Bioactive Compounds Derived from Sargassum wightii Exhibit Antibacterial Activity against Multi-Drug Resistant Acinetobacter baumannii

Bioactive Compounds Derived from Sargassum wightii Exhibit Antibacterial Activity against Multi-Drug Resistant Acinetobacter baumannii | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Bioactive Compounds Derived from Sargassum wightii Exhibit Antibacterial Activity against Multi-Drug Resistant Acinetobacter baumannii Suvaiyarasan Suvaithenamudhan, Sundarraj Dinesh Kumar, Rajendran Thirugnanasambandam, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4641861/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Acinetobacter baumannii ( A. baumannii) is a notorious nosocomial pathogen known for its ability to form biofilms, rendering it highly resistant to conventional antibiotics and immune clearance. In this study, the minimum inhibitory concentration (MIC) ranged from 70 µg/mL to 100 µg/mL remarkably, the tested ethanolic extracts inhibited A. baumannii biofilm development in a concentration-dependent manner. Epifluorescence microscopic analysis revealed a significant reduction in treated biofilm formation compared to the control. Additionally, gas chromatography-mass spectrometry (GC-MS) analysis of the ethanol extract of Sargassum wightii ( S. wightii ) identified 10 major compounds. Molecular docking studies were conducted to explore the interaction of small molecules from S. wightii with the BfmR protein of A. baumannii . The molecular docking of three ligand molecules (CAS No. 002302-12-7, 015120-94-2, and 146397-91-3) with the target BfmR-Ab revealed the lowest binding energies (∆G bind ) of -42.26 and − 50.49 (kcal/mol) for the ligands CAS No. 002302-12-7 and 146397-91-3, respectively, and the lowest Glide score of -4.067 (kcal/mol) for the ligand CAS No. 015120-94-2. These top three hit molecules exhibited the highest affinity as efficient ligands against BfmR of A. baumannii . Nevertheless, S. wightii demonstrated antibiofilm activities against the multidrug-resistant (MDR) pathogen A. baumannii , with bioactive compounds exhibiting promising drug-likeness and pharmacokinetic signatures. Biological sciences/Biotechnology Biological sciences/Computational biology and bioinformatics Acinetobacter baumannii BfmR Biofilm Sargassum wightii brown seaweed molecular docking molecular dynamic simulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Acinetobacter baumannii is a Gram-negative bacterial pathogen implicated in hospital-acquired infections, often leading to significant morbidity and mortality 1 . This pathogen poses a substantial global public health threat due to its Multi-drug resistance (MDR) characteristics 2–4 . The Infectious Diseases Society of America (IDSA) has identified A. baumannii as one of the six critical MDR pathogens 5,6 . Notably, A. baumannii exhibits resistance to carbapenems and is responsible for a range of recalcitrant conditions including meningitis, urinary tract infections, wound infections, and ventilator-associated pneumonia (VAP) 7–9 . Mortality rates associated with A. baumannii VAP vary between 40% and 70% 10–12 . The ability of A. baumannii to spread could be attributed to its inherent capacity to thrive in adverse environmental conditions 13,14 . Consequently, there is an urgent need to develop novel antimicrobial agents against A. baumannii 15–17 . The formation of biofilms is one of the main causes of treatment resistance in A.baumannii infections. Quorum sensing encourages the growth of biofilms, and that is why the search for “quorum quenchers” has increased recently 18 . A.baumannii has the ability to form biofilm, which appears to be one of the major rationales for its solid role in antimicrobial resistance. The two-component system of BfmRS controls both virulence and biofilm development in A.baumannii . The BfmRS two-component system orchestrates virulence and biofilm formation in A. baumannii , with the BfmR regulator, in conjunction with histidine kinase BfmS, constituting the BfmRS system 19–21 . The BfmS sensor is instrumental in biofilm distortion, epithelial cell adhesion, and heightened sensitivity to serum killing. Moreover, the entire bfmRS operon governs the K locus, which regulates capsular exopolysaccharide expression, suggesting the involvement of BfmR in controlling diverse gene sets across various A. baumannii phenotypes 22 . BfmR has been implicated in conferring resistance to colistin, imipenem, erythromycin, and rifampin 23–25 . The pili-mediated biofilm formation in A. baumannii is dependent on gene clustering within the cus operon, resulting in a pilus-like bundled structure 26,27 . BfmR sequence conservation across bacteria and archaea underscores its role in regulating multidrug resistance and modulating pathogenesis, while its absence in mammalian hosts highlights its potential as a therapeutic target 28,29 . Seaweeds represent a vast reservoir of proximate compositions, micronutrients, and trace elements, offering substantial nutritional benefits for humans and other animals 30 . Brown seaweeds, particularly those of the Sargassum genus, harbor a wealth of bioactive compounds with significant pharmacological and biological applications 31,32 . An estimated 35.7 million wet metric tonnes of seaweed were produced worldwide in 2019, with 96.97% originating from cultivation and 3.03% from natural beds 33 . According to the Food and Agriculture Organization (FAO) database, the first-sale value of the 34.7 million metric tonnes of seaweed produced globally for various food and non-food purposes in 2019 was USD 14.7 billion. Seaweed-based functional food components largely provide plenty of medicinal benefits and a variety of bioactivity against various ailments. The great diversity of brown seaweeds of the genus Sargassum provides an undiscovered reservoir of bioactive compounds with considerable pharmacological and biological applications 34 . Fucoidan, fucoxanthin, flavonoids, and polyphenols, including phloroglucinol, phlorotannins, and phenolic acids, are abundant in brown seaweeds, notably Sargassum wightii ( S. wightii ). The rich polysaccharide content and phytochemical derivatives of S. wightii contribute to its diverse biological properties, making it a valuable resource in combating various ailments 35 . The Indian brown seaweed, S.wightii (Fig. 1 A), was collected from Mandapam (Lat- 9° 16' 58.4436'' N; Long- 79° 11' 4.6572'' E), Palk Bay region, India. Among the algal family, S.wightii is a brown macroalga that comes under the family of Sargassaceae and order of Fucales with more polysaccharide contents 36 . Its polysaccharide-rich composition renders it suitable for animal feed, food ingredients, and fertilizer applications 37 . The enzymatic-ligand interactions inherent to S. wightii facilitate the synthesis of novel pharmacophores and nutraceuticals to counter MDR bacterial infections 38 . Over the past century, seaweeds have found utility in various industrial applications, including the production of agar, alginate, and carrageen an, as well as serving as a main food in Asian cuisines. S. wightii stands out as the largest, economically significant, and ecologically dominant algae 39 . This study aims to isolate and evaluate bioactive compounds from aerial parts of S. wightii for their antibacterial and antibiofilm activities against A. baumannii through in silico and in vitro studies. Furthermore, the interaction patterns of these compounds with MDR BfmR from A. baumannii (MDR BfmR-Ab) complexes will be predicted and validated to design novel potential inhibitors against A. baumannii . Materials and Methods The brown seaweed, S.wightii (Fig. 1 A), was collected from Mandapam (Lat- 9° 16' 58.4436'' N; Long- 79° 11' 4.6572'' E), Palk Bay region, India. The species was identified following the references provided by Oza and Zaidu (2003) 40 and Krishnamurthy and Joshi (1970) 41 , and subsequently verified by Dr. M. Ganesan, a Senior Principal Scientist at CSMCRI, Mandapam Camp, Tamil Nadu. The voucher specimen validating this identification was deposited at the Department of Marine Science, Bharathidasan University, with the accession number BDUMSSW20210019. The collected S. wightii sample was shifted to the laboratory, rinsed three times using double distilled water and processed further. The plant's aerial components were broken up, cleaned, and allowed to dry for 10 days in the shadows. The plant components were then processed using a mechanical blender to create a fine powder. Until subsequent studies, the dry powder was kept at 4⁰C. Preparation of solvent extract. Soxhlet extraction was used to make crude plant extract. In a nutshell, 250 mL of ethanol was used to extract 20g of finely ground plant material. Once the solvent in the extractor's syphon tube had turned colourless, the extraction was continued for another 24h. A rotary evaporator was used to condense the extract after it had been collected in a beaker and subjected to reduced pressure. For future use, the crude extract was kept at 4°C. Identification of bioactive compounds from Sargassum wightii (Brown Algae). According to established procedures, a gas chromatography-mass spectrometry (GC-MS) (Agilent GC 7890A/MS5975C) chromatograph outfitted with a Shimadzu QP-500 mass spectrometer was used to investigate the phytochemical composition of an ethanolic extract of S.wightii . About 1L of the sample was used for analysis after the plant extract was dissolved in ethanol (1:25). The stationary phase was a fused-silica column (30 m long, 0.25 mm in diameter, and 0.25 m in film thickness) coated with polydimethyl siloxane. Helium with 1 ml/min was employed as the carrier gas. The injector's temperature was set at 325°C. The oven's temperature was kept at 50°C for 1minute before being raised to 300°C at a pace of 12°C per minute. The split rate was kept at 1:5, and the ionisation voltage was 70eV. 4 The results are shown in Supplementary Table S1 , which details how the mass spectra were used to determine the extract's composition by comparing them to recognised substances or published data. Preparation of protein and ligand structures. The reference protein structure (Biofilm-controlling Response Regulator BfmR from A.baumannii ) was obtained from the Protein Data Bank (PDB ID: 6BR7) (Fig. 2 A) 42–44 . The preparation and optimization process of the protein BfmR-Ab was performed by the Protein Preparation Wizard and Epik module in the Schrödinger suite (Maestro 2023-1) 45,46 . Assigning the hydrogen bonding network was carried out by OPLS3e force field 47 and to acquire the least possible energy minimization 48 . The structure of the small molecules identified from S.wightii was obtained from the National Institute Standard and Technology Chemistry WebBook 49 . Schrödinger Maestro (2023-1) used for geometry optimizations with energy minimization of small molecule structures. Module in the LigPrep builder panel was used to prepare and generate the 3D structures of the small molecules (Fig. 3 A). OPLS4 force field was used for energy minimization and also to generate low-energy ring confirmation per small molecule 50,51 . Binding site prediction. Active site prediction by the SiteMap module was carried out based on the qualitative site score values obtained from the physical and structural properties of the protein (BfmR-Ab) 52 . Glide XP docking. A total of ten ligands identified from S.wightii were tested for their antibacterial properties against A.baumannii using the Glide XP docking studies (Table 1 ). The 3D structure of S. wightii phytocompounds was optimized for the docking conformation studies (Fig. 3 ). The Glide XP docking was performed to determine the BfmR as a potential drug target for A.baumannii (BfmR-Ab) ligand interaction. Docking was carried out after constructing the grid by selecting the amino acid residues present in the binding site within the radius of 3Å as the centroid. Default parameters were selected by keeping the ligands flexible on the docking calculation set. The formation of hydrogen bonds between the ligands and the residues of the active site, its length, and Glide XP (extra-precision) score were recorded 53–55 . Binding free energy calculation. The binding free energy was calculated with prime Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) 56,57 . Prime uses a surface GB model employing a Gaussian surface instead of the van der Waals surface that better represents the solvent-accessible surface area. The following Eqs. (1–4) were used for calculating the binding energy (ΔG bind ). ΔG bind = ΔE + ΔG soly + ΔG SA , ( 1 ) ΔE = E complex – E protein – E ligand , ( 2 ) where E complex , E protein , and E ligand are the minimized energies of the protein-inhibitor complex, protein, and inhibitor, respectively 58 . ΔG solv = G solv (complex) - G solv (protein) - G solv (ligand), ( 3 ) where G solv (complex), G solv (protein), and G solv (ligand) are the solvation-free energies of the complex, protein, and inhibitor, respectively. ΔG SA = G SA (complex) - G SA (protein) - G SA (ligand), ( 4 ) where G SA (complex), G SA (protein), and G SA (ligand) are the surface area energies for the complex, protein, and inhibitor, respectively. The rational criteria for the selection of best compounds based on scoring and interaction parameters are shown in Glide XP docking studies 59,60 . Molecular dynamics simulations. GROMACS 5.1.4 package is used for molecular dynamics (MD) analysis and the topology files are generated using the automated topology builder in the GROMOS96 54a7 force field for protein-ligand complexes 61–63 . PRODRG 2.5 server was used for generating topology files for the ligands 64 . The ligand complex obtained from docking was solvated with a single-point charge water model 65 . The solvated system was subjected to 5000 steps of energy minimization employing the steepest descent algorithm. This step was followed by 1 nanosecond (ns) MD simulation, where the BfmR-Ab with ligands complex were position restrained to equilibrate the water and ions under the influence of the solute. The production run was carried out for all the systems for 100 ns using a 2 femtosecond (fs) time step for the integration of the equation of motion in the NPT ensemble at 300 K and at 1 atmospheric pressure, which was controlled using a V-rescale thermostat and Parrinello Rahman Barostat, respectively. Bond lengths involving hydrogen atoms were constrained by using the Linear Constraint Solver (LINCS) algorithm 66 . The Particle Mesh Ewald (PME) method was used to calculate the electrostatic interaction 67,68 . A 10Å cutoff distance was used for the long-range electrostatic and van der Waals energy terms and a 2 ps interval was used for saving the MD simulation coordinates of all the systems for further analyses. A careful equilibration at a desired temperature and pressure for MD analyses was done after the completion of two equilibration phases. For each ligand, with the available 3D structure (PDB ID: 6BR7) of the BfmR-Ab, the final MD run was set at 100 ns. Trajectory Analysis. Analysis of the trajectories was carried out using gmx rms, gmx rmsf, and gmx hbond utilities of the GROMACS package to obtain the graphs of the RMSD, RMSF values and number of hydrogen bonds using the Gnuplot tool version 5.4 69 . Quantitative analysis of biofilm by crystal violet assay. Biofilm was quantified against different concentrations of ethanolic extract of S. wightii according to minor modification. The overnight grown culture (0.6 at OD 600nm ) was inoculated in Luria-Bertani (LB) broth using a 96-well microtiter plate in the ratio of 1:10 with different concentrations of S. wightii ethanolic extract (70µg/mL, 80µg/mL, 90µg/mL and 100µg/mL) and incubated in static at 37⁰C for 48h. The wells of the plate were rinsed with 1X phosphate-buffered saline (PBS), followed by staining with crystal violet (0.2%) for 10 min. The excess stain was rewashed with 1X PBS and allowed to dry. The stained biofilm ring in the wells at the air-water interface was dissolved with acetic acid (30%), and the culture was measured at OD 560nm with a microplate reader (Synergy H1, BioTek, USA). The percentage (%) of biofilm inhibition was compared based on absorbance between higher and lower levels at particular experimental groups using the following Eqs. (5), 2.10. Antibiofilm activity by epifluorescence microscopic observation. Imaging of biofilm matrix against different concentrations of ethanolic extract of S. wightii (70µg/mL, 80µg/mL, 90µg/mL and 100µg/mL) provide information regarding changes in matrix pattern and their contribution to the biomass for the aggregation, formation and the determination of the size of aggregates 70 . Antibiofilm activity and microscopic observation of S. wightii extract on candidate bacteria A.baumannii were analysed using the epifluorescence microscope (Carl Zeiss Axioscope 5, Germany). Overnight grown bacterial culture (0.6 at OD 600nm ) was diluted to 1:10 in pre-sterilized LB broth medium containing glass slides (1cm x1cm) and incubated in static at 37⁰C for 48h. After incubation, the slides were rinsed with 1X PBS. The specific position and relative amount of matrix components mainly bacterial adherent cell biomass were viewed by staining with specific fluorescent markers (Syto9, Thermo Scientific) 71 . The biofilm-formed glass slides were stained by adding stain solution for 20 mins followed by washing twice using 1X PBS after staining in dark conditions. After the final wash, the coverslip was fixed and examined under the objective lens of the epifluorescence microscope. Results and Discussion The chromatography of the ethanol extract of S. wightii (Fig. 1 B) reveals the identification of bioactive compounds represented by 25 prominent peaks. The list of compounds, along with their retention times and the percentage of area occupied, is presented in Table S1 . Major compounds identified with GC-MS analysis were glycerin and n-heptyl hexanoate (42.64%), octadecanoic acid (15.67%), ethanone ,1-(6-methyl-7-oxabicyclo acid [4.1.0] hept-1-yl) and 9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]- (8.17%), pregn-5- en-20-one,3-(acetyloxy)-acid 20-(1,2-ethanediyl acetal), (3 beta)-, 2-hydroxytetracosanoic acid and pentanoic acid, 4-oxo-, butyl ester (5.28%) and n-decanoic acid and n-hexadecanoic acid (3.94%) (Table 1 and Fig. 1 B). Table 1 Selected bioactive compounds of brown seaweed Sargassum wightii (GC-MS analysis). S. No Retention Time CAS Number Name of the Compound Molecular Formula Molecular Weight Peak Area (%) 1 RT-5.076 000056-81-5 Glycerin C 3 H 8 O 3 92.094 42.64 006976-72-3 n-heptyl hexanoate C 13 H 26 O 2 214.344 2 RT-14.297 002302-12-7 Pregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta)- C 25 H 38 O 4 402.6 5.28 1000336-12-4 2-hydroxytetracosanoic acid C 24 H 48 O 3 384.6 002052-15-5 Pentanoic acid, 4-oxo-, butyl ester C 9 H 16 O 3 172.222 3 RT-14.630 000334-48-5 n-decanoic acid C 10 H 20 O 2 172.265 3.94 000057-10-3 n-hexadecanoic acid C 16 H 32 O 2 256.424 4 RT-16.396 000057-11-4 Octadecanoic acid C 18 H 36 O 2 284.477 15.67 5 RT-20.474 015120-94-2 Ethanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl)- C 9 H 14 O 2 154.21 8.17 146397-91-3 9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]- C 27 H 33 N 3 O 5 479.6 The major compound, with 42.64% occupancy, was observed to be n-heptylhexanoate, which is commonly used as a flavoring agent in the food industry. The essential oil of Bursera schlechtendalii contains 17.6% n-heptylhexanoate 72 . n-hexadecanoic acid has been reported to possess anti-inflammatory 73 , cytotoxic 74 , anticancer 75 , and antibacterial 76 activities. Ethanone, 1-(6-methyl-7-oxobicyclo[4.1.0]hept-1-yl), is a corticosteroid analog, also known as 1-acetyl-1,2,epoxy-2-methylcyclohexane 77 . Corticosteroids are immunosuppressant commonly used in treating inflammation and allergic disorders. ADME properties predictions. Estimating the ADME properties of ligands becomes crucial in drug discovery for considering them as efficient hit compounds. The drug-likeness attributes of identified bioactive compounds from the brown seaweed S. wightii were evaluated for their ADME characteristics using the QikProp module, which included detailed analyses of HB donor/acceptor, central nervous system (CNS) permeation, Rule of Five, Rule of Three, QPlogPw, QPlogPo/w, percent human oral absorption, QPlogBB, and QPlogS, among others. The drug-likeness of ligands, as assessed by the Lipinski Rule of Five, is vital in the rational design of a drug. Notably, the human oral absorption percentage was found to be 100% for n-heptyl hexanoate (CAS No. 006976-72-3) and pregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta-) (CAS No. 002302-12-7). Ligands such as 2-hydroxytetracosanoic acid (CAS No. 1000336-12-4), pentanoic acid, 4-oxo-, butyl ester (CAS No. 002052-15-5), octadecanoic acid (CAS No. 000057-11-4), and ethanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl)- (CAS No. 015120-94-2) exhibited human oral absorption rates of 92.752%, 91.168%, 91.558%, and 96.746%, respectively. These findings indicate excellent absorption properties for the potent hit compounds listed above. The allowable range of solvent-accessible surface area (SASA) for ligands ranged between 300 and 1000. The potent hit compounds analyzed for toxicity prediction exhibited SASA within the admissible range of 270 to 938, indicating the druggable nature of these ligands. The ligands glycerin, n-heptyl hexanoate, pregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta), 2-hydroxytetracosanoic acid, pentanoic acid, 4-oxo-, butyl ester, n-decanoic acid, n-hexadecanoic acid, octadecanoic acid, ethanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl), and 9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]- recorded SASA values of 270.549, 597.82, 686.236, 938.649, 447.219, 480.679, 675.443, 733.206, 348.35, and 852.72, respectively. A high SASA value was observed with 2-hydroxytetracosanoic acid (938.649) and a CNS value of -2. The hydrogen bond donors of the ligand molecules ranged between 0 and 3, and the hydrogen bond acceptors ranged between 2 and 8.5. n-Heptyl hexanoate, n-decanoic acid, n-hexadecanoic acid, and octadecanoic acid had the minimum number of hydrogen acceptors, while glycerin, pentanoic acid, 4-oxo-butyl ester, ethanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl), and 9H-fluoren-9-one,3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]- exhibited the maximum number of hydrogen bond acceptors. More than 60 calculated and predicted properties were considered, but for documentation, we have tabulated certain important properties in Table 2 , where all the compounds were well within the range of drugability. As shown in Table 2 , all 10 compounds (Fig. 3 ) fall within the ranges of known drug properties. From the table, it could be inferred that there is not more than one violation of Lipinski’s Rule of Five. Table 2. Prediction of ADME properties for the selected compounds from brown seaweed Sargassum wightii using QikProp. Compound Name mol_ MW Donor HB Acceptor HB CNS SASA %Human Oral Absorption Rule of five Rule of three QPlogPw QPlogPo/w QPlogS QPlogBB Glycerin 92.094 3 5.1 -1 270.549 65.515 0 0 9.725 -1.193 0.081 -0.881 n-heptyl hexanoate 214.344 0 2 0 597.82 100 0 0 1.308 4.287 -4.871 -0.637 Pregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta)- 402.573 0 3.5 1 686.236 100 1 1 5.106 5.754 -7.226 0.069 2-pydroxytetracosanoic acid 384.641 2 3.7 -2 938.649 92.752 1 1 4.461 7.248 -8.077 -2.724 Pentanoic acid, 4-oxo-, butyl ester 172.224 0 4 0 447.219 91.168 0 0 4.037 1.42 -1.621 -0.608 n-decanoic acid 172.265 1 2 -1 480.679 86.886 0 0 3.283 2.988 -2.956 -0.932 n-hexadecanoic acid 256.424 1 2 -2 675.443 89.022 1 0 2.375 5.34 -5.585 -1.393 Octadecanoic acid 284.477 1 2 -2 733.206 91.558 1 1 2.127 6.002 -6.331 -1.648 Ethanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl)- 154.208 0 4 1 348.35 96.746 0 0 4.559 0.91 -0.561 0.213 9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]- 479.575 0 8.5 1 852.72 74.709 0 0 10.19 3.526 -4.021 -0.725 Property or Descriptor mol_MW Donor HB Acceptor HB CNS SASA % Human Oral Absorption Rule of five Rule of three QPlogPw QPlogPo/w QPlogS QPlogBB Range or recommended values 130.0 – 725.0 0–6 2–20 -2 to +2 300–1000 >80% is high, < 25% is poor Max 4 Max 3 4−45 -2 -6.5 (Neg) -6.5 -0.5 (Neg) -3 -1.2 (Neg) Table 3. SiteMap properties of BfmR from Acinetobacter baumannii (BfmR-Ab) Binding Sites AA Residues Cavity Size (A) 3 Site Score Enclosure Hydrophobic Hydrophilic D Score * Contact Binding site-1 12,14,16,19,22,23,26,29,84,107,108,109,111,114 38 0.779 0.662 1.203 0.605 0.780 0.937 Binding site-2 9,10,11,33,34,35,46,50,52 29 0.666 0.64 1.059 0.815 0.625 0.948 Ligand Binding Site Analysis of BfmR-Ab. Predicting the binding pose of the complex plays a key role in making the docking process precise. The validation of the docking process using Glide XP was carried out by docking the ligand molecules with the active sites of the target protein BfmR-Ab, which is a potential drug target for A. baumannii (Fig. 2 A). Two binding sites were identified in BfmR-Ab (Fig. 2 B). The Site scores (S) of binding sites 1 and 2 were 0.779 and 0.666, respectively, and the drug ability scores (D) were observed as 0.780 and 0.625, respectively. The binding pocket with the maximum D score was selected for further in silico studies (Table 3 ). Since the binding site with a higher D score indicates a more favourable or promising binding site for a ligand or molecule, binding site-1 was chosen for further analysis. Molecular Docking. The hit ligands observed in S.wightii , including glycerin (CAS No. 000056-81-5), n-heptyl hexanoate (CAS No. 006976-72-3), pregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta)- (CAS No. 002302-12-7), 2-hydroxytetracosanoic acid (CAS No. 1000336-12-4), pentanoic acid, 4-oxo-, butyl ester (CAS No. 002052-15-5), n-decanoic acid (CAS No. 000334-48-5), n-hexadecanoic acid (CAS No. 000057-10-3), octadecanoic acid (CAS No. 000057-11-4), ethanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl)- (CAS No. 015120-94-2), and 9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]- (CAS No. 146397-91-3), were investigated for their binding efficiencies with A. baumannii (BfmR-Ab), and their 2D and 3D structures are presented in Figs. 4 and 5 . The interacting amino acids, H-bonded interactions, bond lengths, Glide score (kcal/mol), and MM-GBSA ∆G bind (kcal/mol) of the protein-ligand complexes are shown in Table 4 . Glycerin and n-heptyl hexanoate, which bound at the binding site of the target protein, exhibited a Glide score of -3.812 (kcal/mol) and a binding energy (∆G bind ) of -22.82 kcal/mol. Hydrogen bond interactions were found with the OH group of THR23 and VAL109 amino acid residues with bond lengths of 1.90 Å and 1.85 Å, respectively and a Glide score of -3.812 (kcal/mol) and binding energy (∆G bind ) of -22.82 kcal/mol hydrogen bond interactions were observed with the VAL109 amino acid residues with a bond length of 2.48Å. The interaction of the ligands glycerin and n-heptyl hexanoate with BfmR-Ab is depicted in Fig. 5 . The docking of pregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta)- with BfmR-Ab was mediated by the HO group of VAL109 residue with a bond length of 1.85 Å, Glide score of -0.511 (kcal/mol), and binding energy (∆G bind ) of -42.26 kcal/mol. The docking of 2-hydroxytetracosanoic acid with BfmR-Ab exhibited a Glide score of -3.100 kcal/mol, binding energy (∆G bind ) of -32.64 kcal/mol, and hydrogen bond interactions were found with the OH and HO groups of ASP16 and LYS107 amino acid residues with bond lengths of 1.80 Å and 2.13 Å, respectively. Pentanoic acid, 4-oxo-, butyl ester, and n-decanoic acid are two ligands that commonly interact with the VAL109 residue of BfmR-Ab through HO groups with bond lengths of 1.92 Å and 1.69 Å, respectively. The Glide scores were found to be -4.029 and − 1.502 kcal/mol, and the binding energies (∆G bind ) were − 26.51 and − 15.30 kcal/mol. The ligands n-hexadecanoic acid and octadecanoic acid each make two interactions with the ARG29 residue of BfmR-Ab (c.f. Table 4 ). Based on Table 4 ,it can be observed that the lowest Glide score and binding energy of the two ligands indicate that ethanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl)- and 9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]- acidare potential drug candidates with high affinity towards the BfmR of A. baumannii . From the Glide docking studies, it could be inferred that four compounds, namely pregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta)-, pentanoic acid, 4-oxo-, butyl ester, ethanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl)- and 9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]-, can be considered as potent BfmR-Ab inhibitor candidates, and can be used as potential antibacterial drugs that could regulate or inhibit the biofilm activity in A.baumannii . Table 4 Glide XP results and prime-MM/GBSA binding free energy (ΔG bind ) for the 10 ligands with the BfmR-Ab. S. No. Ligands Amino Acid H-Bond Interaction Bond Length (Å) Glide XP Score (kcal/mol) MM-GBSA ∆G bind (kcal/mol) 1 Glycerin THR 23 VAL 109 O-H O-H 1.90 1.85 -3.812 -22.82 2 n-heptyl hexanoate VAL 109 H-O 2.48 -3.812 -22.82 3 Pregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta)- VAL 109 H-O 1.85 -0.511 -42.26 4 2-hydroxytetracosanoic acid ASP 16 LYS 107 O-H H-O 1.80 2.13 -3.100 -32.64 5 Pentanoic acid, 4-oxo-, butyl ester VAL 109 H-O 1.92 -4.029 -26.51 6 n-decanoic acid VAL 109 H-O 1.69 -1.502 -15.30 7 n-hexadecanoic acid ARG 29 ARG 29 H-O H-O 2.05 2.40 -1.424 -28.09 8 Octadecanoic acid ARG 29 ARG 29 H-O H-O 1.85 1.59 -2.458 -27.46 9 Ethanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl)- VAL 109 H-O 2.02 -4.067 -23.67 10 9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]- VAL 109 H-O 2.03 -2.491 -50.49 Molecular dynamics analysis. The structural behaviour and flexibility of BfmR-Ab, along with the 10 selected ligand molecules, were studied using MD simulation for 100 ns employing GROMACS version 5.1.4. MD simulation provides information regarding the dynamic behaviour of the protein-ligand complexes in an environment containing ions and water molecules. The stability of the complex was determined by Root Mean Square Deviation (RMSD) and Root Mean Square Fluctuation (RMSF) values of the backbone atoms of the protein and ligand complexes. Figure 6 shows the variation of RMSD from the starting conformation. It was observed that throughout the simulation period (1000 ns), the protein backbone of BfmR-Ab exhibited deviations of not more than 0.3 nm. Molecular dynamics evaluation of the protein-ligand complexes. Stability and interaction pattern of the receptor-ligand complex, explicit MD simulations of the accessible configuration space of BfmR-Ab and ten ligand complexes were conducted for 100 ns using GROMACS 4.6.5 63 . The simulation studies of BfmR-Ab with ligand complexes to evaluate stability showed the variation of RMSD from the starting conformation, as depicted in Fig. 7 A. It was observed that throughout the simulation period (100 ns), the BfmR-Ab-ligand complexes showed that the deviation was not more than 0.35 nm throughout the simulation time (Fig. 7 A). The RMSF plot showed that for all 10 complexes, only very little fluctuation was observed in the residue region 85–100. The two ligands, pregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta)- and 9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]-, exhibited little fluctuation throughout the simulation period compared to the other ligands (Fig. 7 B). This suggests that these ligands are tightly or stably bound to their binding sites within the target protein, indicating effective activity against biofilm formation. Table 5 presents the average number of hydrogen bond interactions between BfmR-Ab and the ligands. The interaction of BfmR-Ab with ligand 9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]- (CAS No. 146397-91-3) exhibited the highest average hydrogen bonding of 1.19 (Fig. S1 ). Table 5 Average number of hydrogen bond interactions of BfmR-Ab and ligand complexes at 100 ns. S. No. Ligands CAS Number Average Hydrogen bond 1 Glycerin 000056-81-5 0.37 2 n-heptyl hexanoate 006976-72-3 0.09 3 Pregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta)- 002302-12-7 0.08 4 2-hydroxytetracosanoic acid 1000336-12-4 0.44 5 Pentanoic acid, 4-oxo-, butyl ester 002052-15-5 0.06 6 n-decanoic acid 000334-48-5 0.21 7 n-hexadecanoic acid 000057-10-3 0.28 8 Octadecanoic acid 000057-11-4 0.09 9 Ethanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl)- 015120-94-2 0.01 10 9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]- 146397-91-3 1.19 Antibiofilm Activity. Minimal inhibitory concentration and antibiofilm activity analysis by crystal violet staining. The minimum inhibitory concentration (MIC) using the resazurin assay was performed to determine the minimal concentration of ethanolic extract from S. wightii that can inhibit the growth of A. baumannii 78 . The pictorial representation and observation of growth inhibitory activity of ethanolic extract from S. wightii on candidate bacteria A. baumannii , a highly troublesome pathogen, is illustrated in Fig. 8 . From this approach, the ethanolic extract of S. wightii exhibits bacterial growth inhibition potential at a concentration of 90 µg/mL. The concentration of 90 µg/mL revealed no visible growth, indicated by the absence of colour change (from blue to pink), which is considered the MIC 79 . The change in colour from blue to pink denotes the bacteria's viability and growth, whereas the absence of a change in the blue colour represents no bacterial growth inhibition. Rajivgandhi et al. (2021) reported the MIC value of ethanolic extract from S. wightii was observed at 200 µg/mL on Pseudomonas aeruginosa 80 . A previous study reported that the bioactivity of pyrogallol exhibited MIC at 120 µg/mL 81 . Additionally, a prior study also yielded comparable findings, demonstrating the enhanced bioactivity of a crude extract of S. wightii against gram-negative bacteria. S. wightii possesses a greater abundance of polysaccharide compounds compared to other algae, and these polysaccharides exhibit the capacity to penetrate pathogens and disrupt their nuclei effectively. Once inside the nucleus, they exert an impact on the entire bacterial structure, impeding the cell cycle growth and ultimately resulting in cell demise 82 . In this study, the antibiofilm activities of ethanolic extract from S. wightii on candidate bacteria A. baumannii were measured in a concentration-dependent manner using the absorbance (OD 560nm ) value of crystal violet staining at 48-hour intervals. The concentration of the ethanolic extract from S. wightii for the antibiofilm study was selected based on the MIC value of the objective extract. Accordingly, two steps down and one step-up concentration at a 10 µg/mL difference were selected. However, the concentrations of ethanolic extract from S. wightii were selected as 70 µg/mL, 80 µg/mL, 90 µg/mL, and 100 µg/mL. The ability to form biofilms is unique to each species of bacteria due to differences in genetic makeup, physiology, and other variables; therefore, different microbe species may have varying degrees of proficiency in building biofilms. From this study, the antibiofilm activity based on crystal violet staining of ethanolic extract from S. wightii on A. baumannii was represented in Fig. 9 . Results suggested that two step-down concentrations from MIC (70 and 80 µg/mL) of ethanolic extract exhibited a significant reduction in biofilm biomass formed by A. baumannii . However, MIC (90 µg/mL) and one step higher concentration (100 µg/mL) of ethanolic extract from S. wightii , compared to the MIC value, revealed biofilm eradication potentiality formed by A. baumannii. Thus, ethanolic extract from S. wightii reveals antibiofilm potentiality in a dose-dependent manner compared to a control group without exposure to the objective extract. However, a comparative analysis of the percentage inhibition of biofilm formation by A. baumannii in the presence of ethanolic extract from S. wightii , compared to control groups (without exposure of the objective extract), indicates 27.57%, 43.41%, 54.87%, and 77.74% in T-1 (treated concentration: 70 µg/mL), T2 (treated concentration: 80 µg/mL), T3 (treated concentration: 90 µg/mL) followed by the T4 experimental group (treated concentration: 100 µg/mL). The crystal violet-based antibiofilm activity of S. wightii ethanolic crude extract demonstrated outstanding performance with a remarkably low MIC of just two steps down concentration from MIC. At this concentration, the biofilm was significantly reduced, and its original characteristics underwent gradual changes with increasing concentrations. In a recent development, researchers reported that extracts derived from seaweeds like S. wightii and Halimeda gracilis exhibit remarkable antibiofilm activity even at lower concentrations 83 . Methicillin-Resistant Staphylococcus aureus (MRSA) and A. baumannii (MDRAB) hold a prominent position on the World Health Organization's (WHO) list of high-priority human pathogens. These two microorganisms, responsible for challenging and long-lasting human infections, demand special attention due to their significant impact 84 . A. baumannii induces severe infections in individuals with compromised immune systems, thrives on non-living surfaces within hospital environments, and establishes bacterial colonization on a variety of medical devices 85 . A. baumannii has the capacity to create biofilms on non-living surfaces, making it a significant contributor to hospital-acquired infections. Adherence and biofilm formation are pathogenic mechanisms that contribute to clinical complications 86 . The ability to create biofilms on both living and non-living surfaces is a critical factor and a frequent contributor to persistent infections associated with implanted medical devices, as well as resistance to a broad range of antimicrobial agents. Preventing biofilm formation not only aids bacteria in evading the host's immune defences and antimicrobial treatments but also hinders the progression of infections 87 . However, based on this investigation, we conclude the antibacterial activity of seaweed extract; these findings indicate that the MIC value of the crude extract is comparatively lower. In other cases, an effective concentration of crude extract up to 400 mg/mL has been reported in several studies examining its antibacterial properties (Zammuto et al., 2022) 88,89 . Visualization of the Biofilm in Epifluorescence Microscope. After treatment with an ethanolic extract of S.wightii , representative virtual images of biofilms were captured in fluorescence mode. Further, stained with Syto9, images display the full extent of the biofilms developing at the surface of the cover slips. Figure 9 illustrates biofilm-covered regions (A, B, C) that have been evaluated for control, where Fig. 9 A represents magnification at x20, Fig. 9 B denotes magnification x40 and Fig. 9 C represents a 2.5D illustration. The treated groups are now taking into consideration all scan modes in fluorescence mode after Syto9 staining. Followed by Fig. 10 , T1 to T4, (D-O), notably the extract decreases in the bacterial cells at different concentrations of S. wightii (70 µg/mL, 80 µg/mL, 90 µg/mL and 100 µg/mL), respectively. The attachment of biofilm biomass in the presence of S. wightii extract showed a significant decrease in biofilm-covered area, area, as clearly depicted in Fig. 10 . SYTO9's capacity to attach to nucleic acids, particularly DNA, is the basis for its staining principle in biofilm studies. It is a fluorescent nucleic acid stain often used to mark living bacterial cells in a biofilm. It enters bacterial cells and attaches to their DNA, generating green fluorescence when stimulated by light 90 . The control group showed a higher count of live cells, while the ethanolic extracts from S.wightii demonstrated nearly identical antibiofilm effectiveness, as evidenced by the number of deceased cells. At the mature stage of the biofilm formation process, the effective inhibition was seen. The ethanolic extract of S.wightii prevented the formation of biofilms during the adhesion stage, and the epifluorescence micrograph clearly showed disruption to the bacterial biofilm matrix with few adherent cells. Biofilm provides a shield for microorganisms against traditional drugs and disinfectants. According to information provided by the National Institutes of Health and the Centers for Disease Control, microbial biofilms are responsible for roughly 65–80% of infections 91 . The capacity of A. baumannii to attach to surfaces is a crucial mechanism in the bacterium's pathogenicity. Adherence is influenced by specific factors like adhesins and non-specific factors such as hydrophobicity and electric charge on the cell surface. Cell surface hydrophobicity (CSH) significantly contributes to initial adhesion, biofilm development, pathogenicity, and virulence 92 . In the initial phase of biofilm formation, extracellular polymeric substances (EPS) are produced, comprising polysaccharides, nucleic acids, lipid molecules, and proteins. The EPS plays a crucial role in establishing the three-dimensional, sturdy structure of the biofilm matrix 93 . In this current investigation, we assessed the potentiality of ethanolic extract from S.wightii against A. baumannii biofilm formed on the glass surface. The findings demonstrated the efficacy of ethanolic extracts from the seaweeds in preventing biofilm formation. Numerous reports have documented the antimicrobial properties of marine seaweeds 94,95 . Extracts obtained from various seaweed using chloroform and methanol solvents displayed potent activity against a range of pathogenic bacteria known to affect humans. Additionally, ethanol extracts from S. wightii demonstrated unique anti biofilm properties against clinically significant pathogenic microorganisms like A. baumannii . Conclusions In silico analysis of our present work showed the binding activity of the ten ligands of S.wightii with A.baumannii and identified the drug-likeness property of those ligands to be used as lead molecules in drug discovery. MD simulation studies were used to understand the stability of the ligands in binding with the target BfmR protein of A.baumannii . We also found through an in silico analysis identified pregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta) (CAS No. 002302-12-7), ethanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl) (CAS No. 015120-94-2) and 9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy] (CAS No. 146397-91-3) as potent ligands with strong and stable binding potentials with BfmR-Ab. In the molecular docking of the three ligand molecules (CAS No. 002302-12-7, 015120-94-2 and 146397-91-3) with the target BfmR-Ab, it was found that all three ligands showed good binding efficiency to the biofilm-controlling response regulator BfmR, albeit with differences. The interaction results of the ligand molecules and BfmR of A.baumannii revealed that the amino acid VAL 109 was found to have a crucial role in the formation of hydrogen bonds with most of the selected ligands that contributed to the stability of the complexes. From all the ten ligands, the lowest binding energy (∆G bind ) -42.26 and − 50.49 (kcal/mol) was obtained for the ligands i.e; CAS No. 002302-12-7 and 146397-91-3 respectively and thelowest Glide score of -4.067 (kcal/mol) was obtained for the ligand CAS No. 015120-94-2. These three ligand molecules with the highest affinity can be considered as potent hit molecules against the BfmR of A.baumannii . The ethanol extract of S.wightii showed effective inhibitory activity against A.baumannii. Future research is needed to determine the potential pharmacological and real-time therapeutic applications of the antibiofilm properties of the ethanolic extract of S.wightii against MDR pathogen A.baumannii and discover metabolic components. Especially, the antibiofilm potential of the ethanolic extract of S.wightii enhanced the susceptibility of A.baumannii growth formation was evaluated. The effectiveness and safety of the extract must be fully explored, which demands further studies. Declarations Competing Interests The authors declare no competing interests. Funding S.S. and E.M.S. are supported by a Research Associate Grant (File No: 45/2/2020-DDI/BMS) from the Indian Council of Medical Research (ICMR), New Delhi. Author Contribution Suvaiyarasan Suvaithenamudhan, Esaki M. Shankar and Vanitha Mariappan: Methodology. Suvaiyarasan Suvaithenamudhan, Esaki M. Shankar and Sundarraj Dinesh Kumar: Software. Suvaiyarasan Suvaithenamudhan and Esaki M. Shankar: Validation. Suvaiyarasan Suvaithenamudhan, Esaki M. Shankar, Vanitha Mariappan, Sundarraj Dinesh Kumar and Esaki Muthu Ponmalar : Visualization and Investigation. Esaki M. Shankar and Vanitha Mariappan: Data curation. Esaki M. Shankar and Vanitha Mariappan and Sundarraj Dinesh Kumar: Writing original draft preparation. Suvaiyarasan Suvaithenamudhan, Esaki M. Shankar, Vanitha Mariappan, Rajendran Thirugnanasambandam and Sundarraj Dinesh Kumar: Writing review and Editing. Suvaiyarasan Suvaithenamudhan, Esaki M. Shankar and Vanitha Mariappan, Sundarraj Dinesh Kumar, Esaki Muthu Ponmalar, Parthiban Rudrapathy. and Pitchaipillai Sankar Ganesh: Visualization. Esaki M. Shankar, Vanitha Mariappan and Parthiban Rudrapathy: Supervision. Suvaiyarasan Suvaithenamudhan and Esaki M. Shankar: Project administration. Esaki M. Shankar and Vanitha Mariappan: Funding acquisition. All authors have read and agreed to the published version of the manuscript. Acknowledgement The authors acknowledge and appreciate the management for its support and encouragement Data Availability Data will be available upon reasonable request from the corresponding author. References I. Kyriakidis, E. Vasileiou, Z.D. Pana, A. Tragiannidis, Acinetobacter baumannii Antibiotic Resistance Mechanisms. Pathogens. 10(3), 373. https://doi.org/10.3390/pathogens10030373 (2021). B. Mirzaei, Z.N. Bazgir, H.R. Goli, F. Iranpour, F. 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Microbial pathogenesis. 124, 311-315. https://doi.org/10.1016/j.micpath.2018.08.060 (2018). Additional Declarations No competing interests reported. Supplementary Files SupplementaryTablesFigures.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4641861","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":329876719,"identity":"b8a404be-db71-4b8c-a550-fd3ff61bbc1b","order_by":0,"name":"Suvaiyarasan Suvaithenamudhan","email":"","orcid":"","institution":"Central University of Tamil Nadu","correspondingAuthor":false,"prefix":"","firstName":"Suvaiyarasan","middleName":"","lastName":"Suvaithenamudhan","suffix":""},{"id":329876720,"identity":"28fda055-b618-4d94-a87a-8fd624ec29db","order_by":1,"name":"Sundarraj Dinesh Kumar","email":"","orcid":"","institution":"Bharathidasan University","correspondingAuthor":false,"prefix":"","firstName":"Sundarraj","middleName":"Dinesh","lastName":"Kumar","suffix":""},{"id":329876722,"identity":"6f9ef9e1-1acd-4240-834d-bbe8b1658044","order_by":2,"name":"Rajendran Thirugnanasambandam","email":"","orcid":"","institution":"Sathyabama Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Rajendran","middleName":"","lastName":"Thirugnanasambandam","suffix":""},{"id":329876724,"identity":"3316712a-5c0c-4632-9aec-88be1a58ec3a","order_by":3,"name":"Esaki Muthu Ponmalar","email":"","orcid":"","institution":"Sri Sairam Siddha Medical College and Research Centre","correspondingAuthor":false,"prefix":"","firstName":"Esaki","middleName":"Muthu","lastName":"Ponmalar","suffix":""},{"id":329876725,"identity":"b3d9e7e8-b129-44f7-b550-86a0d8ade8b8","order_by":4,"name":"Pitchaipillai Sankar Ganesh","email":"","orcid":"","institution":"Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Pitchaipillai","middleName":"Sankar","lastName":"Ganesh","suffix":""},{"id":329876726,"identity":"e437fc8e-7b9f-4969-9704-c4e61c55a1e1","order_by":5,"name":"Vanitha Mariappan","email":"","orcid":"","institution":"Universiti Kebangsaan Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Vanitha","middleName":"","lastName":"Mariappan","suffix":""},{"id":329876727,"identity":"37e57430-4e60-4f24-ae59-3083e4fe681a","order_by":6,"name":"Esaki M. Shankar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIiWNgGAWjYDACCQYGxgYQg70BJsTYgEMtuhaeAwwMB0jTIpEA00IAyM9ufvZwRs09Od2Zj59Jf9zBIM/fwNz2AJ8WgzvHzA03HCs2NrudZiZx8AyD4YwDjO0GeLVIJJhJPmBLSNx2O4dN4mAbA+MGBsY2CbwOm5H+TfLBv4T6bTfPgLXYE9TCcCPHTHJjW0KC2Q0esJZEgloMbuSUSc7sSzDcdibN2OJsm0TyjMOEHbZNsudbgrzZ8cMPb1S22dj2t7c/w+8wNABUzEyK+lEwCkbBKBgFWAEAf/pKLJHoH4IAAAAASUVORK5CYII=","orcid":"","institution":"Central University of Tamil Nadu","correspondingAuthor":true,"prefix":"","firstName":"Esaki","middleName":"M.","lastName":"Shankar","suffix":""},{"id":329876728,"identity":"6dd0836d-757a-4bd2-b4ca-1caa5213810c","order_by":7,"name":"Parthiban Rudrapathy","email":"","orcid":"","institution":"Malabar Cancer Centre","correspondingAuthor":false,"prefix":"","firstName":"Parthiban","middleName":"","lastName":"Rudrapathy","suffix":""}],"badges":[],"createdAt":"2024-06-26 10:07:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4641861/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4641861/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60922610,"identity":"25b8c4d9-96ce-4bf6-b427-b9095afc8266","added_by":"auto","created_at":"2024-07-23 15:08:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":798697,"visible":true,"origin":"","legend":"\u003cp\u003eGas chromatography-mass spectrometry (GC-MS) analyses of \u003cem\u003eSargassum wightii\u003c/em\u003e. \u003cstrong\u003e(A)\u003c/strong\u003e Marine seaweed \u003cem\u003eSargassum wightii \u003c/em\u003e(brown seaweed), \u0026nbsp;\u003cstrong\u003e(B)\u003c/strong\u003e GC-MS chromatogram of available phytochemical compounds of \u003cem\u003eSargassum wightii \u003c/em\u003eextract.\u003c/p\u003e","description":"","filename":"Picture1.png","url":"https://assets-eu.researchsquare.com/files/rs-4641861/v1/0b9149dc11b5eab61f24d6b5.png"},{"id":60923742,"identity":"bba6d883-1d5a-430b-8a1c-dbe4f9aff046","added_by":"auto","created_at":"2024-07-23 15:24:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1122537,"visible":true,"origin":"","legend":"\u003cp\u003eSiteMap shows structural complementarities of the BfmR from \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e.\u003cstrong\u003e (A) \u003c/strong\u003e3D-structure of Beryllium fluorinated receiver domain of BfmR from \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e (PDB ID: 6BR7), (\u003cstrong\u003eB)\u003c/strong\u003e shows the solid of the receptor. (i) Red colour for H-bond acceptor sites (ii) Solidblue for H-bond donor sites (iii) Yellow for hydrophobic sites.\u003c/p\u003e","description":"","filename":"Picture2.png","url":"https://assets-eu.researchsquare.com/files/rs-4641861/v1/b278f445ade4928e01c3ffa8.png"},{"id":60922611,"identity":"74f2ed8b-23bb-41b6-826c-211c8ba8a92c","added_by":"auto","created_at":"2024-07-23 15:08:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":432736,"visible":true,"origin":"","legend":"\u003cp\u003eTwo-dimensional structures of the phytochemical compound isolated from the \u003cem\u003eSargassum wightii.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Picture3.png","url":"https://assets-eu.researchsquare.com/files/rs-4641861/v1/55be1f075a51b901f992010d.png"},{"id":60922614,"identity":"3c2277ca-37ea-4dc1-a5ef-024cefcfe691","added_by":"auto","created_at":"2024-07-23 15:08:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1223302,"visible":true,"origin":"","legend":"\u003cp\u003eDocking interactions of ligands (A). 000056-81-5, (B). 006976-72-3, (C). 002302-12-7, (D). 1000336-12-4, (E). 002052-15-5, (F). 000334-48-5, (G). 000057-10-3, (H). 000057-11-4, (I). 015120-94-2 and (J). 146397-91-3with BfmR-Ab ligand docking poses represented in two dimensions (2D).\u003c/p\u003e","description":"","filename":"Picture4.png","url":"https://assets-eu.researchsquare.com/files/rs-4641861/v1/4097fca7779d543d67027da3.png"},{"id":60922613,"identity":"de520e02-55cc-4781-9553-5ef3bc3cef52","added_by":"auto","created_at":"2024-07-23 15:08:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1299734,"visible":true,"origin":"","legend":"\u003cp\u003eDocking interactions of ligands (A). 000056-81-5, (B). 006976-72-3, (C). 002302-12-7, (D). 1000336-12-4, (E). 002052-15-5, (F). 000334-48-5, (G). 000057-10-3, (H). 000057-11-4, (I). 015120-94-2 and (J). 146397-91-3with BfmR-Ab ligand docking poses represented in three dimensions (3D). The ligands are depicted as a ball-stick model and the dots pink lines indicate hydrogen bond interactions with the residues.\u003c/p\u003e","description":"","filename":"Picture5.png","url":"https://assets-eu.researchsquare.com/files/rs-4641861/v1/c61ee476d8c4e3b189318184.png"},{"id":60923236,"identity":"e96784d4-9a41-47b6-af10-bcca710a9a12","added_by":"auto","created_at":"2024-07-23 15:16:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":103416,"visible":true,"origin":"","legend":"\u003cp\u003eRMSD of the backbone of BfmR-Ab shows a stable structure up to1000 ns.\u003c/p\u003e","description":"","filename":"Picture6.png","url":"https://assets-eu.researchsquare.com/files/rs-4641861/v1/cc6b324c225ff86fcc01437d.png"},{"id":60922619,"identity":"1fcd0298-45d4-4ea6-9b69-6a067854f80f","added_by":"auto","created_at":"2024-07-23 15:08:48","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1364168,"visible":true,"origin":"","legend":"\u003cp\u003e(A)\u003cstrong\u003e \u003c/strong\u003eRMSD of the backbone of protein-ligand complexes exhibits a stable form without any significant conformational changes up to 100 ns. (B)\u003cstrong\u003e \u003c/strong\u003eRMSF value of protein-ligand complexes during the trajectory period of simulation.\u003c/p\u003e","description":"","filename":"Picture7.png","url":"https://assets-eu.researchsquare.com/files/rs-4641861/v1/5127f9cfba91c76fda361011.png"},{"id":60922621,"identity":"c9d61ae4-15e9-43db-a496-30ada8f6da1b","added_by":"auto","created_at":"2024-07-23 15:08:49","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":408262,"visible":true,"origin":"","legend":"\u003cp\u003eMIC determination of ethanolic \u003cem\u003eS. wightii\u003c/em\u003e extract on pathogenic bacteria \u003cem\u003eA.baumannii\u003c/em\u003e. In the experimental row, well 1 indicates control without \u003cem\u003eS.wightii\u003c/em\u003eextract (bacteria + LB medium + indicator) Whereas, well 2 to 12 represent different concentrations of ethanolic extracts of \u003cem\u003eS.wightii\u003c/em\u003e (10, 20, 30, 40, 50, 60, 70, 80, 90, 100 and 110 mg/mL). Here well number 10 represents the MIC value (90µg/mL).\u003c/p\u003e","description":"","filename":"Picture8.png","url":"https://assets-eu.researchsquare.com/files/rs-4641861/v1/76d10584ec4f98b125ec9b72.png"},{"id":60922617,"identity":"e59a5c05-4960-4b36-a240-10155276a7e8","added_by":"auto","created_at":"2024-07-23 15:08:48","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":55554,"visible":true,"origin":"","legend":"\u003cp\u003eThe antibiofilm activity analysis by crystal violet staining, Also, T1 to T4 indicated significant decreases in the bacterial cells.\u003c/p\u003e","description":"","filename":"Picture9.png","url":"https://assets-eu.researchsquare.com/files/rs-4641861/v1/7da7b66c84b9696221cba6d8.png"},{"id":60922618,"identity":"ce4bfc53-31b3-440a-b506-d7147225ccd3","added_by":"auto","created_at":"2024-07-23 15:08:48","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":1875066,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence microscopy visualization of biofilms developed on 48-hour culture [Fig. 2 (A, B\u0026amp; C) denotes as a control in the magnification of range of 10x, 100x and two-dimensional (2D) images, respectively]. Whereas, different concentrations (70 µg/mL, 80 µg/mL, 90 µg/mL, 100 µg/mL) of ethanolic extract of \u003cem\u003eS. wightii\u003c/em\u003e tested (T1 to T4) against \u003cem\u003eA. baumannii \u003c/em\u003e(Fig. 2 (D-O).\u003c/p\u003e","description":"","filename":"Picture10.png","url":"https://assets-eu.researchsquare.com/files/rs-4641861/v1/46d9c8fac0def8291044c7eb.png"},{"id":64579769,"identity":"5bfd8dd2-390c-4d63-aa79-633693912cb6","added_by":"auto","created_at":"2024-09-16 05:33:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11382935,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4641861/v1/cffc05f8-0a70-4d16-8c5e-40c5e22e9fc1.pdf"},{"id":60923237,"identity":"27da7032-3486-4646-b000-fc1229212171","added_by":"auto","created_at":"2024-07-23 15:16:48","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":194914,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTablesFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4641861/v1/aa8fad8e000571b2f808937d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bioactive Compounds Derived from Sargassum wightii Exhibit Antibacterial Activity against Multi-Drug Resistant Acinetobacter baumannii","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e is a Gram-negative bacterial pathogen implicated in hospital-acquired infections, often leading to significant morbidity and mortality\u003csup\u003e1\u003c/sup\u003e. This pathogen poses a substantial global public health threat due to its Multi-drug resistance (MDR) characteristics\u003csup\u003e2\u0026ndash;4\u003c/sup\u003e. The Infectious Diseases Society of America (IDSA) has identified \u003cem\u003eA. baumannii\u003c/em\u003e as one of the six critical MDR pathogens\u003csup\u003e5,6\u003c/sup\u003e. Notably, \u003cem\u003eA. baumannii\u003c/em\u003e exhibits resistance to carbapenems and is responsible for a range of recalcitrant conditions including meningitis, urinary tract infections, wound infections, and ventilator-associated pneumonia (VAP)\u003csup\u003e7\u0026ndash;9\u003c/sup\u003e. Mortality rates associated with \u003cem\u003eA. baumannii\u003c/em\u003e VAP vary between 40% and 70% \u003csup\u003e10\u0026ndash;12\u003c/sup\u003e. The ability of \u003cem\u003eA. baumannii\u003c/em\u003e to spread could be attributed to its inherent capacity to thrive in adverse environmental conditions\u003csup\u003e13,14\u003c/sup\u003e. Consequently, there is an urgent need to develop novel antimicrobial agents against \u003cem\u003eA. baumannii\u003c/em\u003e\u003csup\u003e15\u0026ndash;17\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe formation of biofilms is one of the main causes of treatment resistance in \u003cem\u003eA.baumannii\u003c/em\u003e infections. Quorum sensing encourages the growth of biofilms, and that is why the search for \u0026ldquo;quorum quenchers\u0026rdquo; has increased recently\u003csup\u003e18\u003c/sup\u003e. \u003cem\u003eA.baumannii\u003c/em\u003e has the ability to form biofilm, which appears to be one of the major rationales for its solid role in antimicrobial resistance. The two-component system of BfmRS controls both virulence and biofilm development in \u003cem\u003eA.baumannii\u003c/em\u003e. The BfmRS two-component system orchestrates virulence and biofilm formation in \u003cem\u003eA. baumannii\u003c/em\u003e, with the BfmR regulator, in conjunction with histidine kinase BfmS, constituting the BfmRS system\u003csup\u003e19\u0026ndash;21\u003c/sup\u003e. The BfmS sensor is instrumental in biofilm distortion, epithelial cell adhesion, and heightened sensitivity to serum killing. Moreover, the entire bfmRS operon governs the K locus, which regulates capsular exopolysaccharide expression, suggesting the involvement of BfmR in controlling diverse gene sets across various \u003cem\u003eA. baumannii\u003c/em\u003e phenotypes \u003csup\u003e22\u003c/sup\u003e. BfmR has been implicated in conferring resistance to colistin, imipenem, erythromycin, and rifampin\u003csup\u003e23\u0026ndash;25\u003c/sup\u003e. The pili-mediated biofilm formation in \u003cem\u003eA. baumannii\u003c/em\u003e is dependent on gene clustering within the cus operon, resulting in a pilus-like bundled structure\u003csup\u003e26,27\u003c/sup\u003e. BfmR sequence conservation across bacteria and archaea underscores its role in regulating multidrug resistance and modulating pathogenesis, while its absence in mammalian hosts highlights its potential as a therapeutic target\u003csup\u003e28,29\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSeaweeds represent a vast reservoir of proximate compositions, micronutrients, and trace elements, offering substantial nutritional benefits for humans and other animals\u003csup\u003e30\u003c/sup\u003e. Brown seaweeds, particularly those of the \u003cem\u003eSargassum\u003c/em\u003e genus, harbor a wealth of bioactive compounds with significant pharmacological and biological applications\u003csup\u003e31,32\u003c/sup\u003e. An estimated 35.7\u0026nbsp;million wet metric tonnes of seaweed were produced worldwide in 2019, with 96.97% originating from cultivation and 3.03% from natural beds\u003csup\u003e33\u003c/sup\u003e. According to the Food and Agriculture Organization (FAO) database, the first-sale value of the 34.7\u0026nbsp;million metric tonnes of seaweed produced globally for various food and non-food purposes in 2019 was USD 14.7\u0026nbsp;billion. Seaweed-based functional food components largely provide plenty of medicinal benefits and a variety of bioactivity against various ailments. The great diversity of brown seaweeds of the genus \u003cem\u003eSargassum\u003c/em\u003e provides an undiscovered reservoir of bioactive compounds with considerable pharmacological and biological applications\u003csup\u003e34\u003c/sup\u003e. Fucoidan, fucoxanthin, flavonoids, and polyphenols, including phloroglucinol, phlorotannins, and phenolic acids, are abundant in brown seaweeds, notably \u003cem\u003eSargassum wightii\u003c/em\u003e (\u003cem\u003eS. wightii\u003c/em\u003e). The rich polysaccharide content and phytochemical derivatives of \u003cem\u003eS. wightii\u003c/em\u003e contribute to its diverse biological properties, making it a valuable resource in combating various ailments\u003csup\u003e35\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe Indian brown seaweed, \u003cem\u003eS.wightii\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), was collected from Mandapam (Lat- 9\u0026deg; 16' 58.4436'' N; Long- 79\u0026deg; 11' 4.6572'' E), Palk Bay region, India. Among the algal family, \u003cem\u003eS.wightii\u003c/em\u003e is a brown macroalga that comes under the family of \u003cem\u003eSargassaceae\u003c/em\u003e and order of Fucales with more polysaccharide contents\u003csup\u003e36\u003c/sup\u003e. Its polysaccharide-rich composition renders it suitable for animal feed, food ingredients, and fertilizer applications\u003csup\u003e37\u003c/sup\u003e. The enzymatic-ligand interactions inherent to \u003cem\u003eS. wightii\u003c/em\u003e facilitate the synthesis of novel pharmacophores and nutraceuticals to counter MDR bacterial infections\u003csup\u003e38\u003c/sup\u003e. Over the past century, seaweeds have found utility in various industrial applications, including the production of agar, alginate, and carrageen an, as well as serving as a main food in Asian cuisines. \u003cem\u003eS. wightii\u003c/em\u003e stands out as the largest, economically significant, and ecologically dominant algae\u003csup\u003e39\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThis study aims to isolate and evaluate bioactive compounds from aerial parts of \u003cem\u003eS. wightii\u003c/em\u003e for their antibacterial and antibiofilm activities against \u003cem\u003eA. baumannii\u003c/em\u003e through \u003cem\u003ein silico\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e studies. Furthermore, the interaction patterns of these compounds with MDR BfmR from \u003cem\u003eA. baumannii\u003c/em\u003e (MDR BfmR-Ab) complexes will be predicted and validated to design novel potential inhibitors against \u003cem\u003eA. baumannii\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eThe brown seaweed, \u003cem\u003eS.wightii\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), was collected from Mandapam (Lat- 9\u0026deg; 16' 58.4436'' N; Long- 79\u0026deg; 11' 4.6572'' E), Palk Bay region, India. The species was identified following the references provided by Oza and Zaidu (2003)\u003csup\u003e40\u003c/sup\u003e and Krishnamurthy and Joshi (1970)\u003csup\u003e41\u003c/sup\u003e, and subsequently verified by Dr. M. Ganesan, a Senior Principal Scientist at CSMCRI, Mandapam Camp, Tamil Nadu. The voucher specimen validating this identification was deposited at the Department of Marine Science, Bharathidasan University, with the accession number BDUMSSW20210019. The collected \u003cem\u003eS. wightii\u003c/em\u003e sample was shifted to the laboratory, rinsed three times using double distilled water and processed further. The plant's aerial components were broken up, cleaned, and allowed to dry for 10 days in the shadows. The plant components were then processed using a mechanical blender to create a fine powder. Until subsequent studies, the dry powder was kept at 4⁰C.\u003c/p\u003e \u003cp\u003ePreparation of solvent extract.\u003c/p\u003e \u003cp\u003eSoxhlet extraction was used to make crude plant extract. In a nutshell, 250 mL of ethanol was used to extract 20g of finely ground plant material. Once the solvent in the extractor's syphon tube had turned colourless, the extraction was continued for another 24h. A rotary evaporator was used to condense the extract after it had been collected in a beaker and subjected to reduced pressure. For future use, the crude extract was kept at 4\u0026deg;C.\u003c/p\u003e \u003cp\u003eIdentification of bioactive compounds from \u003cem\u003eSargassum wightii\u003c/em\u003e (Brown Algae).\u003c/p\u003e \u003cp\u003eAccording to established procedures, a gas chromatography-mass spectrometry (GC-MS) (Agilent GC 7890A/MS5975C) chromatograph outfitted with a Shimadzu QP-500 mass spectrometer was used to investigate the phytochemical composition of an ethanolic extract of \u003cem\u003eS.wightii\u003c/em\u003e. About 1L of the sample was used for analysis after the plant extract was dissolved in ethanol (1:25). The stationary phase was a fused-silica column (30 m long, 0.25 mm in diameter, and 0.25 m in film thickness) coated with polydimethyl siloxane. Helium with 1 ml/min was employed as the carrier gas. The injector's temperature was set at 325\u0026deg;C. The oven's temperature was kept at 50\u0026deg;C for 1minute before being raised to 300\u0026deg;C at a pace of 12\u0026deg;C per minute. The split rate was kept at 1:5, and the ionisation voltage was 70eV.\u003csup\u003e4\u003c/sup\u003e The results are shown in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, which details how the mass spectra were used to determine the extract's composition by comparing them to recognised substances or published data.\u003c/p\u003e \u003cp\u003ePreparation of protein and ligand structures.\u003c/p\u003e \u003cp\u003eThe reference protein structure (Biofilm-controlling Response Regulator BfmR from \u003cem\u003eA.baumannii\u003c/em\u003e) was obtained from the Protein Data Bank (PDB ID: 6BR7) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA)\u003csup\u003e42\u0026ndash;44\u003c/sup\u003e. The preparation and optimization process of the protein BfmR-Ab was performed by the Protein Preparation Wizard and Epik module in the Schr\u0026ouml;dinger suite (Maestro 2023-1)\u003csup\u003e45,46\u003c/sup\u003e. Assigning the hydrogen bonding network was carried out by OPLS3e force field\u003csup\u003e47\u003c/sup\u003e and to acquire the least possible energy minimization\u003csup\u003e48\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe structure of the small molecules identified from \u003cem\u003eS.wightii\u003c/em\u003e was obtained from the National Institute Standard and Technology Chemistry WebBook\u003csup\u003e49\u003c/sup\u003e. Schr\u0026ouml;dinger Maestro (2023-1) used for geometry optimizations with energy minimization of small molecule structures. Module in the LigPrep builder panel was used to prepare and generate the 3D structures of the small molecules (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). OPLS4 force field was used for energy minimization and also to generate low-energy ring confirmation per small molecule\u003csup\u003e50,51\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBinding site prediction.\u003c/p\u003e \u003cp\u003eActive site prediction by the SiteMap module was carried out based on the qualitative site score values obtained from the physical and structural properties of the protein (BfmR-Ab)\u003csup\u003e52\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGlide XP docking.\u003c/p\u003e \u003cp\u003eA total of ten ligands identified from \u003cem\u003eS.wightii\u003c/em\u003e were tested for their antibacterial properties against \u003cem\u003eA.baumannii\u003c/em\u003e using the Glide XP docking studies (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The 3D structure of \u003cem\u003eS. wightii\u003c/em\u003e phytocompounds was optimized for the docking conformation studies (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The Glide XP docking was performed to determine the BfmR as a potential drug target for \u003cem\u003eA.baumannii\u003c/em\u003e (BfmR-Ab) ligand interaction. Docking was carried out after constructing the grid by selecting the amino acid residues present in the binding site within the radius of 3\u0026Aring; as the centroid. Default parameters were selected by keeping the ligands flexible on the docking calculation set. The formation of hydrogen bonds between the ligands and the residues of the active site, its length, and Glide XP (extra-precision) score were recorded\u003csup\u003e53\u0026ndash;55\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBinding free energy calculation.\u003c/p\u003e \u003cp\u003eThe binding free energy was calculated with prime Molecular Mechanics/Generalized Born Surface Area (MM/GBSA)\u003csup\u003e56,57\u003c/sup\u003e. Prime uses a surface GB model employing a Gaussian surface instead of the van der Waals surface that better represents the solvent-accessible surface area. The following Eqs.\u0026nbsp;(1\u0026ndash;4) were used for calculating the binding energy (ΔG\u003csub\u003ebind\u003c/sub\u003e).\u003c/p\u003e \u003cp\u003eΔG\u003csub\u003ebind\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;ΔE\u0026thinsp;+\u0026thinsp;ΔG\u003csub\u003esoly\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ΔG\u003csub\u003eSA\u003c/sub\u003e, (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eΔE\u0026thinsp;=\u0026thinsp;E\u003csub\u003ecomplex\u003c/sub\u003e \u0026ndash; E\u003csub\u003eprotein\u003c/sub\u003e \u0026ndash; E\u003csub\u003eligand\u003c/sub\u003e, (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e \u003cp\u003ewhere E\u003csub\u003ecomplex\u003c/sub\u003e, E\u003csub\u003eprotein\u003c/sub\u003e, and E\u003csub\u003eligand\u003c/sub\u003e are the minimized energies of the protein-inhibitor complex, protein, and inhibitor, respectively\u003csup\u003e58\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eΔG\u003csub\u003esolv\u003c/sub\u003e = G\u003csub\u003esolv\u003c/sub\u003e(complex) - G\u003csub\u003esolv\u003c/sub\u003e(protein) - G\u003csub\u003esolv\u003c/sub\u003e(ligand), (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e \u003cp\u003ewhere G\u003csub\u003esolv\u003c/sub\u003e(complex), G\u003csub\u003esolv\u003c/sub\u003e(protein), and G\u003csub\u003esolv\u003c/sub\u003e(ligand) are the solvation-free energies of the complex, protein, and inhibitor, respectively.\u003c/p\u003e \u003cp\u003eΔG\u003csub\u003eSA\u003c/sub\u003e = G\u003csub\u003eSA\u003c/sub\u003e(complex) - G\u003csub\u003eSA\u003c/sub\u003e(protein) - G\u003csub\u003eSA\u003c/sub\u003e(ligand), (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)\u003c/p\u003e \u003cp\u003ewhere G\u003csub\u003eSA\u003c/sub\u003e(complex), G\u003csub\u003eSA\u003c/sub\u003e(protein), and G\u003csub\u003eSA\u003c/sub\u003e(ligand) are the surface area energies for the complex, protein, and inhibitor, respectively. The rational criteria for the selection of best compounds based on scoring and interaction parameters are shown in Glide XP docking studies\u003csup\u003e59,60\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMolecular dynamics simulations.\u003c/p\u003e \u003cp\u003eGROMACS 5.1.4 package is used for molecular dynamics (MD) analysis and the topology files are generated using the automated topology builder in the GROMOS96 54a7 force field for protein-ligand complexes\u003csup\u003e61\u0026ndash;63\u003c/sup\u003e. PRODRG 2.5 server was used for generating topology files for the ligands\u003csup\u003e64\u003c/sup\u003e. The ligand complex obtained from docking was solvated with a single-point charge water model\u003csup\u003e65\u003c/sup\u003e. The solvated system was subjected to 5000 steps of energy minimization employing the steepest descent algorithm. This step was followed by 1 nanosecond (ns) MD simulation, where the BfmR-Ab with ligands complex were position restrained to equilibrate the water and ions under the influence of the solute. The production run was carried out for all the systems for 100 ns using a 2 femtosecond (fs) time step for the integration of the equation of motion in the NPT ensemble at 300 K and at 1 atmospheric pressure, which was controlled using a V-rescale thermostat and Parrinello Rahman Barostat, respectively. Bond lengths involving hydrogen atoms were constrained by using the Linear Constraint Solver (LINCS) algorithm \u003csup\u003e66\u003c/sup\u003e. The Particle Mesh Ewald (PME) method was used to calculate the electrostatic interaction\u003csup\u003e67,68\u003c/sup\u003e. A 10\u0026Aring; cutoff distance was used for the long-range electrostatic and van der Waals energy terms and a 2 ps interval was used for saving the MD simulation coordinates of all the systems for further analyses.\u003c/p\u003e \u003cp\u003eA careful equilibration at a desired temperature and pressure for MD analyses was done after the completion of two equilibration phases. For each ligand, with the available 3D structure (PDB ID: 6BR7) of the BfmR-Ab, the final MD run was set at 100 ns.\u003c/p\u003e \u003cp\u003eTrajectory Analysis.\u003c/p\u003e \u003cp\u003eAnalysis of the trajectories was carried out using gmx rms, gmx rmsf, and gmx hbond utilities of the GROMACS package to obtain the graphs of the RMSD, RMSF values and number of hydrogen bonds using the Gnuplot tool version 5.4\u003csup\u003e69\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eQuantitative analysis of biofilm by crystal violet assay.\u003c/p\u003e \u003cp\u003eBiofilm was quantified against different concentrations of ethanolic extract of \u003cem\u003eS. wightii\u003c/em\u003e according to minor modification. The overnight grown culture (0.6 at OD\u003csub\u003e600nm\u003c/sub\u003e) was inoculated in Luria-Bertani (LB) broth using a 96-well microtiter plate in the ratio of 1:10 with different concentrations of \u003cem\u003eS. wightii\u003c/em\u003e ethanolic extract (70\u0026micro;g/mL, 80\u0026micro;g/mL, 90\u0026micro;g/mL and 100\u0026micro;g/mL) and incubated in static at 37⁰C for 48h. The wells of the plate were rinsed with 1X phosphate-buffered saline (PBS), followed by staining with crystal violet (0.2%) for 10 min. The excess stain was rewashed with 1X PBS and allowed to dry. The stained biofilm ring in the wells at the air-water interface was dissolved with acetic acid (30%), and the culture was measured at OD\u003csub\u003e560nm\u003c/sub\u003ewith a microplate reader (Synergy H1, BioTek, USA). The percentage (%) of biofilm inhibition was compared based on absorbance between higher and lower levels at particular experimental groups using the following Eqs.\u0026nbsp;(5),\u003c/p\u003e\u003cp\u003e\u003cimg 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\" style=\"width: 477px; height: 58.3288px;\" width=\"477\" height=\"58.3288\"\u003e\u003c/p\u003e \u003cp\u003e2.10. Antibiofilm activity by epifluorescence microscopic observation.\u003c/p\u003e \u003cp\u003eImaging of biofilm matrix against different concentrations of ethanolic extract of \u003cem\u003eS. wightii\u003c/em\u003e (70\u0026micro;g/mL, 80\u0026micro;g/mL, 90\u0026micro;g/mL and 100\u0026micro;g/mL) provide information regarding changes in matrix pattern and their contribution to the biomass for the aggregation, formation and the determination of the size of aggregates\u003csup\u003e70\u003c/sup\u003e. Antibiofilm activity and microscopic observation of \u003cem\u003eS. wightii\u003c/em\u003e extract on candidate bacteria \u003cem\u003eA.baumannii\u003c/em\u003e were analysed using the epifluorescence microscope (Carl Zeiss Axioscope 5, Germany). Overnight grown bacterial culture (0.6 at OD\u003csub\u003e600nm\u003c/sub\u003e) was diluted to 1:10 in pre-sterilized LB broth medium containing glass slides (1cm x1cm) and incubated in static at 37⁰C for 48h. After incubation, the slides were rinsed with 1X PBS. The specific position and relative amount of matrix components mainly bacterial adherent cell biomass were viewed by staining with specific fluorescent markers (Syto9, Thermo Scientific)\u003csup\u003e71\u003c/sup\u003e. The biofilm-formed glass slides were stained by adding stain solution for 20 mins followed by washing twice using 1X PBS after staining in dark conditions. After the final wash, the coverslip was fixed and examined under the objective lens of the epifluorescence microscope.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eThe chromatography of the ethanol extract of \u003cem\u003eS. wightii\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB) reveals the identification of bioactive compounds represented by 25 prominent peaks. The list of compounds, along with their retention times and the percentage of area occupied, is presented in Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e. Major compounds identified with GC-MS analysis were glycerin and n-heptyl hexanoate (42.64%), octadecanoic acid (15.67%), ethanone ,1-(6-methyl-7-oxabicyclo acid [4.1.0] hept-1-yl) and 9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]- (8.17%), pregn-5- en-20-one,3-(acetyloxy)-acid 20-(1,2-ethanediyl acetal), (3 beta)-, 2-hydroxytetracosanoic acid and pentanoic acid, 4-oxo-, butyl ester (5.28%) and n-decanoic acid and n-hexadecanoic acid (3.94%) (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eSelected bioactive compounds of brown seaweed \u003cem\u003eSargassum wightii\u003c/em\u003e (GC-MS analysis).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eS. No\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRetention Time\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCAS Number\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eName of the Compound\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMolecular Formula\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMolecular Weight\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePeak Area (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eRT-5.076\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e000056-81-5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlycerin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e3\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e92.094\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" rowspan=\"2\"\u003e\n \u003cp\u003e42.64\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e006976-72-3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en-heptyl hexanoate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e13\u003c/sub\u003eH\u003csub\u003e26\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e214.344\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eRT-14.297\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e002302-12-7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta)-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e25\u003c/sub\u003eH\u003csub\u003e38\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e402.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e5.28\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1000336-12-4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2-hydroxytetracosanoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e24\u003c/sub\u003eH\u003csub\u003e48\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e384.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e002052-15-5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePentanoic acid, 4-oxo-, butyl ester\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e172.222\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eRT-14.630\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e000334-48-5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en-decanoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e172.265\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e3.94\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e000057-10-3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en-hexadecanoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e32\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e256.424\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRT-16.396\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e000057-11-4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOctadecanoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e36\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e284.477\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15.67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eRT-20.474\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e015120-94-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEthanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl)-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e154.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" rowspan=\"2\"\u003e\n \u003cp\u003e8.17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e146397-91-3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e27\u003c/sub\u003eH\u003csub\u003e33\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e479.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThe major compound, with 42.64% occupancy, was observed to be n-heptylhexanoate, which is commonly used as a flavoring agent in the food industry. The essential oil of \u003cem\u003eBursera schlechtendalii\u003c/em\u003e contains 17.6% n-heptylhexanoate\u003csup\u003e72\u003c/sup\u003e. n-hexadecanoic acid has been reported to possess anti-inflammatory\u003csup\u003e73\u003c/sup\u003e, cytotoxic\u003csup\u003e74\u003c/sup\u003e, anticancer\u003csup\u003e75\u003c/sup\u003e, and antibacterial\u003csup\u003e76\u003c/sup\u003e activities. Ethanone, 1-(6-methyl-7-oxobicyclo[4.1.0]hept-1-yl), is a corticosteroid analog, also known as 1-acetyl-1,2,epoxy-2-methylcyclohexane\u003csup\u003e77\u003c/sup\u003e. Corticosteroids are immunosuppressant commonly used in treating inflammation and allergic disorders.\u003c/p\u003e\n\u003cp\u003eADME properties predictions.\u003c/p\u003e\n\u003cp\u003eEstimating the ADME properties of ligands becomes crucial in drug discovery for considering them as efficient hit compounds. The drug-likeness attributes of identified bioactive compounds from the brown seaweed \u003cem\u003eS. wightii\u003c/em\u003e were evaluated for their ADME characteristics using the QikProp module, which included detailed analyses of HB donor/acceptor, central nervous system (CNS) permeation, Rule of Five, Rule of Three, QPlogPw, QPlogPo/w, percent human oral absorption, QPlogBB, and QPlogS, among others. The drug-likeness of ligands, as assessed by the Lipinski Rule of Five, is vital in the rational design of a drug. Notably, the human oral absorption percentage was found to be 100% for n-heptyl hexanoate (CAS No. 006976-72-3) and pregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta-) (CAS No. 002302-12-7). Ligands such as 2-hydroxytetracosanoic acid (CAS No. 1000336-12-4), pentanoic acid, 4-oxo-, butyl ester (CAS No. 002052-15-5), octadecanoic acid (CAS No. 000057-11-4), and ethanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl)- (CAS No. 015120-94-2) exhibited human oral absorption rates of 92.752%, 91.168%, 91.558%, and 96.746%, respectively. These findings indicate excellent absorption properties for the potent hit compounds listed above. The allowable range of solvent-accessible surface area (SASA) for ligands ranged between 300 and 1000. The potent hit compounds analyzed for toxicity prediction exhibited SASA within the admissible range of 270 to 938, indicating the druggable nature of these ligands.\u003c/p\u003e\n\u003cp\u003eThe ligands glycerin, n-heptyl hexanoate, pregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta), 2-hydroxytetracosanoic acid, pentanoic acid, 4-oxo-, butyl ester, n-decanoic acid, n-hexadecanoic acid, octadecanoic acid, ethanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl), and 9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]- recorded SASA values of 270.549, 597.82, 686.236, 938.649, 447.219, 480.679, 675.443, 733.206, 348.35, and 852.72, respectively. A high SASA value was observed with 2-hydroxytetracosanoic acid (938.649) and a CNS value of -2. The hydrogen bond donors of the ligand molecules ranged between 0 and 3, and the hydrogen bond acceptors ranged between 2 and 8.5. n-Heptyl hexanoate, n-decanoic acid, n-hexadecanoic acid, and octadecanoic acid had the minimum number of hydrogen acceptors, while glycerin, pentanoic acid, 4-oxo-butyl ester, ethanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl), and 9H-fluoren-9-one,3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]- exhibited the maximum number of hydrogen bond acceptors. More than 60 calculated and predicted properties were considered, but for documentation, we have tabulated certain important properties in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, where all the compounds were well within the range of drugability. As shown in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, all 10 compounds (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e) fall within the ranges of known drug properties. From the table, it could be inferred that there is not more than one violation of Lipinski\u0026rsquo;s Rule of Five.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003ePrediction of ADME properties for the selected compounds from brown seaweed \u003cem\u003eSargassum wightii\u003c/em\u003e using QikProp.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"left\" width=\"1052\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.792578496669837%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCompound\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eName\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.421503330161751%\"\u003e\n \u003cp\u003e\u003cstrong\u003emol_ MW\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDonor\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eHB\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAcceptor\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eHB\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.137963843958135%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCNS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.992388201712655%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSASA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.705042816365367%\"\u003e\n \u003cp\u003e\u003cstrong\u003e%Human\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eOral\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAbsorption\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e\u003cstrong\u003eRule of five\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.279733587059943%\"\u003e\n \u003cp\u003e\u003cstrong\u003eRule of three\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e\u003cstrong\u003eQPlogPw\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.992388201712655%\"\u003e\n \u003cp\u003e\u003cstrong\u003eQPlogPo/w\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e\u003cstrong\u003eQPlogS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e\u003cstrong\u003eQPlogBB\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.792578496669837%\"\u003e\n \u003cp\u003eGlycerin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.421503330161751%\"\u003e\n \u003cp\u003e92.094\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e5.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.137963843958135%\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.992388201712655%\"\u003e\n \u003cp\u003e270.549\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.705042816365367%\"\u003e\n \u003cp\u003e65.515\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.279733587059943%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e9.725\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.992388201712655%\"\u003e\n \u003cp\u003e-1.193\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e0.081\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e-0.881\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.792578496669837%\"\u003e\n \u003cp\u003en-heptyl hexanoate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.421503330161751%\"\u003e\n \u003cp\u003e214.344\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.137963843958135%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.992388201712655%\"\u003e\n \u003cp\u003e597.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.705042816365367%\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.279733587059943%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e1.308\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.992388201712655%\"\u003e\n \u003cp\u003e4.287\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e-4.871\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e-0.637\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.792578496669837%\"\u003e\n \u003cp\u003ePregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta)-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.421503330161751%\"\u003e\n \u003cp\u003e402.573\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.137963843958135%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.992388201712655%\"\u003e\n \u003cp\u003e686.236\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.705042816365367%\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.279733587059943%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e5.106\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.992388201712655%\"\u003e\n \u003cp\u003e5.754\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e-7.226\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e0.069\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.792578496669837%\"\u003e\n \u003cp\u003e2-pydroxytetracosanoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.421503330161751%\"\u003e\n \u003cp\u003e384.641\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e3.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.137963843958135%\"\u003e\n \u003cp\u003e-2\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.992388201712655%\"\u003e\n \u003cp\u003e938.649\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.705042816365367%\"\u003e\n \u003cp\u003e92.752\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.279733587059943%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e4.461\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.992388201712655%\"\u003e\n \u003cp\u003e7.248\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e-8.077\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e-2.724\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.792578496669837%\"\u003e\n \u003cp\u003ePentanoic acid, 4-oxo-, butyl ester\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.421503330161751%\"\u003e\n \u003cp\u003e172.224\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.137963843958135%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.992388201712655%\"\u003e\n \u003cp\u003e447.219\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.705042816365367%\"\u003e\n \u003cp\u003e91.168\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.279733587059943%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e4.037\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.992388201712655%\"\u003e\n \u003cp\u003e1.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e-1.621\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e-0.608\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.792578496669837%\"\u003e\n \u003cp\u003en-decanoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.421503330161751%\"\u003e\n \u003cp\u003e172.265\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.137963843958135%\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.992388201712655%\"\u003e\n \u003cp\u003e480.679\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.705042816365367%\"\u003e\n \u003cp\u003e86.886\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.279733587059943%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e3.283\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.992388201712655%\"\u003e\n \u003cp\u003e2.988\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e-2.956\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e-0.932\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.792578496669837%\"\u003e\n \u003cp\u003en-hexadecanoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.421503330161751%\"\u003e\n \u003cp\u003e256.424\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.137963843958135%\"\u003e\n \u003cp\u003e-2\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.992388201712655%\"\u003e\n \u003cp\u003e675.443\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.705042816365367%\"\u003e\n \u003cp\u003e89.022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.279733587059943%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e2.375\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.992388201712655%\"\u003e\n \u003cp\u003e5.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e-5.585\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e-1.393\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.792578496669837%\"\u003e\n \u003cp\u003eOctadecanoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.421503330161751%\"\u003e\n \u003cp\u003e284.477\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.137963843958135%\"\u003e\n \u003cp\u003e-2\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.992388201712655%\"\u003e\n \u003cp\u003e733.206\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.705042816365367%\"\u003e\n \u003cp\u003e91.558\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.279733587059943%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e2.127\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.992388201712655%\"\u003e\n \u003cp\u003e6.002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e-6.331\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e-1.648\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.792578496669837%\"\u003e\n \u003cp\u003eEthanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl)-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.421503330161751%\"\u003e\n \u003cp\u003e154.208\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.137963843958135%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.992388201712655%\"\u003e\n \u003cp\u003e348.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.705042816365367%\"\u003e\n \u003cp\u003e96.746\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.279733587059943%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e4.559\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.992388201712655%\"\u003e\n \u003cp\u003e0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e-0.561\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e0.213\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.792578496669837%\"\u003e\n \u003cp\u003e9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.421503330161751%\"\u003e\n \u003cp\u003e479.575\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e8.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.137963843958135%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.992388201712655%\"\u003e\n \u003cp\u003e852.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.705042816365367%\"\u003e\n \u003cp\u003e74.709\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.279733587059943%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e10.19\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.992388201712655%\"\u003e\n \u003cp\u003e3.526\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.708848715509039%\"\u003e\n \u003cp\u003e-4.021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.850618458610847%\"\u003e\n \u003cp\u003e-0.725\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"1069\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.176800748362957%\"\u003e\n \u003cp\u003eProperty or Descriptor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.541627689429374%\"\u003e\n \u003cp\u003emol_MW\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.612722170252573%\"\u003e\n \u003cp\u003eDonor HB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.17399438727783%\"\u003e\n \u003cp\u003eAcceptor HB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.612722170252573%\"\u003e\n \u003cp\u003eCNS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.735266604303087%\"\u003e\n \u003cp\u003eSASA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.980355472404115%\"\u003e\n \u003cp\u003e% Human Oral Absorption\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.17399438727783%\"\u003e\n \u003cp\u003eRule of five\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.612722170252573%\"\u003e\n \u003cp\u003eRule of three\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.17399438727783%\"\u003e\n \u003cp\u003eQPlogPw\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.296538821328344%\"\u003e\n \u003cp\u003eQPlogPo/w\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.735266604303087%\"\u003e\n \u003cp\u003eQPlogS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.17399438727783%\"\u003e\n \u003cp\u003eQPlogBB\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.176800748362957%\"\u003e\n \u003cp\u003eRange or recommended values\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.541627689429374%\"\u003e\n \u003cp\u003e130.0 \u0026ndash; 725.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.612722170252573%\"\u003e\n \u003cp\u003e0\u0026ndash;6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.17399438727783%\"\u003e\n \u003cp\u003e2\u0026ndash;20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.612722170252573%\"\u003e\n \u003cp\u003e-2 to +2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.735266604303087%\"\u003e\n \u003cp\u003e300\u0026ndash;1000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.980355472404115%\"\u003e\n \u003cp\u003e\u0026gt;80% is high,\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026lt; 25% is poor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.17399438727783%\"\u003e\n \u003cp\u003eMax 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.612722170252573%\"\u003e\n \u003cp\u003eMax 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.17399438727783%\"\u003e\n \u003cp\u003e4\u0026minus;45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.296538821328344%\"\u003e\n \u003cp\u003e-2 -6.5\u003c/p\u003e\n \u003cp\u003e(Neg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.735266604303087%\"\u003e\n \u003cp\u003e-6.5 -0.5\u003c/p\u003e\n \u003cp\u003e(Neg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.17399438727783%\"\u003e\n \u003cp\u003e-3 -1.2\u003c/p\u003e\n \u003cp\u003e(Neg)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u003c/strong\u003e SiteMap properties of BfmR from \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e (BfmR-Ab)\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"1015\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.738916256157635%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBinding Sites\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.586206896551722%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAA Residues\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.064039408866995%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCavity\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eSize (A)\u003csup\u003e3\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.320197044334975%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSite\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eScore\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.275862068965518%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eEnclosure\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.64039408866995%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eHydrophobic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.049261083743842%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eHydrophilic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.458128078817735%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eD Score\u003csup\u003e*\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.866995073891626%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eContact\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.738916256157635%\"\u003e\n \u003cp\u003eBinding site-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.586206896551722%\"\u003e\n \u003cp\u003e12,14,16,19,22,23,26,29,84,107,108,109,111,114\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.064039408866995%\"\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.320197044334975%\"\u003e\n \u003cp\u003e0.779\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.275862068965518%\"\u003e\n \u003cp\u003e0.662\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.64039408866995%\"\u003e\n \u003cp\u003e1.203\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.049261083743842%\"\u003e\n \u003cp\u003e0.605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.458128078817735%\"\u003e\n \u003cp\u003e0.780\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.866995073891626%\"\u003e\n \u003cp\u003e0.937\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.738916256157635%\"\u003e\n \u003cp\u003eBinding site-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.586206896551722%\"\u003e\n \u003cp\u003e9,10,11,33,34,35,46,50,52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.064039408866995%\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.320197044334975%\"\u003e\n \u003cp\u003e0.666\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.275862068965518%\"\u003e\n \u003cp\u003e0.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.64039408866995%\"\u003e\n \u003cp\u003e1.059\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.049261083743842%\"\u003e\n \u003cp\u003e0.815\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.458128078817735%\"\u003e\n \u003cp\u003e0.625\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.866995073891626%\"\u003e\n \u003cp\u003e0.948\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eLigand Binding Site Analysis of BfmR-Ab.\u003c/p\u003e\n\u003cp\u003ePredicting the binding pose of the complex plays a key role in making the docking process precise. The validation of the docking process using Glide XP was carried out by docking the ligand molecules with the active sites of the target protein BfmR-Ab, which is a potential drug target for \u003cem\u003eA. baumannii\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Two binding sites were identified in BfmR-Ab (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). The Site scores (S) of binding sites 1 and 2 were 0.779 and 0.666, respectively, and the drug ability scores (D) were observed as 0.780 and 0.625, respectively. The binding pocket with the maximum D score was selected for further \u003cem\u003ein silico\u003c/em\u003e studies (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Since the binding site with a higher D score indicates a more favourable or promising binding site for a ligand or molecule, binding site-1 was chosen for further analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular Docking.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe hit ligands observed in \u003cem\u003eS.wightii\u003c/em\u003e, including glycerin (CAS No. 000056-81-5), n-heptyl hexanoate (CAS No. 006976-72-3), pregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta)- (CAS No. 002302-12-7), 2-hydroxytetracosanoic acid (CAS No. 1000336-12-4), pentanoic acid, 4-oxo-, butyl ester (CAS No. 002052-15-5), n-decanoic acid (CAS No. 000334-48-5), n-hexadecanoic acid (CAS No. 000057-10-3), octadecanoic acid (CAS No. 000057-11-4), ethanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl)- (CAS No. 015120-94-2), and 9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]- (CAS No. 146397-91-3), were investigated for their binding efficiencies with \u003cem\u003eA. baumannii\u003c/em\u003e (BfmR-Ab), and their 2D and 3D structures are presented in Figs. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e. The interacting amino acids, H-bonded interactions, bond lengths, Glide score (kcal/mol), and MM-GBSA ∆G\u003csub\u003ebind\u003c/sub\u003e (kcal/mol) of the protein-ligand complexes are shown in Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. Glycerin and n-heptyl hexanoate, which bound at the binding site of the target protein, exhibited a Glide score of -3.812 (kcal/mol) and a binding energy (∆G\u003csub\u003ebind\u003c/sub\u003e) of -22.82 kcal/mol. Hydrogen bond interactions were found with the OH group of THR23 and VAL109 amino acid residues with bond lengths of 1.90 \u0026Aring; and 1.85 \u0026Aring;, respectively and a Glide score of -3.812 (kcal/mol) and binding energy (∆G\u003csub\u003ebind\u003c/sub\u003e) of -22.82 kcal/mol hydrogen bond interactions were observed with the VAL109 amino acid residues with a bond length of 2.48\u0026Aring;.\u003c/p\u003e\n\u003cp\u003eThe interaction of the ligands glycerin and n-heptyl hexanoate with BfmR-Ab is depicted in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e. The docking of pregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta)- with BfmR-Ab was mediated by the HO group of VAL109 residue with a bond length of 1.85 \u0026Aring;, Glide score of -0.511 (kcal/mol), and binding energy (∆G\u003csub\u003ebind\u003c/sub\u003e) of -42.26 kcal/mol. The docking of 2-hydroxytetracosanoic acid with BfmR-Ab exhibited a Glide score of -3.100 kcal/mol, binding energy (∆G\u003csub\u003ebind\u003c/sub\u003e) of -32.64 kcal/mol, and hydrogen bond interactions were found with the OH and HO groups of ASP16 and LYS107 amino acid residues with bond lengths of 1.80 \u0026Aring; and 2.13 \u0026Aring;, respectively. Pentanoic acid, 4-oxo-, butyl ester, and n-decanoic acid are two ligands that commonly interact with the VAL109 residue of BfmR-Ab through HO groups with bond lengths of 1.92 \u0026Aring; and 1.69 \u0026Aring;, respectively. The Glide scores were found to be -4.029 and \u0026minus;\u0026thinsp;1.502 kcal/mol, and the binding energies (∆G\u003csub\u003ebind\u003c/sub\u003e) were \u0026minus;\u0026thinsp;26.51 and \u0026minus;\u0026thinsp;15.30 kcal/mol. The ligands n-hexadecanoic acid and octadecanoic acid each make two interactions with the ARG29 residue of BfmR-Ab (c.f. Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eBased on Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e,it can be observed that the lowest Glide score and binding energy of the two ligands indicate that ethanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl)- and 9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]- acidare potential drug candidates with high affinity towards the BfmR of \u003cem\u003eA. baumannii\u003c/em\u003e. From the Glide docking studies, it could be inferred that four compounds, namely pregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta)-, pentanoic acid, 4-oxo-, butyl ester, ethanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl)- and 9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]-, can be considered as potent BfmR-Ab inhibitor candidates, and can be used as potential antibacterial drugs that could regulate or inhibit the biofilm activity in \u003cem\u003eA.baumannii\u003c/em\u003e.\u003c/p\u003e\n\u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eGlide XP results and prime-MM/GBSA binding free energy (\u0026Delta;G\u003csub\u003ebind\u003c/sub\u003e) for the 10 ligands with the BfmR-Ab.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eS. No.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLigands\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAmino Acid\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eH-Bond\u003c/p\u003e\n \u003cp\u003eInteraction\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBond Length\u003c/p\u003e\n \u003cp\u003e(\u0026Aring;)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGlide XP Score\u003c/p\u003e\n \u003cp\u003e(kcal/mol)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMM-GBSA\u003c/p\u003e\n \u003cp\u003e∆G\u003csub\u003ebind\u003c/sub\u003e\u003c/p\u003e\n \u003cp\u003e(kcal/mol)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlycerin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTHR 23\u003c/p\u003e\n \u003cp\u003eVAL 109\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eO-H\u003c/p\u003e\n \u003cp\u003eO-H\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.90\u003c/p\u003e\n \u003cp\u003e1.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-3.812\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-22.82\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en-heptyl hexanoate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVAL 109\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH-O\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-3.812\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-22.82\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta)-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVAL 109\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH-O\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.511\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-42.26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2-hydroxytetracosanoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eASP 16\u003c/p\u003e\n \u003cp\u003eLYS 107\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eO-H\u003c/p\u003e\n \u003cp\u003eH-O\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.80\u003c/p\u003e\n \u003cp\u003e2.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-3.100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-32.64\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePentanoic acid, 4-oxo-, butyl ester\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVAL 109\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH-O\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-4.029\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-26.51\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en-decanoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVAL 109\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH-O\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.502\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-15.30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en-hexadecanoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eARG 29\u003c/p\u003e\n \u003cp\u003eARG 29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH-O\u003c/p\u003e\n \u003cp\u003eH-O\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.05\u003c/p\u003e\n \u003cp\u003e2.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.424\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-28.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOctadecanoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eARG 29\u003c/p\u003e\n \u003cp\u003eARG 29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH-O\u003c/p\u003e\n \u003cp\u003eH-O\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.85\u003c/p\u003e\n \u003cp\u003e1.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.458\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-27.46\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEthanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl)-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVAL 109\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH-O\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-4.067\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-23.67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVAL 109\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH-O\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.491\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-50.49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eMolecular dynamics analysis.\u003c/p\u003e\n\u003cp\u003eThe structural behaviour and flexibility of BfmR-Ab, along with the 10 selected ligand molecules, were studied using MD simulation for 100 ns employing GROMACS version 5.1.4. MD simulation provides information regarding the dynamic behaviour of the protein-ligand complexes in an environment containing ions and water molecules. The stability of the complex was determined by Root Mean Square Deviation (RMSD) and Root Mean Square Fluctuation (RMSF) values of the backbone atoms of the protein and ligand complexes.\u003c/p\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e shows the variation of RMSD from the starting conformation. It was observed that throughout the simulation period (1000 ns), the protein backbone of BfmR-Ab exhibited deviations of not more than 0.3 nm.\u003c/p\u003e\n\u003cp\u003eMolecular dynamics evaluation of the protein-ligand complexes.\u003c/p\u003e\n\u003cp\u003eStability and interaction pattern of the receptor-ligand complex, explicit MD simulations of the accessible configuration space of BfmR-Ab and ten ligand complexes were conducted for 100 ns using GROMACS 4.6.5\u003csup\u003e63\u003c/sup\u003e. The simulation studies of BfmR-Ab with ligand complexes to evaluate stability showed the variation of RMSD from the starting conformation, as depicted in Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA. It was observed that throughout the simulation period (100 ns), the BfmR-Ab-ligand complexes showed that the deviation was not more than 0.35 nm throughout the simulation time (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA). The RMSF plot showed that for all 10 complexes, only very little fluctuation was observed in the residue region 85\u0026ndash;100. The two ligands, pregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta)- and 9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]-, exhibited little fluctuation throughout the simulation period compared to the other ligands (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB). This suggests that these ligands are tightly or stably bound to their binding sites within the target protein, indicating effective activity against biofilm formation.\u003c/p\u003e\n\u003cp\u003eTable \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e presents the average number of hydrogen bond interactions between BfmR-Ab and the ligands. The interaction of BfmR-Ab with ligand 9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]- (CAS No. 146397-91-3) exhibited the highest average hydrogen bonding of 1.19 (Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\n\u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eAverage number of hydrogen bond interactions of BfmR-Ab and ligand complexes at 100 ns.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eS. No.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLigands\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCAS Number\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAverage Hydrogen bond\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlycerin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e000056-81-5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en-heptyl hexanoate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e006976-72-3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta)-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e002302-12-7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2-hydroxytetracosanoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1000336-12-4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.44\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePentanoic acid, 4-oxo-, butyl ester\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e002052-15-5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en-decanoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e000334-48-5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en-hexadecanoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e000057-10-3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOctadecanoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e000057-11-4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEthanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl)-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e015120-94-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy]-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e146397-91-3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eAntibiofilm Activity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMinimal inhibitory concentration and antibiofilm activity analysis by crystal violet staining.\u003c/p\u003e\n\u003cp\u003eThe minimum inhibitory concentration (MIC) using the resazurin assay was performed to determine the minimal concentration of ethanolic extract from \u003cem\u003eS. wightii\u003c/em\u003e that can inhibit the growth of \u003cem\u003eA. baumannii\u003c/em\u003e\u003csup\u003e78\u003c/sup\u003e. The pictorial representation and observation of growth inhibitory activity of ethanolic extract from \u003cem\u003eS. wightii\u003c/em\u003e on candidate bacteria \u003cem\u003eA. baumannii\u003c/em\u003e, a highly troublesome pathogen, is illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e. From this approach, the ethanolic extract of \u003cem\u003eS. wightii\u003c/em\u003e exhibits bacterial growth inhibition potential at a concentration of 90 \u0026micro;g/mL. The concentration of 90 \u0026micro;g/mL revealed no visible growth, indicated by the absence of colour change (from blue to pink), which is considered the MIC\u003csup\u003e79\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe change in colour from blue to pink denotes the bacteria\u0026apos;s viability and growth, whereas the absence of a change in the blue colour represents no bacterial growth inhibition. Rajivgandhi \u003cem\u003eet al.\u003c/em\u003e (2021) reported the MIC value of ethanolic extract from \u003cem\u003eS. wightii\u003c/em\u003e was observed at 200 \u0026micro;g/mL on \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e\u003csup\u003e80\u003c/sup\u003e. A previous study reported that the bioactivity of pyrogallol exhibited MIC at 120 \u0026micro;g/mL\u003csup\u003e81\u003c/sup\u003e. Additionally, a prior study also yielded comparable findings, demonstrating the enhanced bioactivity of a crude extract of \u003cem\u003eS. wightii\u003c/em\u003e against gram-negative bacteria. \u003cem\u003eS. wightii\u003c/em\u003e possesses a greater abundance of polysaccharide compounds compared to other algae, and these polysaccharides exhibit the capacity to penetrate pathogens and disrupt their nuclei effectively. Once inside the nucleus, they exert an impact on the entire bacterial structure, impeding the cell cycle growth and ultimately resulting in cell demise\u003csup\u003e82\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn this study, the antibiofilm activities of ethanolic extract from \u003cem\u003eS. wightii\u003c/em\u003e on candidate bacteria \u003cem\u003eA. baumannii\u003c/em\u003e were measured in a concentration-dependent manner using the absorbance (OD\u003csub\u003e560nm\u003c/sub\u003e) value of crystal violet staining at 48-hour intervals. The concentration of the ethanolic extract from \u003cem\u003eS. wightii\u003c/em\u003e for the antibiofilm study was selected based on the MIC value of the objective extract. Accordingly, two steps down and one step-up concentration at a 10 \u0026micro;g/mL difference were selected. However, the concentrations of ethanolic extract from \u003cem\u003eS. wightii\u003c/em\u003e were selected as 70 \u0026micro;g/mL, 80 \u0026micro;g/mL, 90 \u0026micro;g/mL, and 100 \u0026micro;g/mL. The ability to form biofilms is unique to each species of bacteria due to differences in genetic makeup, physiology, and other variables; therefore, different microbe species may have varying degrees of proficiency in building biofilms. From this study, the antibiofilm activity based on crystal violet staining of ethanolic extract from \u003cem\u003eS. wightii\u003c/em\u003e on \u003cem\u003eA. baumannii\u003c/em\u003e was represented in Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e. Results suggested that two step-down concentrations from MIC (70 and 80 \u0026micro;g/mL) of ethanolic extract exhibited a significant reduction in biofilm biomass formed \u003cem\u003eby A. baumannii\u003c/em\u003e. However, MIC (90 \u0026micro;g/mL) and one step higher concentration (100 \u0026micro;g/mL) of ethanolic extract from \u003cem\u003eS. wightii\u003c/em\u003e, compared to the MIC value, revealed biofilm eradication potentiality formed by \u003cem\u003eA. baumannii.\u003c/em\u003e Thus, ethanolic extract from \u003cem\u003eS. wightii\u003c/em\u003e reveals antibiofilm potentiality in a dose-dependent manner compared to a control group without exposure to the objective extract. However, a comparative analysis of the percentage inhibition of biofilm formation by \u003cem\u003eA. baumannii\u003c/em\u003e in the presence of ethanolic extract from \u003cem\u003eS. wightii\u003c/em\u003e, compared to control groups (without exposure of the objective extract), indicates 27.57%, 43.41%, 54.87%, and 77.74% in T-1 (treated concentration: 70 \u0026micro;g/mL), T2 (treated concentration: 80 \u0026micro;g/mL), T3 (treated concentration: 90 \u0026micro;g/mL) followed by the T4 experimental group (treated concentration: 100 \u0026micro;g/mL).\u003c/p\u003e\n\u003cp\u003eThe crystal violet-based antibiofilm activity of \u003cem\u003eS. wightii\u003c/em\u003e ethanolic crude extract demonstrated outstanding performance with a remarkably low MIC of just two steps down concentration from MIC. At this concentration, the biofilm was significantly reduced, and its original characteristics underwent gradual changes with increasing concentrations. In a recent development, researchers reported that extracts derived from seaweeds like \u003cem\u003eS. wightii\u003c/em\u003e and \u003cem\u003eHalimeda gracilis\u003c/em\u003e exhibit remarkable antibiofilm activity even at lower concentrations\u003csup\u003e83\u003c/sup\u003e. Methicillin-Resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) and \u003cem\u003eA. baumannii\u003c/em\u003e (MDRAB) hold a prominent position on the World Health Organization\u0026apos;s (WHO) list of high-priority human pathogens. These two microorganisms, responsible for challenging and long-lasting human infections, demand special attention due to their significant impact\u003csup\u003e84\u003c/sup\u003e. \u003cem\u003eA. baumannii\u003c/em\u003e induces severe infections in individuals with compromised immune systems, thrives on non-living surfaces within hospital environments, and establishes bacterial colonization on a variety of medical devices\u003csup\u003e85\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eA. baumannii\u003c/em\u003e has the capacity to create biofilms on non-living surfaces, making it a significant contributor to hospital-acquired infections. Adherence and biofilm formation are pathogenic mechanisms that contribute to clinical complications\u003csup\u003e86\u003c/sup\u003e. The ability to create biofilms on both living and non-living surfaces is a critical factor and a frequent contributor to persistent infections associated with implanted medical devices, as well as resistance to a broad range of antimicrobial agents. Preventing biofilm formation not only aids bacteria in evading the host\u0026apos;s immune defences and antimicrobial treatments but also hinders the progression of infections\u003csup\u003e87\u003c/sup\u003e. However, based on this investigation, we conclude the antibacterial activity of seaweed extract; these findings indicate that the MIC value of the crude extract is comparatively lower. In other cases, an effective concentration of crude extract up to 400 mg/mL has been reported in several studies examining its antibacterial properties (Zammuto et al., 2022)\u003csup\u003e88,89\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eVisualization of the Biofilm in Epifluorescence Microscope.\u003c/p\u003e\n\u003cp\u003eAfter treatment with an ethanolic extract of \u003cem\u003eS.wightii\u003c/em\u003e, representative virtual images of biofilms were captured in fluorescence mode. Further, stained with Syto9, images display the full extent of the biofilms developing at the surface of the cover slips. Figure \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e illustrates biofilm-covered regions (A, B, C) that have been evaluated for control, where Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eA represents magnification at x20, Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eB denotes magnification x40 and Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eC represents a 2.5D illustration. The treated groups are now taking into consideration all scan modes in fluorescence mode after Syto9 staining. Followed by Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e, T1 to T4, (D-O), notably the extract decreases in the bacterial cells at different concentrations of \u003cem\u003eS. wightii\u003c/em\u003e (70 \u0026micro;g/mL, 80 \u0026micro;g/mL, 90 \u0026micro;g/mL and 100 \u0026micro;g/mL), respectively. The attachment of biofilm biomass in the presence of \u003cem\u003eS. wightii\u003c/em\u003e extract showed a significant decrease in biofilm-covered area, area, as clearly depicted in Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e. SYTO9\u0026apos;s capacity to attach to nucleic acids, particularly DNA, is the basis for its staining principle in biofilm studies. It is a fluorescent nucleic acid stain often used to mark living bacterial cells in a biofilm. It enters bacterial cells and attaches to their DNA, generating green fluorescence when stimulated by light\u003csup\u003e90\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe control group showed a higher count of live cells, while the ethanolic extracts from \u003cem\u003eS.wightii\u003c/em\u003e demonstrated nearly identical antibiofilm effectiveness, as evidenced by the number of deceased cells. At the mature stage of the biofilm formation process, the effective inhibition was seen. The ethanolic extract of \u003cem\u003eS.wightii\u003c/em\u003e prevented the formation of biofilms during the adhesion stage, and the epifluorescence micrograph clearly showed disruption to the bacterial biofilm matrix with few adherent cells. Biofilm provides a shield for microorganisms against traditional drugs and disinfectants. According to information provided by the National Institutes of Health and the Centers for Disease Control, microbial biofilms are responsible for roughly 65\u0026ndash;80% of infections\u003csup\u003e91\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe capacity of \u003cem\u003eA. baumannii\u003c/em\u003e to attach to surfaces is a crucial mechanism in the bacterium\u0026apos;s pathogenicity. Adherence is influenced by specific factors like adhesins and non-specific factors such as hydrophobicity and electric charge on the cell surface. Cell surface hydrophobicity (CSH) significantly contributes to initial adhesion, biofilm development, pathogenicity, and virulence\u003csup\u003e92\u003c/sup\u003e. In the initial phase of biofilm formation, extracellular polymeric substances (EPS) are produced, comprising polysaccharides, nucleic acids, lipid molecules, and proteins. The EPS plays a crucial role in establishing the three-dimensional, sturdy structure of the biofilm matrix\u003csup\u003e93\u003c/sup\u003e. In this current investigation, we assessed the potentiality of ethanolic extract from \u003cem\u003eS.wightii\u003c/em\u003e against \u003cem\u003eA. baumannii\u003c/em\u003e biofilm formed on the glass surface. The findings demonstrated the efficacy of ethanolic extracts from the seaweeds in preventing biofilm formation. Numerous reports have documented the antimicrobial properties of marine seaweeds\u003csup\u003e94,95\u003c/sup\u003e. Extracts obtained from various seaweed using chloroform and methanol solvents displayed potent activity against a range of pathogenic bacteria known to affect humans. Additionally, ethanol extracts from \u003cem\u003eS. wightii\u003c/em\u003e demonstrated unique anti biofilm properties against clinically significant pathogenic microorganisms like \u003cem\u003eA. baumannii\u003c/em\u003e.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003e \u003cem\u003eIn silico\u003c/em\u003e analysis of our present work showed the binding activity of the ten ligands of \u003cem\u003eS.wightii\u003c/em\u003e with \u003cem\u003eA.baumannii\u003c/em\u003e and identified the drug-likeness property of those ligands to be used as lead molecules in drug discovery. MD simulation studies were used to understand the stability of the ligands in binding with the target BfmR protein of \u003cem\u003eA.baumannii\u003c/em\u003e. We also found through an \u003cem\u003ein silico\u003c/em\u003e analysis identified pregn-5-en-20-one, 3-(acetyloxy)-, cyclic 20-(1,2-ethanediyl acetal), (3beta) (CAS No. 002302-12-7), ethanone, 1-(6-methyl-7-oxabicyclo[4.1.0]hept-1-yl) (CAS No. 015120-94-2) and 9H-fluoren-9-one, 3-nitro-2,7-bis[2-(1-piperidinyl)ethoxy] (CAS No. 146397-91-3) as potent ligands with strong and stable binding potentials with BfmR-Ab.\u003c/p\u003e \u003cp\u003eIn the molecular docking of the three ligand molecules (CAS No. 002302-12-7, 015120-94-2 and 146397-91-3) with the target BfmR-Ab, it was found that all three ligands showed good binding efficiency to the biofilm-controlling response regulator BfmR, albeit with differences. The interaction results of the ligand molecules and BfmR of \u003cem\u003eA.baumannii\u003c/em\u003e revealed that the amino acid VAL 109 was found to have a crucial role in the formation of hydrogen bonds with most of the selected ligands that contributed to the stability of the complexes. From all the ten ligands, the lowest binding energy (∆G\u003csub\u003ebind\u003c/sub\u003e) -42.26 and \u0026minus;\u0026thinsp;50.49 (kcal/mol) was obtained for the ligands i.e; CAS No. 002302-12-7 and 146397-91-3 respectively and thelowest Glide score of -4.067 (kcal/mol) was obtained for the ligand CAS No. 015120-94-2. These three ligand molecules with the highest affinity can be considered as potent hit molecules against the BfmR of \u003cem\u003eA.baumannii\u003c/em\u003e. The ethanol extract of \u003cem\u003eS.wightii\u003c/em\u003e showed effective inhibitory activity against \u003cem\u003eA.baumannii.\u003c/em\u003e Future research is needed to determine the potential pharmacological and real-time therapeutic applications of the antibiofilm properties of the ethanolic extract of \u003cem\u003eS.wightii\u003c/em\u003e against MDR pathogen \u003cem\u003eA.baumannii\u003c/em\u003e and discover metabolic components. Especially, the antibiofilm potential of the ethanolic extract of \u003cem\u003eS.wightii\u003c/em\u003e enhanced the susceptibility of \u003cem\u003eA.baumannii\u003c/em\u003e growth formation was evaluated. The effectiveness and safety of the extract must be fully explored, which demands further studies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eS.S. and E.M.S. are supported by a Research Associate Grant (File No: 45/2/2020-DDI/BMS) from the Indian Council of Medical Research (ICMR), New Delhi.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSuvaiyarasan Suvaithenamudhan, Esaki M. Shankar and Vanitha Mariappan: Methodology. Suvaiyarasan Suvaithenamudhan, Esaki M. Shankar and Sundarraj Dinesh Kumar: Software. Suvaiyarasan Suvaithenamudhan and Esaki M. Shankar: Validation. Suvaiyarasan Suvaithenamudhan, Esaki M. Shankar, Vanitha Mariappan, Sundarraj Dinesh Kumar and Esaki Muthu Ponmalar : Visualization and Investigation. Esaki M. Shankar and Vanitha Mariappan: Data curation. Esaki M. Shankar and Vanitha Mariappan and Sundarraj Dinesh Kumar: Writing original draft preparation. Suvaiyarasan Suvaithenamudhan, Esaki M. Shankar, Vanitha Mariappan, Rajendran Thirugnanasambandam and Sundarraj Dinesh Kumar: Writing review and Editing. Suvaiyarasan Suvaithenamudhan, Esaki M. Shankar and Vanitha Mariappan, Sundarraj Dinesh Kumar, Esaki Muthu Ponmalar, Parthiban Rudrapathy. and Pitchaipillai Sankar Ganesh: Visualization. Esaki M. Shankar, Vanitha Mariappan and Parthiban Rudrapathy: Supervision. Suvaiyarasan Suvaithenamudhan and Esaki M. Shankar: Project administration. Esaki M. Shankar and Vanitha Mariappan: Funding acquisition. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors acknowledge and appreciate the management for its support and encouragement\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData will be available upon reasonable request from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eI. Kyriakidis, E. Vasileiou, Z.D. Pana, A. Tragiannidis, \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e Antibiotic Resistance Mechanisms. Pathogens. 10(3), 373. https://doi.org/10.3390/pathogens10030373 (2021).\u003c/li\u003e\n\u003cli\u003eB. Mirzaei, Z.N. Bazgir, H.R. Goli, F. Iranpour, F. 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Sasidharan, I. Darah, M.K.M.J. Noordin, In vitro antimicrobial activity against Pseudomonas aeruginosa and acute oral toxicity of marine algae Gracilariachangii. New Biotechnology. 27(4), 390-396. https://doi.org/10.1016/j.nbt.2010.02.002 (2010).\u003c/li\u003e\n\u003cli\u003eS. Suganya, R. Ishwarya, R. Jayakumar, M. Govindarajan, N.S. Alharbi, S. Kadaikunnan, B. Vaseeharan, New insecticides and antimicrobials derived from Sargassum wightii and Halimeda gracillis seaweeds: Toxicity against mosquito vectors and antibiofilm activity against microbial pathogens. South African Journal of Botany. 125, 466-480. https://doi.org/10.1016/j.sajb.2019.08.006 (2019).\u003c/li\u003e\n\u003cli\u003eM. Kumaresan, K.V. Anand, K. Govindaraju, S. Tamilselvan, V.G. Kumar, Seaweed Sargassum wightii mediated preparation of zirconia (ZrO2) nanoparticles and their antibacterial activity against gram positive and gram-negative bacteria. Microbial pathogenesis. 124, 311-315. https://doi.org/10.1016/j.micpath.2018.08.060 (2018).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Acinetobacter baumannii, BfmR, Biofilm, Sargassum wightii, brown seaweed, molecular docking, molecular dynamic simulation","lastPublishedDoi":"10.21203/rs.3.rs-4641861/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4641861/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e (\u003cem\u003eA. baumannii)\u003c/em\u003e is a notorious nosocomial pathogen known for its ability to form biofilms, rendering it highly resistant to conventional antibiotics and immune clearance. In this study, the minimum inhibitory concentration (MIC) ranged from 70 \u0026micro;g/mL to 100 \u0026micro;g/mL remarkably, the tested ethanolic extracts inhibited \u003cem\u003eA. baumannii\u003c/em\u003e biofilm development in a concentration-dependent manner. Epifluorescence microscopic analysis revealed a significant reduction in treated biofilm formation compared to the control. Additionally, gas chromatography-mass spectrometry (GC-MS) analysis of the ethanol extract of \u003cem\u003eSargassum wightii\u003c/em\u003e (\u003cem\u003eS. wightii\u003c/em\u003e) identified 10 major compounds. Molecular docking studies were conducted to explore the interaction of small molecules from \u003cem\u003eS. wightii\u003c/em\u003e with the BfmR protein of \u003cem\u003eA. baumannii\u003c/em\u003e. The molecular docking of three ligand molecules (CAS No. 002302-12-7, 015120-94-2, and 146397-91-3) with the target BfmR-Ab revealed the lowest binding energies (∆G\u003csub\u003ebind\u003c/sub\u003e) of -42.26 and \u0026minus;\u0026thinsp;50.49 (kcal/mol) for the ligands CAS No. 002302-12-7 and 146397-91-3, respectively, and the lowest Glide score of -4.067 (kcal/mol) for the ligand CAS No. 015120-94-2. These top three hit molecules exhibited the highest affinity as efficient ligands against BfmR of \u003cem\u003eA. baumannii\u003c/em\u003e. Nevertheless, \u003cem\u003eS. wightii\u003c/em\u003e demonstrated antibiofilm activities against the multidrug-resistant (MDR) pathogen \u003cem\u003eA. baumannii\u003c/em\u003e, with bioactive compounds exhibiting promising drug-likeness and pharmacokinetic signatures.\u003c/p\u003e","manuscriptTitle":"Bioactive Compounds Derived from Sargassum wightii Exhibit Antibacterial Activity against Multi-Drug Resistant Acinetobacter baumannii","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-23 15:08:43","doi":"10.21203/rs.3.rs-4641861/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a69de04e-73ed-4995-a348-b045aec731c7","owner":[],"postedDate":"July 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":34916507,"name":"Biological sciences/Biotechnology"},{"id":34916508,"name":"Biological sciences/Computational biology and bioinformatics"}],"tags":[],"updatedAt":"2024-11-11T03:08:23+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-23 15:08:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4641861","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4641861","identity":"rs-4641861","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}