. 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In: Efflux-Mediated Antimicrobial Resistance in Bacteria: Mechanisms, Regulation and Clinical Implications [Internet] 2016. Switzerland: Springer International Publishing; Available from: https://doi.org/10.1007/978-3-319-39658-3 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8939866","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":596684033,"identity":"c16a6fc6-f3ff-4966-9a6d-86ee1e1e14ad","order_by":0,"name":"Nara Juliana Santos Araújo","email":"","orcid":"","institution":"Federal University of Cariri","correspondingAuthor":false,"prefix":"","firstName":"Nara","middleName":"Juliana Santos","lastName":"Araújo","suffix":""},{"id":596684034,"identity":"a01006ad-17b6-4f4e-b33d-a080da174f6e","order_by":1,"name":"Camila Aparecida Pereira Silva","email":"","orcid":"","institution":"Federal University of Cariri","correspondingAuthor":false,"prefix":"","firstName":"Camila","middleName":"Aparecida Pereira","lastName":"Silva","suffix":""},{"id":596684035,"identity":"5c7528e1-bf67-4ec2-b7ee-1c5a7a5548df","order_by":2,"name":"Cicera Datiane Morais Oliveira-Tintino","email":"","orcid":"","institution":"Regional University of Cariri, Crato - URCA","correspondingAuthor":false,"prefix":"","firstName":"Cicera","middleName":"Datiane Morais","lastName":"Oliveira-Tintino","suffix":""},{"id":596684036,"identity":"0011cf13-45dd-4abd-9f7a-ad6a7358f54e","order_by":3,"name":"Gabriel Gonçalves Alencar","email":"","orcid":"","institution":"Regional University of Cariri, Crato - URCA","correspondingAuthor":false,"prefix":"","firstName":"Gabriel","middleName":"Gonçalves","lastName":"Alencar","suffix":""},{"id":596684037,"identity":"45c8fe6d-57e4-4ea2-a2be-c0b53d1afc20","order_by":4,"name":"Maria do Socorro Costa","email":"","orcid":"","institution":"Regional University of Cariri, Crato - URCA","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"do Socorro","lastName":"Costa","suffix":""},{"id":596684038,"identity":"335694b8-067d-4ffb-b69f-a5b1dc7ac0a2","order_by":5,"name":"Ana Raquel Pereira da Silva","email":"","orcid":"","institution":"Regional University of Cariri, Crato - URCA","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"Raquel Pereira da","lastName":"Silva","suffix":""},{"id":596684039,"identity":"aaff4977-402d-4fed-911a-d310cb726f06","order_by":6,"name":"Juliete Bezerra Soares","email":"","orcid":"","institution":"Federal University of Cariri","correspondingAuthor":false,"prefix":"","firstName":"Juliete","middleName":"Bezerra","lastName":"Soares","suffix":""},{"id":596684040,"identity":"a78eb309-a850-427a-aa45-a2508d96f5da","order_by":7,"name":"Janaína Esmeraldo Rocha","email":"","orcid":"","institution":"Regional University of Cariri, Crato - URCA","correspondingAuthor":false,"prefix":"","firstName":"Janaína","middleName":"Esmeraldo","lastName":"Rocha","suffix":""},{"id":596684041,"identity":"1632bb41-3fc5-48c0-a257-85f192a06f09","order_by":8,"name":"Saulo Relison Tintino","email":"","orcid":"","institution":"Regional University of Cariri, Crato - URCA","correspondingAuthor":false,"prefix":"","firstName":"Saulo","middleName":"Relison","lastName":"Tintino","suffix":""},{"id":596684042,"identity":"b9b028ab-ce75-45a7-b72d-9a3f04d5eeb1","order_by":9,"name":"Vanessa Lima-Bezerra","email":"","orcid":"","institution":"Federal University of Cariri","correspondingAuthor":false,"prefix":"","firstName":"Vanessa","middleName":"","lastName":"Lima-Bezerra","suffix":""},{"id":596684044,"identity":"c0f0c780-46a2-4f41-bffd-c1f202b6da71","order_by":10,"name":"Henrique Douglas Melo Coutinho","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYBACA4YEMM3DIMHA+ADE4CNSiwFIC7MBSAsbsVoYgFrYJEBMglrM2bMTP3xs+yNjLt18rPJrjp0MGwPzw0c38Gix7Hm7WXJmmwGP5ZxjabdltyUDHcZmbJyDz2E3crcx8wK1GNzIMbstuY0ZqIWHTZpoLcWS2+pJ1ML4cdthIrScAfplxjljoJa0ZGnGbcd52JgJ+eV47sYPH8rk7A1uJB/8+HNbtT0/e/PDx/i0oABmHjBJrHIQYPxBiupRMApGwSgYMQAAJldESAKX6YYAAAAASUVORK5CYII=","orcid":"","institution":"Regional University of Cariri, Crato - URCA","correspondingAuthor":true,"prefix":"","firstName":"Henrique","middleName":"Douglas Melo","lastName":"Coutinho","suffix":""},{"id":596684045,"identity":"6f36aba1-cbd6-4494-871c-c95952bba45e","order_by":11,"name":"Vanessa Leopoldino Coelho Rodrigues","email":"","orcid":"","institution":"Teresina Technological Education Center – CET","correspondingAuthor":false,"prefix":"","firstName":"Vanessa","middleName":"Leopoldino Coelho","lastName":"Rodrigues","suffix":""},{"id":596684046,"identity":"462c974b-9f13-47f6-ad2c-bda1d320a0e5","order_by":12,"name":"José Maria Barbosa-Filho","email":"","orcid":"","institution":"Federal University of Paraiba - UFPB","correspondingAuthor":false,"prefix":"","firstName":"José","middleName":"Maria","lastName":"Barbosa-Filho","suffix":""},{"id":596684048,"identity":"4419e88e-ee60-4bf8-8ede-50f7bbbb8913","order_by":13,"name":"José Bezerra de Araújo-Neto","email":"","orcid":"","institution":"Regional University of Cariri, Crato - URCA","correspondingAuthor":false,"prefix":"","firstName":"José","middleName":"Bezerra","lastName":"de Araújo-Neto","suffix":""},{"id":596684049,"identity":"a25f4f88-ca73-4944-a798-adb26949f5ce","order_by":14,"name":"Ana Carolina Ferreira Araujo","email":"","orcid":"","institution":"Regional University of Cariri, Crato - URCA","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"Carolina Ferreira","lastName":"Araujo","suffix":""},{"id":596684050,"identity":"51b4c11e-86f3-4b89-8959-a09335105146","order_by":15,"name":"João Arthur de Oliveira Borges","email":"","orcid":"","institution":"Regional University of Cariri, Crato - URCA","correspondingAuthor":false,"prefix":"","firstName":"João","middleName":"Arthur de Oliveira","lastName":"Borges","suffix":""},{"id":596684051,"identity":"fbf00055-e9fe-4f92-9d8e-ebbfb35e637e","order_by":16,"name":"Gildênia Alves de Araújo","email":"","orcid":"","institution":"Federal University of Cariri","correspondingAuthor":false,"prefix":"","firstName":"Gildênia","middleName":"Alves","lastName":"de Araújo","suffix":""},{"id":596684052,"identity":"b8d60c29-f778-4268-b572-fc230ecd0108","order_by":17,"name":"José Thyálisson da Costa Silva","email":"","orcid":"","institution":"Regional University of Cariri, Crato - URCA","correspondingAuthor":false,"prefix":"","firstName":"José","middleName":"Thyálisson da Costa","lastName":"Silva","suffix":""},{"id":596684053,"identity":"0b76112b-fed8-475d-80fb-6f3370d069b9","order_by":18,"name":"Jacqueline Cosmo Andrade-Pinheiro","email":"","orcid":"","institution":"Federal University of Cariri","correspondingAuthor":false,"prefix":"","firstName":"Jacqueline","middleName":"Cosmo","lastName":"Andrade-Pinheiro","suffix":""}],"badges":[],"createdAt":"2026-02-22 14:53:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8939866/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8939866/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103516629,"identity":"a2885c30-cc02-421d-8964-f9db3de9454a","added_by":"auto","created_at":"2026-02-26 14:29:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":92214,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of the inhibition of the efflux pump MepA by Lupeol against the strain \u003cem\u003eS. aureus\u003c/em\u003e K2068. Substance associated with ciprofloxacin (A) and ethidium bromide (B). Statistical analysis performed with one-way ANOVA followed by the Bonferroni post-hoc test. **** = p \u0026lt; 0.0001 compared to the control, in the reduction of MIC.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8939866/v1/d1b6e126b051c934f5027c26.png"},{"id":103515986,"identity":"4af8d298-9128-41f5-83a4-6d38d3e7bfbc","added_by":"auto","created_at":"2026-02-26 14:25:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":77121,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of Lupeol action on the permeability of the bacterial membrane of \u003cem\u003eS. aureus\u003c/em\u003eK2068. **** p \u0026lt; 0.0001 vs Sytox Green; ns = not significant vs Sytox Green. Results are expressed as mean fluorescence intensity. One-way ANOVA followed by Tukey's test.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8939866/v1/0ed486479745de72736ff566.png"},{"id":103516004,"identity":"cbe29e4e-6e16-45b1-8b45-a901d5e19afe","added_by":"auto","created_at":"2026-02-26 14:26:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":66139,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of NorA and MepA efflux pump inhibition by measuring fluorescence emission in \u003cem\u003eS. aureus\u003c/em\u003e 1199B (A) and K2068 (B) strains treated with Lupeol at concentrations of 50, 20, and 10 µg/mL. EtBr = ethidium bromide; **** = p \u0026lt; 0.0001 vs EtBr; ** = p \u0026lt; 0.01 vs EtBr; ns = not significant vs EtBr.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8939866/v1/198d1a67e6bd2b43560b2d3f.png"},{"id":103515994,"identity":"5fbc2b36-a951-4623-a340-6507f124805c","added_by":"auto","created_at":"2026-02-26 14:25:54","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":34896,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of mepA gene expression by RT-qPCR (ΔΔCt method) in the presence of ciprofloxacin alone and in combination with lupeol against the K2068 strain of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. “**” indicates a statistically significant difference between the groups (Student's t-test; p = 0.0063).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8939866/v1/669e3a1e6389ea44f600aed7.jpg"},{"id":103515996,"identity":"3d03f2c6-6e83-4997-8a6a-cb96f73e0647","added_by":"auto","created_at":"2026-02-26 14:25:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":69881,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentation of the conformation (A) and interactions (B) of Lupeol in the MepA binding site\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8939866/v1/d65b6edc80639ca60ef58ee8.png"},{"id":105562487,"identity":"5805647f-dca7-40b3-9305-02aae2ddf15b","added_by":"auto","created_at":"2026-03-27 12:41:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1204785,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8939866/v1/2ea69e09-e0fc-469b-9abe-f81bd79859a8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Lupeol as a Modulator of Bacterial Resistance Mediated by the MepA Efflux Pump in Staphylococcus aureus","fulltext":[{"header":"1.INTRODUCTION","content":"\u003cp\u003eBacterial resistance to antibiotics has become a growing challenge for healthcare institutions globally, representing a direct threat to human life [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This phenomenon is intensified by factors such as the indiscriminate use of antimicrobials\u0026mdash;widely observed during the COVID-19 pandemic\u0026mdash;and by genetic mechanisms that favor the maintenance and spread of resistance among bacterial strains [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven this alarming scenario, investment in research aimed at identifying compounds capable of effectively and safely combating multidrug-resistant organisms becomes urgent [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Among emerging strategies, the development of efflux pump inhibitors stands out, whose action consists of blocking bacterial systems that reduce the intracellular concentration of drugs, compromising their effectiveness. Efflux pumps are transmembrane proteins that act in the active extrusion of toxic substances and antimicrobials into the extracellular environment. The MepA pump, belonging to the MATE (Multidrug and Toxic Compound Extrusion) family, is a relevant example due to its ability to expel various compounds from the cell interior [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this context, secondary metabolites have gained prominence due to their diverse chemical structures and promising mechanisms of action for the development of new therapeutic agents [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Among these compounds, terpenoids have attracted interest for their antinociceptive, antiviral, anti-inflammatory, and antibacterial properties [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Lupeol, a triterpene widely distributed in nature, stands out for its broad range of pharmacological activities, including antibacterial, anti-inflammatory, and wound-healing effects [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTherefore, the present study aimed to evaluate the inhibitory capacity of the triterpene Lupeol on the MepA efflux pump, seeking to explore its potential as a modulator of bacterial resistance. The K2068 strain of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, used in this study, is a clinical strain characterized by exhibiting resistance to multiple antimicrobials and expressing the MepA efflux pump.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1- Lupeol and bacterial strain\u003c/h2\u003e \u003cp\u003eThe triterpene Lupeol (C\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e50\u003c/sub\u003eO), used in this study, was acquired from Sigma-Aldrich (St. Louis, MO, USA).\u003c/p\u003e \u003cp\u003eThe bacterial strain employed was the K2068 strain of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, known to express the MepA efflux pump, acquired from the microorganism collection of the Microbiology and Molecular Biology Laboratory of the Regional University of Cariri (LMBM/URCA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2-Determination of minimum inhibitory concentration and efflux pump inhibitory potential\u003c/h2\u003e \u003cp\u003eThe minimum inhibitory concentration (MIC) was determined by the microdilution method in 96-well plates, according to the guidelines established by CLSI [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The experiments were performed in triplicate, using resazurin as an indicator of bacterial viability. Lupeol dilutions and standard inhibitors were prepared in BHI medium and the plates were incubated at 35\u0026deg;C for 24 hours. The MIC reading was performed by observing the change in resazurin color, which indicated the presence or absence of bacterial growth.\u003c/p\u003e \u003cp\u003eThe evaluation of efflux pump inhibition was conducted using a subinhibitory concentration (MIC/8) of standard inhibitors and Lupeol. A solution containing 10% bacterial inoculum, the inhibitors, and Lupeol was prepared and completed with BHI medium, which was transferred to 96-well microdilution plates. Then, 100 \u0026micro;L of ciprofloxacin or ethidium bromide (EtBr) were added in serial dilutions, ranging from 0.5 to 512 \u0026micro;g/mL. The plates were incubated at 35\u0026deg;C for 24 hours, and resazurin was again used to detect bacterial viability [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3-Membrane permeability assessment\u003c/h2\u003e \u003cp\u003eMembrane permeability was assessed using SYTOX Green dye. Inocula of \u003cem\u003eS. aureus\u003c/em\u003e K2068, adjusted to the 0.5 standard of the McFarland scale, were distributed in 96-well black plates. Lupeol was added at final concentrations of 200, 100, and 50 \u0026micro;g/mL, while polymyxin B was used as a positive control at concentrations of 100, 50, and 25 \u0026micro;g/mL, and PBS as a negative control.\u003c/p\u003e \u003cp\u003eAfter 1 hour of incubation, 100 \u0026micro;L of SYTOX Green (1 \u0026micro;M) were added, followed by another 30 minutes of incubation. Fluorescence was then measured using the Cytation 1 reader (BioTek\u003csup\u003e\u0026reg;\u003c/sup\u003e) with Gen5\u0026trade; 3.11 software, using 485 nm excitation and 528 nm emission filters. The tests were performed in triplicate, as described by Yuen \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4-Fluorescence assessment of ethidium bromide\u003c/h2\u003e \u003cp\u003e \u003cem\u003eS. aureus\u003c/em\u003e K2068 strains were cultured and incubated at 37\u0026deg;C. The bacterial inoculum was adjusted to the 0.5 standard of the McFarland scale in PBS and combined with Lupeol at concentrations of 10, 20, and 50 \u0026micro;g/mL. CCCP (50 \u0026micro;g/mL) was used as a positive control, and PBS containing only the inoculum was the negative control.\u003c/p\u003e \u003cp\u003eAfter incubation for 1 hour and 30 minutes, EtBr (100 \u0026micro;g/mL) was added, followed by a new incubation. The samples were then centrifuged, washed with PBS, and resuspended. The resulting pellet was transferred to 96-well black plates, and fluorescence was measured using the Cytation 1 reader (BioTek\u0026reg;) with Gen5\u0026trade; 3.22 software, using excitation filters at 530 nm and emission filters at 590 nm. The tests were performed in triplicate, as described by Oliveira-Tintino and colleagues [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5-Quantification of MepA gene expression by RT-Qpcr\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eStaphylococcus aureus\u003c/em\u003e K2068 strain was cultured in BHI medium at 37\u0026deg;C for 24 h. Subsequently, the bacterial suspension was incubated in liquid BHI medium under two experimental conditions: control, containing ciprofloxacin at a sub-inhibitory concentration (MIC/8), and test, containing ciprofloxacin (MIC/8) associated with Lupeol (MIC/8). All conditions were prepared in triplicate, with a final volume of up to 5 mL, and incubated at 37\u0026deg;C for 24 h.\u003c/p\u003e \u003cp\u003eAfter incubation, total RNA was extracted using TRIzol\u0026trade; (Thermo Fisher Scientific), preceded by cell lysis with lysozyme and Tris-EDTA buffer. The RNA obtained was quantified and used for cDNA synthesis using the SuperScript\u0026trade; VILO\u0026trade; Master Mix kit (Thermo Fisher Scientific), according to the manufacturer's instructions.\u003c/p\u003e \u003cp\u003eThe expression of the mepA gene was evaluated by RT-qPCR using the SYBR\u0026trade; Green PCR Master Mix (Thermo Fisher Scientific) on the qTowerG3 instrument (Analytik Jena). The 23S gene was used as an endogenous control. Reactions were performed with an annealing temperature of 61\u0026deg;C, and relative gene expression was determined by the ΔΔCt method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6-Molecular docking\u003c/h2\u003e \u003cp\u003eThe structures of lupeol, ethidium bromide, ciprofloxacin, and CCCP were generated in MarvInSketch software (Chemaxon Ltd., version 23.14) and optimized with Open Babel (version 2.4.1) using the MMFF94 force field. The three-dimensional structure of the MepA protein was modeled by homology in SWISS-MODEL, with the UniProt Q2G140 sequence from Staphylococcus aureus (NCTC 8325/PS 47) and the 3W4T model from PDB [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMolecular docking was performed in AutoDock Vina (version 1.2.5) using AutoDock Tools (MGLTools, version 1.5.7), configuring a 50 \u0026Aring; grid box centered on the conveyor cavity (coordinates: x = -32.554; y\u0026thinsp;=\u0026thinsp;0.012; z\u0026thinsp;=\u0026thinsp;11.605). The fit validation followed the methodology of Martin et al. (2024), with a limit of 2.0 \u0026Aring; for the root mean square deviation (RMSD) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7-Statistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using GraphPad Prism 8.01 software. Comparisons between multiple groups were made using two-way ANOVA, followed by the Bonferroni post-hoc test. For comparisons between independent groups with a single variable, one-way ANOVA was used, followed by Tukey's test. The significance level adopted was p\u0026thinsp;\u0026lt;\u0026thinsp;0.01. Results are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean. RT-qPCR results were expressed as geometric mean\u0026thinsp;+\u0026thinsp;standard deviation, statistically evaluated using the T-test, using GraphPad Prism 9.2.0 software. Differences were considered significant when p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1-Determination of the minimum inhibitory concentration and evaluation of efflux pump inhibition\u003c/h2\u003e \u003cp\u003eAfter the minimum inhibitory concentration (MIC) assays and evaluation of the inhibitory potential of the MepA efflux pump, it was observed that Lupeol showed isolated antibacterial activity, but without clinical relevance, with an MIC\u0026thinsp;\u0026ge;\u0026thinsp;1024 \u0026micro;g/mL.\u003c/p\u003e \u003cp\u003eIn the efflux pump inhibition tests, Lupeol promoted a significant reduction in the MIC of ciprofloxacin (from 64 \u0026micro;g/mL to 20.1 \u0026micro;g/mL) and ethidium bromide (from 256 \u0026micro;g/mL to 64 \u0026micro;g/mL), compared to the results obtained with these compounds in isolation. This reduction was more significant than that observed with the positive control CCCP, which showed an MIC of 32 \u0026micro;g/mL for ciprofloxacin and 101.6 \u0026micro;g/mL for ethidium bromide (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These results suggest that Lupeol exhibits more efficient inhibition of the MepA efflux pump compared to the standard CCCP inhibitor.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2-Membrane permeability assessment and fluorescence assessment of ethidium bromide\u003c/h2\u003e \u003cp\u003eIn the membrane permeability assessment, it was observed that, in the presence of the K2068 strain, none of the Lupeol concentrations used in the assay significantly altered the fluorescence emission of Sytox green, indicating that the metabolite does not exhibit cell wall activity. The positive control of the test showed a change in the fluorescence level, validating the assay performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the fluorescence assessment of ethidium bromide, it was possible to perceive that, in the presence of K2068, a dose-dependent effect occurred, in which it is possible to see that the higher the concentration of Lupeol used, the greater its effect on increasing the fluorescence intensity of bromide, indicating inhibition of the MepA efflux pump. The CCCP control considerably increased fluorescence emission compared to the negative control, indicating the reproducibility of the assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3- Quantification of mepA gene expression by RT-qPCR\u003c/h2\u003e \u003cp\u003eRT-qPCR analysis revealed that the combination of ciprofloxacin with Lupeol significantly reduced mepA gene expression compared to the control, showing ΔCt\u0026thinsp;\u0026asymp;\u0026thinsp;\u0026minus;\u0026thinsp;2.5, which corresponds to a decrease of approximately 5.7 times in transcription levels, suggesting a modulating effect of Lupeol on the bacterial efflux system (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4-Molecular docking\u003c/h2\u003e \u003cp\u003eMolecular docking of Lupeol with the MepA model resulted in a binding energy equivalent to -9,010 Kcal/mol. The triterpenoid conformation corresponding to this result ensures the establishment of a hydrogen bond with Glu156 (1.90 \u0026Aring; distance) and three hydrophobic (alkyl) interactions with Ala247 (3.47 \u0026Aring;), Val74 (4.17 \u0026Aring;) and Met250 (4.86 \u0026Aring;). Van der Waals interactions (with a maximum distance of 5.00 \u0026Aring;) were also identified, with a predominance of methionine, phenylalanine and serine residues (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on the data mentioned above and those described in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, it is clear that, among the ligands analyzed, Lupeol has the highest affinity for the multidrug transporter. Furthermore, the residues with which these compounds interacted also indicate that the respective conformations occur in the same portion of MepA, revealing its importance for the efflux pump and corroborating the potential of the triterpenoid in question as an efflux inhibitor.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDocking results with ciprofloxacin, ethidium bromide and CCCP\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eResults\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiprofloxacin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEthidium Bromide\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCCCP\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBinding Energy\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-8.236 Kcal/mol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-8.596 Kcal/mol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-7.493 Kcal/mol\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eConventional Hydrogen Bond\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAla247 (1.70)\u003c/p\u003e \u003cp\u003eGln380 (2.32)\u003c/p\u003e \u003cp\u003eGlu156 (2.12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAla247 (2.51)\u003c/p\u003e \u003cp\u003eGln157 (2.71)\u003c/p\u003e \u003cp\u003eGlu295 (2.50)\u003c/p\u003e \u003cp\u003eSer251 (2.78)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eArg160 (2.33)\u003c/p\u003e \u003cp\u003eAsn70 (3.00)\u003c/p\u003e \u003cp\u003eGln157 (2.49)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCarbon-Hydrogen Bond\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGlu295 (3.28)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e----\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e----\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePi-Anion Interaction\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGlu295 (4.01)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eArg160 (3.76)\u003c/p\u003e \u003cp\u003eGlu295 (3.89)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGlu295 (4.24)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAlkyl Interaction\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVal74 (4.21)\u003c/p\u003e \u003cp\u003eVal298 (5.21)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVal74 (4.47)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVal298 (3.93)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePi-Alkyl Interaction\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAla247 (5.05)\u003c/p\u003e \u003cp\u003eVal74 (5.11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVal74 (5.40)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAla247 (5.11)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePi-Sigma Interaction\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e----\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e----\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVal74 (3.44)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePi-Sulfur Interaction\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e----\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMet250 (5.02)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e----\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e*Distances (in parentheses) are expressed in Angstrom (\u0026Aring;).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4.DISCUSSION","content":"\u003cp\u003eAccording to Rosandy \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], Lupeol, a natural pentacyclic triterpene, exhibits antibacterial activity against Gram-positive and Gram-negative strains. The variation in susceptibility between these groups can be attributed to structural differences in their cell walls: Gram-positive bacteria have a thick peptidoglycan layer, while Gram-negative bacteria have an outer membrane rich in lipopolysaccharides (LPS), which acts as a selective barrier, restricting the entry of many antimicrobial agents [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, the antimicrobial activity of triterpenoids is not universal. Al-Ansi et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] observed that compounds of this class, extracted from the latex of \u003cem\u003eEuphorbia arbuscula\u003c/em\u003e, showed weak or absent antibacterial activity. These discrepancies may be related to structural variations among triterpenes, their bioavailability, affinity for specific molecular targets, and the presence of bacterial resistance mechanisms, such as efflux pumps and modifying enzymes [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Lupeol activity appears to be concentration-dependent, potentially exerting bactericidal or bacteriostatic effects, as highlighted by Adnan \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eKwon \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] demonstrated that this triterpene is capable of inhibiting bacterial protein synthesis, which may represent an additional mechanism to its effect on efflux pumps. This inhibition, by compromising translation and the functional integrity of ribosomes, is in agreement with the findings of Adnan \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], who reported the negative regulation of ribosomal genes of the 30S and 50S subunits and RNA polymerase, directly interfering with bacterial growth.\u003c/p\u003e \u003cp\u003eThe absence of significant changes in cell membrane permeability, observed in the experiments of the present study, is in agreement with the data of Nonato and colleagues [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], who describe Lupeol as a membrane stabilizer, limiting the permeabilization of lipid bilayers. Furthermore, Souza [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] suggests that its structural similarity to cholesterol may favor interactions with specific lipid domains, affecting the fluidity and functional activity of transmembrane proteins, including active transporters such as MATE pumps.\u003c/p\u003e \u003cp\u003eAt the molecular level, Labbozzetta, Pomo and Notarbartolo [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] showed that Lupeol can inhibit the transcription of P-gp protein mRNA in acute myeloid leukemia cell lines, also reducing the expression of other oncoproteins. These findings support the hypothesis that Lupeol exerts modulatory effects on high functional weight membrane proteins, including bacterial efflux pumps.\u003c/p\u003e \u003cp\u003eDocking assays performed with abietic acid, a diterpene structurally distinct from Lupeol, against the MepA pump, revealed predominantly hydrophobic interactions with nonpolar residues, such as methionine and phenylalanine, in addition to hydrogen bonding interactions with polar residues such as asparagine, glutamine, serine, threonine, and glutamic acid [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Similar patterns were described by Almeida \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] with safrole, reinforcing the relevance of hydrophobic domains in the structure of MATE family pumps.\u003c/p\u003e \u003cp\u003eThis interaction pattern is consistent with the functional model of MepA, whose architecture includes binding sites rich in nonpolar residues, fundamental for the recognition and translocation of lipophilic compounds [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In particular, the observed interaction between Lupeol and the Glu156 residue\u0026mdash;previously described as essential for MepA function\u0026mdash;may be crucial for both blocking the active site and modulating the pump's conformation, directly impacting its ability to expel antimicrobial compounds [28, 29].\u003c/p\u003e"},{"header":"5.CONCLUSIONS","content":"\u003cp\u003eThis research proposes an innovative approach by exploring isolated Lupeol as a promising alternative in combating bacterial resistance. The results obtained highlight its ability to inhibit the MepA efflux pump, a central mechanism in bacterial resistance. Although the minimum inhibitory concentration of Lupeol did not show clinical relevance, the data regarding membrane permeability and fluorescence with ethidium bromide confirm its specific action, independent of interactions in the cell wall.\u003c/p\u003e \u003cp\u003eFurthermore, molecular docking results revealed a robust interaction between Lupeol and the target protein, with a binding energy of -9,010 Kcal/mol. The conformation of Lupeol, with key interactions such as hydrogen bonds and hydrophobic interactions with specific residues, reinforces its potential as a valuable tool in the modulation of efflux pumps.\u003c/p\u003e \u003cp\u003eThese findings highlight the need for further investigations, both to optimize the structure of Lupeol in search of greater efficacy and to expand the understanding of its possible therapeutic implications. The role of natural products, such as Lupeol, is crucial in tackling bacterial resistance, offering new perspectives for the development of innovative and sustainable drugs capable of overcoming the challenges posed by this global phenomenon.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCONFLICT OF INTEREST\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFUNDING\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: JCA-P, HDMC, JMB-F, NJSA. Method: NJSA, CAPS, VLB, CDMO-T, GGA, MSC, ARPS, JBA-N, JBS, VLCR, JMB-F, JER, ACFA, JAOB, GAA. Writing: NJSA, VLB, JBA-N, JCA-P, JTCS.Review: JCA-P, HDMC, SRT.Supervision: JCA-P, JER, HDMC.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGMENTS\u003c/h2\u003e \u003cp\u003eTo the Laboratory of Microbiology and Molecular Biology - LMBM, Regional University of Cariri (URCA) Brazil; Laboratory of Applied Microbiology - LAMAP, Federal University of Cariri - UFCA, to the National Council for Scientific and Technological Development - CNPq and to the Cear\u0026aacute; Foundation for Support of Scientific and Technological Development - FUNCAP, for financing the project \u0026ldquo;Pentacyclic Triterpenes: \u003cem\u003eIn Vitro\u003c/em\u003e and \u003cem\u003eIn Silico\u003c/em\u003e Evaluation in Single-Species and Multi-Species Bacterial Biofilms (BP6-0241-00330.01.00/25)\u0026rdquo;.\u003c/p\u003e\u003ch2\u003eDATA AVAILABILITY STATEMENT\u003c/h2\u003e \u003cp\u003eThe data that suport the findings of this study are avaliable from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGuedes MK Brometo de et\u0026iacute;dio na identifica\u0026ccedil;\u0026atilde;o de bombas de efluxo em bact\u0026eacute;rias para controle de mastite bovina: Revis\u0026atilde;o integrativa [Internet] 2021. 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Switzerland: Springer International Publishing; Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-3-319-39658-3\u003c/span\u003e\u003cspan address=\"10.1007/978-3-319-39658-3\" 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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Triterpenes, molecular docking, RT-qPCR, membrane permeability, efflux inhibitor","lastPublishedDoi":"10.21203/rs.3.rs-8939866/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8939866/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIncreasing bacterial resistance has stimulated the search for alternative therapeutic strategies, particularly efflux pump inhibitors, which enhance antimicrobial activity by increasing intracellular drug concentration. Natural compounds such as Lupeol have emerged as promising resistance modulators. This study investigated the ability of Lupeol to inhibit the MepA efflux pump in the K2068 strain of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. Minimum inhibitory concentration, efflux inhibition by microdilution, membrane permeability (Sytox Green), ethidium bromide fluorescence, gene expression by RT-qPCR, and molecular docking analyses were performed. Lupeol showed no intrinsic antibacterial or cell wall activity but effectively inhibited the MepA efflux pump. Its effect on ethidium bromide fluorescence was dose-dependent, and RT-qPCR confirmed significant suppression of mepA gene expression. Molecular docking revealed strong binding affinity (\u0026minus;\u0026thinsp;9.010 kcal/mol) involving van der Waals, hydrophobic, and hydrogen interactions. These findings indicate that Lupeol is a promising efflux pump inhibitor with potential to combat bacterial resistance.\u003c/p\u003e","manuscriptTitle":"Lupeol as a Modulator of Bacterial Resistance Mediated by the MepA Efflux Pump in Staphylococcus aureus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-26 14:05:47","doi":"10.21203/rs.3.rs-8939866/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-14T12:24:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-10T18:49:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-09T08:00:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-02T11:17:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"138305388428618190649211079757972242815","date":"2026-03-01T12:38:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"218614577471326592661853546544527688221","date":"2026-03-01T12:20:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"9376928744151001239288595778010432465","date":"2026-02-25T05:45:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"149674175878087215899647141996497332362","date":"2026-02-24T11:25:37+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-24T11:12:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-24T06:09:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-24T06:07:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"Naunyn-Schmiedeberg's Archives of Pharmacology","date":"2026-02-22T14:49:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b7079305-84e5-4b91-bbd6-8ed2bb7ca1d5","owner":[],"postedDate":"February 26th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-11T21:09:00+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-26 14:05:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8939866","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8939866","identity":"rs-8939866","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}