Deciphering the Therapeutic Promise of β-Lapachone against Pathogenic Multidrug Resistant Microbes and Breast Cancer Cell

Deciphering the Therapeutic Promise of β-Lapachone against Pathogenic Multidrug Resistant Microbes and Breast Cancer Cell | 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 Deciphering the Therapeutic Promise of β-Lapachone against Pathogenic Multidrug Resistant Microbes and Breast Cancer Cell Rawan Amer Asiri, Irfan Ahmad, Yasser Alraey, Safia Obaidur Rab, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7982878/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The growing rise of multidrug-resistant (MDR) infections, as well as the worldwide prevalence of cancer, one of the major causes of mortality, are two of the most serious and significant issues facing modern medicine. This study evaluated the antimicrobial and anticancer properties of beta lapachone in vitro and in silico. Antimicrobial activity was assessed using established assays such as diffusion methods, MIC and MBC determinations, CFU reduction, biofilm inhibition, oxidative stress analysis, and membrane integrity disruption. The beta lapachone investigated showed strong antimicrobial properties, significantly decreasing microbial viability, preventing biofilm formation, and generating membrane damage and oxidative stress. Anticancer activity was tested on breast cancer cell lines using MTT viability assays, Annexin V/PI flow cytometry, and cell cycle analysis. The beta lapachone had dose-dependent cytotoxic effects, with triggering S-phase arrest, which led to increase apoptosis. Molecular docking investigations showed their affinity for important microbial and cellular targets, proving the hypothesized mechanisms of action. These findings emphasize the beta lapachone therapeutic promise as dual-function medicines capable of treating both MDR infections and malignant tumors, thereby tackling two of the most serious dangers to world health. Biological sciences/Cancer Biological sciences/Computational biology and bioinformatics Biological sciences/Drug discovery Biological sciences/Microbiology Beta lapachone Antimicrobial Anticancer MDR pathogens Breast cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Figure 17 Figure 18 Figure 19 Figure 20 Introduction Multidrug-resistant (MDR) bacteria are a significant risk to public health because they can adapt to multiple antibiotic treatments. Resistance to at least one of three or more distinct classes of antibiotics, including ampicillin, sulfonamides, and chloramphenicol, is commonly used to identify MDR strains. On the other hand, strains that are extensively drug-resistant (XDR) are resistant to nearly every antibiotic that is currently on the market, meaning that only one or two effective treatment options remain. These consist of azithromycin, carbapenems, and piperacillin. XDR strains that are resistant to third-generation cephalosporins such as ceftazidime, cefuroxime, and ceftriaxone, as well as the fluoroquinolone ciprofloxacin, are noteworthy examples ( 1 ). When bacteria change in ways that make drugs used to treat infections less effective, this is known as bacterial antimicrobial resistance, or AMR ( 2 ). According to epidemiological studies acquiring extra-chromosomal genetic material through horizontal gene transfer, antibiotic use is directly associated with the development of resistant bacterial strains ( 3 ). In Gram-negative bacteria, reduced outer membrane permeability, efflux pump activity, break down antibiotics by the synthesis of enzymes, the formation of biofilms, and changes to antibiotic targets that allow them to avoid the effects of antibiotics ( 4 ). AMR is one of the most pressing public health issues of our time due to decrease antibiotic effect on multidrug-resistant (MDR) bacteria, which present a growing threat to global health ( 4 ). Many antimicrobials have been developed and marketed over the years with the shared objective of treating and curing mild to severe infections ( 5 ). Beyond the severe human impact, AMR has a significant financial cost that will only increase if resistance is not addressed. Failure to act will result in a massive loss of world production of $ 100 trillion by 2050 ( 6 ). The development of extremely virulent MDR bacteria highlights the urgent demand for substitute therapies in order to successfully stop MDR infections and their spread ( 7 ). A natural product is a chemical that is made by living things, such as microbes, plants, mushrooms, and animals. In addition to being used for centuries to treat serious illnesses, plants are still frequently employed to create novel therapeutic candidates ( 8 ). Scientists are paying attention to natural chemicals because they may make useful models for creating new medication compounds ( 9 ). Medicinal plants are regarded for their therapeutic characteristics and synergistic effects, which help with illness treatment and prevention resulting in greater manufacturing and a decrease in synthetic drug use ( 10 , 11 ). The evolution of drug resistance in cancer cells, as well as the limits of current chemotherapeutic drugs, have highlighted the critical need to identify compounds with significant anticancer properties and novel modes of action. Natural compounds attract the interest of researchers as viable sources of new anticancer therapies due to their various biological features and ability to overcome resistance mechanisms. β-LP, has been extensively researched for its capacity to produce oxidative stress, impair DNA replication, and promote apoptosis in multiple cancer types. The purpose of this study is to evaluate the antimicrobial and anticancer potential of β-LP by using in vitro biological studies and in silico molecular docking approach against pathogenic and multidrug-resistant bacteria and cancer cell. Materials and Methods Materials This study included β-lapachone (10 mM) as natural antimicrobial agents was first dissolved in dimethyl sulfoxide (DMSO) to ensure optimum dilution and application. For culture and sensitivity testing, microbiological media and reagents included Mueller-Hinton agar (MHA) and Mueller-Hinton broth (MHB). Phosphate-buffered saline (PBS), crystal violet solution, catalase test reagents, and bacterial strains. Antimicrobial activity was determined with 96-well microdilution plates. Methods In-Vitro antimicrobial activity Bacterial Strains and Growth Conditions This study included a variety of microbial strains, including multidrug-resistant (MDR) organisms such VRE, MRSA, Acinetobacter baumannii, Pseudomonas aeruginosa, and Neisseria spp. , as well as other clinically relevant pathogens like Salmonella spp., Escherichia coli, Staphylococcus aureus, Mycobacterium smegmatis, and Candida albicans . All strains were stored as glycerol stocks in the freezer at -80 °C. All strains were grown in Mueller-Hinton broth (MHB). Bacterial suspensions for experimental testing were prepared by diluting the cultures in MHB to an optical density (O.D.) of 0.05 at a wavelength of 610 nm. Following adjustment, the bacterial suspensions were incubated at 37°C for 20-25 minutes to stabilize the inoculum prior to testing. Preparation of Compounds The β-lapachone was synthesized at a 10 mM stock concentration. For experimental purposes, β-lapachone was diluted to a working concentration of 2.5 mM with dimethyl sulfoxide (DMSO) as the solvent. The dilution was performed according to the formula C1V1 = C2V2 , where C1 and C2 represents the beginning and final concentrations respectively, and V1 and V2 represent the beginning and final volumes. Evaluation of antibacterial efficacy by agar well diffusion The β-lapachone was tested for antibacterial activity using the agar well diffusion method (12). Mueller-Hinton agar (MHA) plates were prepared, and wells of 6 mm in diameter were made with a sterilized syringe cap. The surface of each plate was equally inoculated with sterile cotton swabs soaked in the prepared bacterial suspension. Following this, 20 μL of dimethyl sulfoxide (DMSO) was added to the control well, and 20 μL of β-lapachone was added to the corresponding wells. Plates were incubated aerobically at 37°C overnight. After incubation, the diameter of the zone of inhibition (ZOI) along with well was measured in millimeters (mm) to determine antimicrobial activity. Determination of MICs and MBC s Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) were determined for β-lapachone against selected microbial strains. β-lapachone was tested against MRSA, Staphylococcus aureus , Mycobacterium smegmatis , Candida albicans , and Pseudomonas aeruginosa . β-lapachone was serially diluted two-fold from a starting concentration of 2.5 mM. Microbial cultures were cultured in Mueller-Hinton broth (MHB) and adjusted to have an optical density of 0.05. The adjusted suspension (180 μL) was added to each well of a 96-well microdilution plate, followed by 20 μL of β-lapachone. The control wells received 180 μL of microbial solution and 20 μL of MHB. Plates were incubated at 37°C for overnight. The MIC was defined as the lowest concentration that produced no observable growth. To determine MBC, 10 μL of clear wells were subcultured on Mueller-Hinton agar and incubated overnight at 37°C. The MBC had the lowest concentration with no colony formation (13). Determination Colony forming unit To assess bacterial viability after treatment, colony forming units (CFUs) were performed using ten-fold serial dilutions from the MIC wells (14). From each MIC well, 62.5 μL was transferred into 562.5 μL of Mueller-Hinton broth (MHB), and the dilutions were plated onto Mueller-Hinton agar plates (MHA). For control samples, 62.5 μL of bacterial culture and 562.5 μL of MHB were similarly plated. Plates were incubated overnight at 37°C to compare bacterial growth between treated and control samples. Evaluation of growth inhibition The growth inhibition assay assessed the antibacterial activity of β-lapachone against certain microorganisms. In a 96-well microtiter plate, 20 μL of β-lapachone at MIC concentration was added to 180 μL of bacterial culture. Control wells held 180 μL of bacterial suspension and 20 μL of Mueller-Hinton broth (MHB). The plate was incubated at 37°C, and absorbance at 600 nm was monitored after 24 hrs using a FLUOstar Omega plate reader (BMG LABTECH, Germany) (15). Growth inhibition was estimated by comparing the absorbance values of treatment and control wells using the following formula: Inhibition (%) = ((μC – μT) / μC) × 100 Where μC is Mean absorbance of control wells (O.D. 600) and μT is Mean absorbance of treated wells (O.D.600) Antibiofilm activity of β-lapachone Selected microbial strains were used to test the prevention of biofilm development. In 96-well polystyrene microplates, 180 μL of bacterial suspension (OD 600 = 0.05) was added to each well, followed by 20 μL of β-lapachone at their respective MIC values. The strains studied were Staphylococcus aureus , MRSA, Mycobacterium smegmatis, Pseudomonas aeruginosa and Candida albicans . Plates were incubated at 37°C without shaking for 24 hours to generate biofilms. Control wells contained 180 μL of bacterial suspension and 20 μL of Mueller-Hinton broth (MHB). Plates were incubated overnight and then put in the oven at 60°C for 30-45 minutes. Next, 50 μL of 1% crystal violet solution was added to each well (16). Biofilm formation was measured at 610 nm with a BMG LABTECH FLUOstar Omega plate reader (BMG LABTECH, Allmendgrün, Ortenberg, Germany). Kinetic assay for time killing performance A kinetic assay was performed to assess the antibacterial activity of β-lapachone against several pathogens, including Staphylococcus aureus, MRSA, Mycobacterium smegmatis, Pseudomonas aeruginosa and Candida albicans . In a 96-well microplate, 180 μL of bacterial suspension (adjusted to 0.05 OD at 610 nm in Mueller-Hinton broth) was combined with 20 μL of β-lapachone at three concentrations: MIC x 0.5, MIC x 1, and MIC x 2. Control wells contained 180 μL of bacterial culture and 20 μL of MHB. The plates were incubated at 37°C and absorbance was measured at every 2 hours with the FLUOstar Omega plate reader (BMG LABTECH, Allmendgrün, Ortenberg, Germany). By comparing absorbance measurements between treated and control wells, growth kinetics were tracked over the incubation period in order to identify the compounds' time-dependent bactericidal or fungicidal effects (17). Catalase Activity Assay Bacterial cultures treated with β-lapachone at their respective MIC values were incubated overnight at 37°C in a shaking incubator at 200 RPM in order to measure catalase activity. The same conditions were used to incubate untreated bacterial cultures that were produced in the medium alone as a control. Following incubation, each culture received a direct addition of 100 μL of hydrogen peroxide (H 2 O 2 ), and the mixture was promptly checked to produce bubbles. To assess catalase activity, the height of the ensuing air bubble column was measured with a ruler. Cytoplasmic leakage Assay The cytoplasmic leakage assay was used to evaluate membrane integrity after compound treatment. A 24 hour bacterial suspension of each examined organism was centrifuged at 3500 rpm for 15 minutes. To eliminate any remaining media, the bacterial pellets were rinsed with phosphate-buffered saline (PBS, pH 7.0). After adding 1mL of PBS, the pellets were shaken and incubated at 37°C for 24 hours. After incubation, 50 μL of β-LP at a final concentration of 2.5 mM was added to the corresponding samples and kept for 30 minutes at room temperature. The treated and the control suspensions were centrifuged at 3500 rpm for an additional 15 minutes. The supernatant was collected and examined spectrophotometrically at 595 nm with the FLUOstar Omega plate reader (BMG LABTECH, Allmendgrun, Ortenberg, Germany) to quantify the level of DNA released by the cells (18). In-Vitro Anti-Cancer Activity Cell Culture The human breast cancer cell line BT-474 from ATCC was cultured in Dulbecco’s Modified Eagle’s Medium: Nutrient Mix F-12 (D-MEM/F-12 1:1) medium supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin and 100 mg/ml streptomycin in a 37°C incubator with 5% CO2. The cells were passaged by harvesting with trypsin/ EDTA and seeding into flask. MTT assay The cytotoxicity of the test samples was evaluated using the MTT assay (19). Briefly, cells were seeded in a 96-well plate at a density of 1 × 10 4 cells per well and allowed to adhere overnight under standard culture conditions. After incubation, the cells were treated with various concentrations of the β-LP and incubated for an additional 24 hours. Following treatment, 20 µL of MTT solution (5 mg/mL in PBS) was added to each well, and the plates were incubated for 4 hours at 37°C. Subsequently, the culture medium was carefully aspirated, and 150 µL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals. The absorbance was measured at 570 nm using a microplate reader. Cell viability was expressed as a percentage relative to untreated control cells, and all experiments were performed in triplicate to ensure reproducibility. Flow cytometry Flow cytometry was used to assess apoptosis and necrosis after β-lapachone treatment. Cells were seeded in 6-well plates at a density of approximately 2 × 10 5 cells per well and allowed to adhere overnight at 37°C in a humidified atmosphere with 5% CO 2 . Cells were then treated with various concentrations of β-LP (0, 2, 5, 10, and 20 µM) for 24 hours, with untreated cells serving as the control. After treatment, both adherent and floating cells were collected, washed twice with cold phosphate-buffered saline (PBS), and resuspended in 100 µL of binding buffer. Cells were stained by adding 5 µL of Annexin V-FITC and 5 µL of Propidium Iodide (PI) and incubated for 15 minutes at room temperature in the dark. After staining, 400 µL of binding buffer was added to each sample, and flow cytometric analysis was performed immediately using a BD FACSCalibur flow cytometer. Annexin V-FITC and PI signals were detected in FL1 (green) and FL2 (red) channels, respectively, with proper compensation settings to minimize signal overlap. A minimum of 10,000 events were acquired per sample. The cell population was analyzed into four quadrants: viable cells (Annexin V - /PI - ), early apoptotic cells (Annexin V + /PI - ), late apoptotic/secondary necrotic cells (Annexin V + /PI + ), and necrotic cells (Annexin V - /PI + ). Data were analyzed using FlowJo software, and results were expressed as mean ± standard deviation from three independent experiments. Cell Cycle arrest For cell cycle analysis, cells were seeded in 6-well plates at a density of approximately 2 × 10 5 cells per well and incubated overnight at 37°C in a humidified atmosphere containing 5% CO 2 . After adhesion, cells were treated with various concentrations of β-LP (0, 2, 5, 10, and 20 µg/mL) for 24 hours, while untreated cells served as the control. Following treatment, both floating and adherent cells were harvested by trypsinization, washed twice with cold phosphate-buffered saline (PBS), and centrifuged at 300 × g for 5 minutes at 4°C. The resulting pellets were fixed in ice-cold 70% ethanol, added dropwise while gently vortexing, and incubated at -20°C for at least 2 hours. After fixation, cells were washed with PBS, and resuspended in a staining solution containing 50 µg/mL propidium iodide (PI) and 100 µg/mL RNase A. The samples were incubated at room temperature in the dark for 30 minutes to ensure complete staining of DNA. Flow cytometry was performed using a BD FACSCalibur system, with excitation at 488 nm and emission detected in the FL2 channel. For each sample, 10,000–20,000 events were collected. Doublets were excluded by appropriate gating strategies. DNA content histograms were generated, and the distribution of cells across the G0/G1, S, and G2/M phases was analyzed using FlowJo software (20). The results were expressed as the percentage of cells in each phase and reported as mean ± standard deviation (SD) from three independent experiments. In-Silico study Ligand and Target Selection Beta-Lapachone (PubChem CID: 3885) was selected based on their pharmacological relevance and compliance with Lipinski’s Rule of Five. Their 3D structures were retrieved from PubChem in SDF format, converted to PDB using Open Babel, and energy-minimized using the MMFF94 force field. Ten protein targets from five pathogenic microorganisms were selected based on their roles in microbial survival, virulence, and antibiotic resistance. These include: Candida albicans : CYP51 (PDB: 5TZ1), DHFR (PDB: 1AI9), MRSA: PBP2a (PDB: 1VQQ), Sortase A (PDB: 1T2P), Mycobacterium smegmatis : InhA (PDB: 2NV6), Cysteine Desulfurase (PDB: 8KG1), Pseudomonas aeruginosa : LasR (PDB: 3IX3), OprF (PDB: 4RLC), Staphylococcus aureus : Alpha-Hemolysin (PDB: 7AHL), TarO (PDB: 5EZM). Protein structures were obtained from the RCSB PDB. Preparation of Proteins and Ligands Protein structures were preprocessed using AutoDock Tools by removing water molecules and heteroatoms, adding polar hydrogens, and assigning Gasteiger charges. Ligands were prepared and converted into PDBQT format using AutoDock 4.2. Potential binding pockets were predicted using DogSiteScorer for grid box configuration. Molecular Docking and Analysis Molecular docking was performed using AutoDock Vina v1.2.x. Binding affinities (in kcal/mol) were recorded for all ligand–protein complexes. Top-ranked complexes were further analyzed for molecular interactions using Biovia Discovery Studio. Statistical Analysis All statistical analyses and graphical representations were conducted using GraphPad Prism (version 10.4.2). Statistical analyses were performed using SPSS software (version 20) and the value of p < 0.05 was taken as statistically noteworthy. Results In-Vitro Antimicrobial Activity Evaluation of antibacterial efficacy by zone of inhibition The antimicrobial activity of β-lapachone was investigated against MDR as shown in Figure 1. The zone of inhibition (ZOI) is against MDR bacteria using the well diffusion method. The bacteria studied were (A) MRSA, (B) Neisseria spp., (C) VRE, (D) Acinetobacter baumannii, and (E) Pseudomonas aeruginosa. Well number 2 consisted of β-lapachone and common pathogenic microbial strains shown in Figure 2. The zone of inhibition (ZOI) is against pathogenic microbes using the well diffusion method. The bacteria studied were (A) Salmonella spp., (B) Pseudomonas aeruginosa, (C) Escherichia coli, (D) Staphylococcus aureus, (E) Mycobacterium smegmatis, and (F) Candida albicans. Well number 2 consisted of β-lapachone. using the agar well diffusion method. β-LP demonstrated measurable activity against MRSA, with inhibition zones of 19 mm Figure 8. Among the remaining MDR strains, such as Neisseria, VRE, Acinetobacter, and Pseudomonas, none of the compounds showed significant antimicrobial activity. At the same time, β-LP showed the strongest inhibitory effect against common pathogenic microorganisms in Figure 3. Effects of β-lapachone on microbial strains. Included MRSA, Pseudomonas aeruginosa , Staphylococcus aureus , Mycobacterium smegmatis , and Candida albicans . It produced distinct zones of inhibition (ZOI) of 19 mm against Pseudomonas and S. aureus , 20 mm against M. smegmatis , and a strong antimicrobial effect with ZOI of 38 mm against Candida albicans . Determination of MICs and MBCs β-lapachone shown strong antimicrobial effect against Gram-positive bacteria and Candida albicans. The MIC values for MRSA, S. aureus, and M. smegmatis were all 0.156 mM, while P. aeruginosa had a slightly higher MIC of 0.312 mM. Accordingly, MBC values for MRSA, S. aureus, M. smegmatis, and P. aeruginosa were 1.25 mM, 0.312 mM, and 0.625 mM respectively, indicating efficient bactericidal activity shown in Figure 4. MIC and MBC values β-LP against different microbes. (A) For β-LP: MRSA, Pseudomonas aeruginosa, S. aureus, and M. smegmatis. (B) The MIC and MBC of β-LP against Candida albicans. C. albicans showed remarkable sensitivity to β-LP, with MIC of 0.004 mM and MBC of 0.009 mM, indicating promising antifungal characteristics ( Figure 4). Determination Colony forming unit Treatment with β-lapachone (β-LP) significantly reduced colony-forming units (CFUs) in all examined microbes compared to untreated controls as showen in Figure 5. CFU assay showing the effect of (β-LP) on microbial viability. Upper panels (A–E) represent untreated controls, and lower panels (F–J) show β-LP-treated samples. MRSA (A vs. F), Pseudomonas aeruginosa (B vs. G), Staphylococcus aureus (C vs. H), Mycobacterium smegmatis (D vs. I), and Candida albicans (E vs. J). In MRSA, the control plate contained a high density of colonies, whereas the treated sample had a significant drop in CFUs. Pseudomonas aeruginosa developed densely and widely in the control plate, but the treated plate had fewer and more distributed colonies. Similarly, β-LP therapy significantly reduced colony count in Staphylococcus aureus compared to the control group. Mycobacterium smegmatis exhibited pigmented growth in the control, but treatment with β-LP significantly reduced colony density and distribution. The most pronounced effect was seen in Candida albicans , where the treated plate showed a significant drop in CFU count compared to the heavy growth in untreated control. β-LP exhibits substantial antibacterial and antifungal activity, reducing viable microbial populations across many species. Evaluation percentage of growth inhibition The percentage of growth inhibition evaluates the antibacterial and antifungal capacity of β-LP by inhibiting the growth of specific bacterial and fungal strains. The experiment was designed to determine the proportion of growth inhibition caused by every compound under standardized conditions as shown in Figure 6. The growth inhibition percentage of selected bacteria after treatment with β-lapachone. All the organisms tested showed measurable growth suppression after being treated with β-LP. β-LP specifically inhibited MRSA by 42.21%, Pseudomonas aeruginosa by 57.20%, Staphylococcus aureus by 50.96%, Mycobacterium smegmatis by 34.05%, and Candida albicans by 58.3%. These findings suggest that β-LP has moderate antibacterial and antifungal action, with the strongest inhibition found against P. aeruginosa and C. albicans. Percentage of antibiofilm activity of β-lapachone This experiment evaluated the effectiveness of β-lapachone in inhibiting biofilm formation in for medicinal purposes relevant microbes. Biofilm production is a crucial role in microbes persistence and resistance, making it an important target for antimicrobial drugs. As illustrated in Figure 7. The percentage of biofilm inhibition of selected bacteria after treatment with formed by β-lapachone. β-LP treatment resulted in essential levels of biofilm inhibition across all tested strains. The inhibition percentages for MRSA were around 91.8%, 83.7% for Pseudomonas aeruginosa , 76.3% for Staphylococcus aureus , 82.5% for Mycobacterium smegmatis , and 85.7% for Candida albicans . These findings suggest that β-LP has excellent antibiofilm action. Growth kinetic assay for time killing performance In order to assess the antibacterial and antifungal activities of β-LP over a 20-hour period, a kinetic growth experiment was conducted. In this experiment, growth was tracked using absorbance measurements to evaluate the inhibitory effect of β-LP on microbial proliferation at various concentrations (MIC x 0.5, MIC x 1 and MIC x 2). Regarding β-LP, each microbe that was examined, including MRSA, Pseudomonas aeruginosa, Staphylococcus aureus, Mycobacterium smegmatis, and Candida albicans growth were recorded at MIC x 0.5, MIC x 1 and MIC x 2 concentrations, except for C. albicans , where only MIC x 1 and MIC x 2 as shown in Figure 8. Effect of β-LP on microbes growth kinetics. Representative bacterial strains of (A) MRSA, (B) Pseudomonas aeruginosa, (C) Staphylococcus aureus, (D) Mycobacterium smegmatis, and (E) Candida albicans were treated with different concentrations (MIC x 0.5, MIC x 1 and MIC x 2) of β-LP. Growth cycle of untreated organisms served as growth control. Optical density at 600 nm was measured at regular time intervals of 2 hours. All untreated control groups showed normal development progression over time. In contrast, β-LP treatment caused a concentration-dependent decrease in growth across all strains. At MIC x 0.5, considerable inhibition was detected, whereas MIC x 1 induced severe suppression and MIC x 2 led in near-complete inhibition of growth. These results demonstrate that β-LP, efficiently suppresses microbial growth in all strains studied in a way that is dependent on both time and dose. Catalase Activity Assay The purpose of the catalase activity experiment was to measure the catalase enzyme activity and assess the effects of β-LP therapy on the oxidative stress response of different microbes as shown in Figure 9. Catalase activity in both untreated and treated bacteria. Treatment with β-LP Black bars represents control (untreated) samples, while gray bars represent treated samples. The height of the air bubble column (mm) represents the catalase activity levels in each strain. Catalase activity was determined by measuring the height of the oxygen bubble column that developed upon exposure to H 2 O 2 . In this assay, β-LP treatment resulted in a widespread decrease of catalase activity across the majority of tested strains. MRSA showed a moderate reduction (5 mm bubble height), Staphylococcus aureus showed limited activity (1 mm), while P. aeruginosa, M. smegmatis , and C. albicans produced no bubbles at all. Either the substance directly inhibiting the enzyme or severe oxidative stress that destroys the enzyme itself can cause a decrease in catalase activity. These data show that β-LP limit microbial catalase activity, either directly or by causing oxidative stress, and that bubble height is a functional indicator of how bacteria respond to compounds-induced oxidative imbalances. Cytoplasmic leakage Assay The cytoplasmic leakage assay, which measures the release of intracellular contents as indicated by absorbance at 595 nm, was used to assess the membrane-disruptive effects of β-LP on different microbes. Greater cytoplasmic leakage and an impaired membrane are indicated by an increase in absorbance. In the β-LP-treated group shown in Figure 10. Cytoplasmic leakage assay for microbes treated with β-LP. The absorbance at 595 nm was used to measure intracellular leakage. Displays the results for β-LP -treated cells. Black bars represent untreated controls, while gray bars represent treated samples., all tested strains (MRSA, Pseudomonas aeruginosa, Staphylococcus aureus, Mycobacterium smegmatis , and Candida albicans ) showed slightly higher absorbance than their respective untreated controls, suggesting slight membrane disruption. M. smegmatis and C. albicans showed the most marked increases. In-Vitro Anti-Cancer Activity MTT Assay The microscopic images shown in Figure 11. Effect of different concentrations of β-LP on cell viability as assessed by the MTT assay. illustrate the morphological changes in cells after treatment with varying concentrations of β-LP during the MTT assay. In the control group, the cells appeared healthy, densely populated, and exhibited typical spindle-shaped morphology with good adherence to the culture surface, indicating normal cell viability and proliferation. Upon treatment with 125 µg/mL, the cells largely retained their structure, although slight morphological alterations such as mild rounding and a slight reduction in cell density were observed, suggesting early signs of cytotoxic stress. At 250 ug/mL, more pronounced changes were evident; cells exhibited partial rounding, shrinkage, and decreased density compared to the control, indicating moderate cytotoxic effects. Exposure to 500 µg/mL resulted in significant cytotoxicity, characterized by severe cell rounding, detachment from the substrate, and reduced cellular spread. At the highest concentration of 1000 µg/mL, the cells were highly compromised, with extensive rounding, loss of normal structure, and a marked reduction in cell number, demonstrating near-complete cytotoxicity. Overall, these observations confirm a dose-dependent decrease in cell viability and health, with increasing concentrations of β-LP leading to progressive and severe morphological damage, consistent with mitochondrial dysfunction detected in the MTT assay. The MTT assay results demonstrate a clear concentration-dependent decrease in cell viability upon treatment with β-LP as shown in Figure 12 . Effect of different concentrations of β-LP on cell viability as assessed by the MTT assay. The bar graph shows a concentration-dependent decrease in cell viability (%), with the highest. The untreated cell control exhibited 100% viability, confirming normal cellular health in the absence of any toxic exposure. As the concentration of the test compound increased, a progressive decline in cell viability was observed. At 125 µg/mL, the mean cell viability dropped to 29.84%, indicating early signs of cytotoxicity. Further reduction was noted at 250 µg/mL with 22.56% viability, and a sharper decline at 500 µg/mL, where viability reduced to 14.37%. The most substantial cytotoxic effect was recorded at the highest concentration of 1000 µg/mL, where mean cell viability plummeted to just 5.23%. These results strongly indicate that β-LP exerts dose-dependent cytotoxic effects on the treated cells, with higher concentrations causing significant impairment of cell metabolic activity and survival, as reflected by the decreased formazan production measured in the MTT assay. Flow cytometry The effect of β-lapachone on inducing apoptosis and necrosis was evaluated using flow cytometry, and the results in Table 1 . Effect of β-lapachone on early apoptosis, late apoptosis, necrosis, and total cell death percentages as determined by flow cytometry analysis. Cells were treated with increasing concentrations of β-lapachone (0–20 µM) for 24 hours. Data are presented as mean ± standard deviation (SD) from three independent experiments. demonstrated a clear dose-dependent increase in cell death. In the control group (0 µM), minimal cell death was observed, with early apoptosis at 2.1%, late apoptosis at 1.2%, necrosis at 1.0%, and a total cell death of only 4.3%, confirming the baseline health of untreated cells. Upon treatment with 2 µM β-lapachone, early apoptosis increased significantly to 15.5%, while late apoptosis and necrosis rose to 5.4% and 2.5%, respectively, leading to a total cell death of 23.4%. At 5 µM, the early apoptotic population expanded to 32.8%, accompanied by 10.1% late apoptosis and 3.0% necrosis, resulting in a total of 45.9% cell death. Increasing the concentration to 10 µM further elevated early apoptosis to 48.6% and late apoptosis to 18.7%, with a slight rise in necrosis (4.5%), producing a total cell death of 71.8%. At the highest tested concentration (20 µM), β-lapachone induced a profound cytotoxic effect, with early apoptosis reaching 61.2%, late apoptosis 28.5%, and necrosis 5.8%, culminating in a near-complete total cell death of 95.5%. These results clearly demonstrate that β-lapachone triggers apoptosis predominantly, with necrosis contributing minimally, and that its cytotoxic effect on cells is both concentration-dependent and primarily apoptotic in nature. The flow cytometry quadrant plots display the effect of β-lapachone treatment on cell viability, apoptosis, and necrosis, as measured by Annexin V-FITC and Propidium Iodide (PI) staining shown in Figure 13 . Flow cytometry quadrant plots illustrate the effect of β-lapachone at doses of 0 (Control), 2, 5, 10, and 20 µM. Annexin V-FITC and Propidium Iodide (PI) staining are used to identify viable, apoptotic, and necrotic populations. Each quadrant represents a distinct cell population: Lower left (Annexin V - /PI - ): Viable (live) cells, Lower right (Annexin V + /PI - ): Early apoptotic cells, Upper right (Annexin V + /PI + ): Late apoptotic or secondary necrotic cells and Upper left (Annexin V - /PI + ): Necrotic cells. In the control group (0 µM β-lapachone), the vast majority of cells (95.7%) remained viable, with only minimal early apoptosis (2.1%), late apoptosis (1.2%), and necrosis (1.0%), indicating excellent baseline cell health. Upon treatment with 2 µM β-lapachone, there was a noticeable increase in early apoptosis (15.5%) and late apoptosis (5.4%), with viability dropping to 76.6%. This early shift toward apoptosis shows the initial cytotoxic effect of the compound at a low concentration. At 5 µM, apoptosis became more pronounced, with early apoptosis rising to 32.8% and late apoptosis to 10.1%, reducing viable cells to 54.1%. A minor increase in necrosis (3.0%) was also observed. Treatment with 10 µM β-lapachone caused a dramatic shift: only 28.2% of cells remained viable, while early apoptosis reached 48.6% and late apoptosis 18.7%. Necrosis slightly increased to 4.5%, indicating that β-lapachone at this concentration predominantly induces apoptosis rather than direct necrotic death. At the highest concentration, 20 µM, the majority of cells underwent apoptosis: early apoptosis was 61.2%, late apoptosis was 28.5%, and necrosis was slightly elevated at 5.8%. Only 4.5% of cells remained viable, indicating almost complete cell death primarily through the apoptotic pathway. Overall, these results clearly demonstrate a dose-dependent increase in apoptosis, with β-lapachone inducing both early and late apoptosis efficiently at higher concentrations, while necrosis remains relatively low even at 20 µM. This highlights β-lapachone’s strong apoptotic-inducing potential with minimal necrotic effects. Table 1 : Effect of β-lapachone on early apoptosis, late apoptosis, necrosis, and total cell death percentages as determined by flow cytometry analysis. Cells were treated with increasing concentrations of β-lapachone (0–20 µM) for 24 hours. Data are presented as mean ± standard deviation (SD) from three independent experiments. Concentration of β-lapachone (µM) Early Apoptosis (%) Late Apoptosis (%) Necrosis (%) Total Cell Death (%) 0 (Control) 2.1 ± 0.3 1.2 ± 0.2 1.0 ± 0.2 4.3 ± 0.5 2 15.5 ± 1.2 5.4 ± 0.8 2.5 ± 0.6 23.4 ± 1.8 5 32.8 ± 2.0 10.1 ± 1.1 3.0 ± 0.7 45.9 ± 2.4 10 48.6 ± 2.5 18.7 ± 1.8 4.5 ± 0.8 71.8 ± 3.2 20 61.2 ± 3.1 28.5 ± 2.5 5.8 ± 0.9 95.5 ± 4.0 Cell Cycle Arrest Flow cytometry analysis based on DNA content (propidium iodide staining) revealed that β-lapachone induces a dose-dependent S-phase arrest in treated cells. On the X-axis, DNA content is displayed, with 2N DNA content corresponding to the G0/G1 phase (resting or initial growth phase), DNA content between 2N and 4N representing the S-phase (where DNA synthesis and replication occur), and 4N DNA content corresponding to the G2/M phase (mitotic preparation). The Y-axis represents the number of cells detected at each DNA content level. In the control group, cells exhibited a normal distribution, characterized by a strong G0/G1 peak around 2N, a moderate S-phase spread, and a clear G2/M peak around 4N. Upon treatment with 2 µM β-lapachone, a slight reduction in the G0/G1 peak was observed along with a minor increase in the S-phase shoulder, indicating early signs of S-phase accumulation. At 5 µM, the G0/G1 peak further decreased, and the S-phase region broadened, suggesting more cells were arrested during DNA synthesis. With 10 µM β-lapachone, a significant S-phase accumulation was evident around 3.5 PI intensity, accompanied by a notable reduction in the G0/G1 population. The effect was most pronounced at 20 µM, where the S-phase peak became broad and dominant, and the G0/G1 peak was minimal, indicating that the majority of the cells were blocked in S-phase. Throughout the increasing concentrations, the G2/M peak remained relatively small and stable, confirming that cells were not progressing into mitosis Table 2 . Flow cytometry was used to analyze the cell cycle distribution after 24 hours of treatment with β-LP at various concentrations (0 (Control), 2, 5, 10, and 20 µM) using propidium iodide (PI). Based on DNA content, cells were divided into three phases: G0/G1, S, and G2/M. and Figure 14 . Cell cycle analysis histograms demonstrate DNA content (PI intensity) of cells treated with increasing concentrations of β-lapachone (0 (Control), 2, 5, 10, and 20 µM) during 24 hours. The peaks indicate the distribution of G0/G1, S, and G2/M phases, as measured by flow cytometry. These observations suggest that β-lapachone blocks cell cycle progression by arresting cells in the S-phase, likely through inhibition of DNA replication or replication fork stalling mechanisms. Overall, the study demonstrated that β-lapachone treatment led to a progressive and concentration-dependent S-phase arrest, with the S-phase population increasing from approximately 29.1% in the control to 50.1% at 20 µM, while the G0/G1 population decreased correspondingly. Table 2: Flow cytometry was used to analyze the cell cycle distribution after 24 hours of treatment with β-LP at various concentrations (0 (Control), 2, 5, 10, and 20 µM) using propidium iodide (PI). Based on DNA content, cells were divided into three phases: G0/G1, S, and G2/M. Concentration G0/G1 Phase (%) S Phase (%) G2/M Phase (%) Control 53.5 ± 2.0 29.1 ± 1.7 17.4 ± 1.3 2 µM 50.2 ± 2.1 32.8 ± 1.8 17.0 ± 1.4 5 µM 45.8 ± 2.4 37.5 ± 2.1 16.7 ± 1.5 10 µM 38.2 ± 2.6 44.0 ± 2.3 17.8 ± 1.6 20 µM 32.4 ± 2.8 50.1 ± 2.5 17.5 ± 1.5 In-Silico Study The docking study evaluated the binding affinities and molecular interactions of Beta Lapachone with multiple target proteins from various organisms. Docking scores and detailed molecular interactions are presented in Table 3 along with corresponding figures. Table 3: Docking Score of Beta Lapachone with multiple target proteins Interaction Analysis: Candida albicans ( CYP51 & DHFR) and Beta-Lapachone Complex Docking analysis of Beta-Lapachone with Candida albicans CYP51 (PDB ID: 5TZ1) revealed multiple hydrophobic interactions stabilizing the ligand within the active site. A key π–π T-shaped interaction was observed between the aromatic ring of Beta-Lapachone and PHE228 (4.92 Å), enhancing binding via aromatic stacking. Additional pi–alkyl contacts with TYR118 (4.59 Å), LEU121 (3.97 Å), PHE233 (4.96 Å), LEU376 (5.38 Å), and MET508 (5.06 Å) contribute to a non-polar binding pocket, facilitating van der Waals stabilization. These interactions underscore the role of hydrophobic complementarity in ligand accommodation and support Beta-Lapachone’s potential as a CYP51 inhibitor targeting ergosterol biosynthesis (Figure 15A ). Docking of Beta-Lapachone with Candida albicans DHFR (PDB ID: 1AI9) revealed a robust interaction network involving both polar and hydrophobic contacts. ALA11 formed two strong hydrogen bonds (1.82 Å and 2.74 Å), alongside a pi–alkyl interaction (5.30 Å), anchoring the ligand within the active site. MET25 contributed additional stabilization via two pi–alkyl contacts (4.60 Å and 5.30 Å), a pi–sigma interaction (3.69 Å), and a unique pi–sulfur interaction (3.83 Å), reflecting diverse van der Waals and soft acid–base interactions. This combination of directional hydrogen bonds and multipoint hydrophobic contacts underscores Beta-Lapachone’s high binding affinity and supports its potential to inhibit DHFR-mediated folate biosynthesis in C. albicans (Figure 15B). Interaction Analysis: MRSA (PBP2a & Sortase A) and Beta-Lapachone Complex Docking of Beta-Lapachone with MRSA PBP2a (PDB ID: 1VQQ) revealed a strong interaction network comprising both polar and hydrophobic contacts. TYR441 formed two conventional hydrogen bonds (1.96 Å and 2.25 Å), and GLU602 contributed an additional hydrogen bond (2.28 Å), anchoring the ligand via strong electrostatic interactions. TYR519 engaged in a π–π T-shaped interaction (5.36 Å) and a π–alkyl contact (5.54 Å), while ALA601 formed three π–alkyl interactions (3.95 Å, 4.43 Å, and 5.45 Å), reflecting extensive hydrophobic complementarity. This diverse interaction profile supports the stable accommodation of Beta-Lapachone within the PBP2a active site and highlights its potential as an effective inhibitor against β-lactam resistance in MRSA (Figure 16A) . Docking of Beta-Lapachone with MRSA Sortase A (PDB ID: 1T2P) revealed a robust interaction network involving both polar and hydrophobic contacts. Conventional hydrogen bonds were formed with ASN114 (1.78 Å), SER116 (2.53 Å), and ARG197 (2.76 Å and 2.88 Å), anchoring the ligand via strong polar interactions. Hydrophobic stabilization was provided by π–alkyl interactions with PRO163 (5.05 Å) and ILE199 (4.95 Å), along with π–sigma interactions involving THR180 (4.00 Å) and ILE199 (3.94 Å). A π–donor hydrogen bond with VAL168 (2.91 Å) further contributed to binding specificity. This diverse interaction profile suggests that Beta-Lapachone is well-accommodated within the SrtA active site and may effectively inhibit its function, highlighting its potential as an anti-virulence therapeutic against MRSA (Figure 16B) . Interaction Analysis: Mycobacterium smegmatis ( Cysteine Desulfurase & InhA) and Beta-Lapachone Complex Docking of Beta-Lapachone with Mycobacterium smegmatis cysteine desulfurase (PDB ID: 8KG1) revealed a robust interaction profile comprising both polar and hydrophobic contacts. ARG532 forms two strong hydrogen bonds (1.89 Å and 2.55 Å), and ASN328 contributes an additional hydrogen bond (2.42 Å), anchoring the ligand through directional electrostatic interactions. Hydrophobic stabilization is driven by ALA184, which engages in four π–alkyl interactions (4.01 Å, 4.19 Å, 4.71 Å, and 4.87 Å), and ARG512, which forms a π–alkyl contact at 4.57 Å. This combination of hydrogen bonding and extensive van der Waals forces supports a stable and high-affinity binding conformation for Beta-Lapachone, suggesting its potential to inhibit cysteine desulfurase activity and interfere with sulfur metabolism in M. smegmatis (Figure 17A). Molecular docking of Beta-Lapachone with Mycobacterium smegmatis enoyl-acyl carrier protein reductase (InhA; PDB ID: 2NV6) revealed a highly stabilized binding conformation supported by both polar and hydrophobic interactions. A strong conventional hydrogen bond with GLY14 (2.46 Å) provides directional polar anchoring. Aromatic stabilization is mediated by two π–π stacking interactions with PHE41 (3.89 Å and 4.82 Å), while π–alkyl contacts with VAL65, ILE16, and ILE95 (4.39–5.30 Å) enhance hydrophobic compatibility. Additionally, π–sigma interactions with ILE95 (3.71 Å, 3.92 Å) and ILE122 (3.93 Å) contribute further orbital overlap and van der Waals stabilization. This diverse interaction profile suggests that Beta-Lapachone effectively occupies the InhA active site, with the potential to inhibit mycolic acid biosynthesis and exert antimycobacterial activity against M. smegmatis (Figure 17B) . Interaction Analysis: Pseudomonas aeruginosa (LasR & OprF) and Beta-Lapachone Complex Docking of Beta-Lapachone with the LasR transcriptional regulator of Pseudomonas aeruginosa (PDB ID: 3IX3) reveals a robust and multifaceted interaction network. Two strong conventional hydrogen bonds with ARG61 (1.99 Å, 2.77 Å) provide electrostatic anchoring. A π–anion interaction with ASP73 (3.94 Å) contributes additional charge-based stabilization. Aromatic interactions include two π–π stacking contacts with TYR64 (4.91 Å, 5.52 Å), reinforcing ligand orientation. Extensive hydrophobic interactions further stabilize the complex: LEU36 forms both π–alkyl (4.67 Å) and π–sigma (3.87 Å) interactions; TYR47 (5.25 Å), ALA50 (5.32 Å), and VAL76 (3.96–5.33 Å) contribute additional π–alkyl contacts; and ALA127 engages in four π–alkyl interactions (3.77–5.04 Å), supporting van der Waals compatibility. This rich interaction profile positions Beta-Lapachone securely within the LasR binding pocket, suggesting its potential to inhibit quorum sensing, impair virulence factor expression, and reduce biofilm formation, thus making it a promising antivirulence agent against P. aeruginosa (Figure 18A). Docking of Beta-Lapachone with the outer membrane protein OprF of Pseudomonas aeruginosa (PDB ID: 4RLC) reveals a robust interaction network involving electrostatic, aromatic, hydrophobic, and hydrogen bonding contacts. Central to the stabilization are multiple π–anion interactions: LYS13 (3.28 Å, 3.59 Å), ASP72 (3.43 Å), and ASP134 (4.63 Å), anchoring the ligand via charge–π electron attraction. A π–π T-shaped interaction with PHE11 (5.49 Å) adds aromatic complementarity. Hydrophobic stabilization is achieved through alkyl contacts with ALA44 (3.01 Å, 5.15 Å) and ILE74 (5.18 Å, 5.27 Å), enhancing van der Waals compatibility. Two strong hydrogen bonds with SER46 (2.24 Å) and SER89 (2.59 Å) provide directional polar stabilization and aid in precise ligand positioning. This diverse interaction profile positions Beta-Lapachone securely within the OprF binding site, suggesting potential to disrupt porin-mediated transport or membrane integrity and highlighting its promise as an antimicrobial agent targeting P. aeruginosa ( (Figure 18B ). Interaction Analysis: Staphylococcus aureus ( α-hemolysin & TarO) and Beta-Lapachone Complex Docking analysis of Beta-Lapachone with α-hemolysin (PDB ID: 7AHL) from Staphylococcus aureus reveals a triad of stabilizing interactions that contribute to a strong and specific ligand fit. A key conventional hydrogen bond is formed with LYS21 (2.51 Å), providing directional polar anchoring. LYS21 also engages in a carbon–hydrogen bond (3.35 Å), adding complementary electrostatic stabilization. Additionally, a π–anion interaction with GLU289 (3.82 Å) reinforces ligand binding through attraction between the aromatic π-system and the negatively charged carboxylate side chain. Together, these interactions suggest a robust and energetically favorable binding mode for Beta-Lapachone within the AHL cavity, highlighting its potential to inhibit the pore-forming activity of α-hemolysin and function as an antivirulence agent against S. aureus (Figure 19A) . Molecular docking of Beta-Lapachone with the TarO protein of Staphylococcus aureus (PDB ID: 5EZM) reveals a highly stabilized binding mode dominated by π-system-mediated interactions. Two key π–anion interactions with ASP55 (3.65 Å, 4.53 Å) anchor the ligand through electrostatic attraction between the aspartate’s carboxylate group and the aromatic π-electrons of the ligand. A dense hydrophobic network reinforces binding affinity through multiple π–alkyl interactions involving ALA151 (4.12 Å, 4.59 Å), PHE154 (4.88 Å), LEU342 (5.31 Å), PRO343 (4.13 Å, 4.86 Å), and VAL347 (4.31 Å, 4.94 Å). Notably, PHE154 also engages in a π–π stacking interaction (4.83 Å), enhancing aromatic complementarity within the cavity. This rich profile of electrostatic and hydrophobic contacts supports a robust and energetically favorable conformation for Beta-Lapachone, suggesting its potential to inhibit TarO and disrupt wall teichoic acid biosynthesis, a critical pathway in S. aureus cell wall integrity and virulence (Figure 19B). Discussion The prevalence of multidrug-resistant (MDR) microorganisms has become a major worldwide health concern, decreasing the efficacy of many traditional antibiotics and requiring the urgent development of alternative therapeutic options. This study evaluated the antimicrobial and antifungal effects of β-LP against clinically important MDR organisms. A range of microbiological and biochemical studies were conducted to examine their antimicrobial efficacy and the underlying mechanisms of action. The results showed that β-LP had consistent antimicrobial action across many experiments. β-LP demonstrated broad-spectrum antibacterial and antifungal action, with large inhibition zones and low MIC/MBC values, especially against MRSA, S. aureus , and C. albicans . Previous research has shown that β-LP causes intracellular reactive oxygen species (ROS), resulting in redox disorder, membrane damage, and reduced cellular metabolism ( 21 ). In CFU assays, β-LP significantly decreased viable bacterial and fungal populations, with growth almost eliminated in numerous treatment groups. Growth kinetic assay confirmed these findings by demonstrating dose-dependent inhibition of growth over 20 hour, emphasizing the compounds’ concentration- and time-dependent death properties. These findings were similar with prior research Mir et al., 2023 ( 22 ), indicating that they had effective bactericidal and fungicidal capabilities. β-LP demonstrated antibiofilm action in all strains studied, with β-LP reducing biofilm formation by more than 70% in most species. This is a significant feature since biofilm-associated cells are known to have increased resistance to antimicrobial treatments. β-LP also significantly inhibited catalase function in treated bacteria and fungus, indicating oxidative stress as a key mechanism of action. Catalase inhibition by β-LP is consistent with previous work demonstrating its potential to interfere with antioxidant defenses, therefore enhancing ROS the formation and cell death ( 23 , 24 ). Cytoplasmic leakage experiments provided additional confirmation of membrane damage. As a result of this data, it seems that β-LP promotes intracellular oxidative collapse. β-LP has powerful anticancer effects on breast cancer cells in a dose-dependent manner, as demonstrated by MTT viability assays and flow cytometry. β-LP drastically reduced cell viability while inducing apoptosis with minimal necrosis, indicating that apoptosis is the predominant mechanism of cytotoxicity. Flow cytometry analysis showed a gradual increase in early and late apoptotic populations with increasing concentrations of β-LP, reaching 95.5% and 89.4% total cell death, respectively, at the maximum tested dose (20 µM). Calahorra et al. (2024) Click or tap here to enter text.found that β-LP improved apoptotic activity in triple-negative breast cancer (TNBC) cell lines, especially when combined with hydroxytyrosol ( 25 ). Although our study did not include combination therapy, β-LP 's individual efficacy is consistent with their findings, showing its pro-apoptotic capabilities in breast cancer models. Furthermore, β-lapachone significantly disrupted the cell cycle. Our study found that β-lapachone administration resulted in a concentration-dependent S-phase arrest. The S-phase population increased from 29.1% in the control group to 50.1% at 20 µM, whereas the G0/G1 population decreased. Previous research on colon and breast cancer found that β-lapachone impaired DNA replication ( 26 ). Comparative literature backs up our findings. Recent research with oxaliplatin-resistant colorectal cancer cells (HCT116-OxPt-R) revealed that β-lapachone induces more cytotoxicity in resistant cells than in non-resistant cells. These findings highlight β-lapachone's potential for treating sensitive cancer types and overcoming chemoresistance by inducing multi-targeted apoptosis ( 27 ). These findings support the therapeutic benefits of β-lapachone in cancer treatment. Their capacity to cause apoptosis at different cell cycle arrest points complementary mechanisms that could be investigated in combination treatments. Future studies should optimize the use of β-lapachone in resistant or aggressive cancer types by analyzing synergistic combinations and biological pathways. β-LP consistently exhibited lower docking scores, indicating stronger interactions with target proteins. Notably, β-LP showed exceptional binding to dihydrofolate reductase of Candida albicans and PBP2a of MRSA, both critical targets for antifungal and antibacterial treatments. Conclusion The study emphasizes the therapeutic potential of β-lapachone (β-LP) as multiple-function compound having antimicrobial and anticancer properties. The compound was highly effective in inhibiting multidrug-resistant microbes, disrupting biofilms, and inducing oxidative stress in vitro. These findings are especially important in light of developing antimicrobial resistance, which remains one of the most serious global health challenges. It is noteworthy that anticancer evaluations of β-LP against breast cancer cells have shown promising results. The in-silico docking research confirmed these findings by demonstrating the compounds' high binding affinities to important microbial and cellular targets, confirming their proposed modes of action. Nevertheless, it is imperative to assess the potential toxicity, pharmacokinetic properties, and side effects of the compounds. The study results suggest that β-LP could be used as future candidate drugs for the treatment of microbial diseases and breast cancer and might be recommended to biomedical and pharmaceutical applications. Declarations Acknowledgements The authors express their gratitude to the Deanship of Research and Graduate Studies at King Khalid University for financial support. Author Contributions : RAA, IA, YA: Concept, Data Collection, Writing Manuscript . IA, SOR, AD, MSA: Data Analysis . RAA, AKM, MS: Writing Manuscript . MAA, MYQ, MMAS, MS: Data Curation, Project Management . IA, AD, YA, MAA: Concept . IA: Funding. All authors reviewed the manuscript. Funding: The authors express their gratitude to the Deanship of Research and Graduate Studies at King Khalid University for funding this work through the Large Research Group Project under grant number RGP.02/503/46 and the authors would like to express their thanks to Researchers supporting project number (ORF-2025_1326) King Saud University, Riyadh, Saudi Arabia. Competing Interest: The authors declare no competing interests. Data Availability: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Conflict of Interest The authors declare no conflict of interest. References Almutairy B. 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Antimicrobial resistance: Risk associated with antibiotic overuse and initiatives to reduce the problem. Ther. Adv. Drug Saf. 2014, 5, 229–241. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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. 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2","display":"","copyAsset":false,"role":"figure","size":273770,"visible":true,"origin":"","legend":"\u003cp\u003eThe zone of inhibition (ZOI) is against MDR bacteria using the well diffusion method. The bacteria studied were \u003cstrong\u003e(A)\u003c/strong\u003e MRSA, \u003cstrong\u003e(B)\u003c/strong\u003e Neisseria spp., \u003cstrong\u003e(C)\u003c/strong\u003e VRE, \u003cstrong\u003e(D)\u003c/strong\u003e Acinetobacter baumannii, and \u003cstrong\u003e(E)\u003c/strong\u003e Pseudomonas aeruginosa. Wells consisted of the following compounds: \u003cstrong\u003e(1)\u003c/strong\u003e gingerol, \u003cstrong\u003e(2)\u003c/strong\u003eβ-lapachone, \u003cstrong\u003e(3)\u003c/strong\u003e allicin, \u003cstrong\u003e(4)\u003c/strong\u003e benzalkonium chloride (BKC), and \u003cstrong\u003e(5)\u003c/strong\u003ephyscion.\u003c/p\u003e","description":"","filename":"Figure2.ZOIPATHO.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7982878/v1/20df78d4546d7b7634c50c74.jpg"},{"id":97898125,"identity":"2a418be1-c237-4f59-96bf-ccb70ff6969e","added_by":"auto","created_at":"2025-12-10 15:38:42","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":156707,"visible":true,"origin":"","legend":"\u003cp\u003eThe zone of inhibition (ZOI) is against pathogenic microbes using the well diffusion method. The bacteria studied were \u003cstrong\u003e(A)\u003c/strong\u003e Salmonella spp., \u003cstrong\u003e(B)\u003c/strong\u003e Pseudomonas aeruginosa, \u003cstrong\u003e(C)\u003c/strong\u003e Escherichia coli, \u003cstrong\u003e(D)\u003c/strong\u003e Staphylococcus aureus, \u003cstrong\u003e(E)\u003c/strong\u003e Mycobacterium smegmatis, and \u003cstrong\u003e(F)\u003c/strong\u003e Candida albicans. Wells consisted of the following compounds: \u003cstrong\u003e(1)\u003c/strong\u003egingerol, \u003cstrong\u003e(2)\u003c/strong\u003e β-lapachone, \u003cstrong\u003e(3)\u003c/strong\u003e allicin, \u003cstrong\u003e(4)\u003c/strong\u003e benzalkonium chloride (BKC), and \u003cstrong\u003e(5)\u003c/strong\u003e physcion.\u003c/p\u003e","description":"","filename":"Figure3.LPZOI.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7982878/v1/bdbd9d49ff18c62a332f5c8d.jpg"},{"id":97896250,"identity":"d0672b31-4251-4f33-87a9-de761a1a4c1c","added_by":"auto","created_at":"2025-12-10 15:36:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":102954,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of β-lapachone on microbial strains.\u003c/p\u003e","description":"","filename":"Figure4.BlapachoneMICandMBC.png","url":"https://assets-eu.researchsquare.com/files/rs-7982878/v1/703144524e893083b3bef8bb.png"},{"id":97898032,"identity":"e6698b12-968d-416f-ade5-1f1e7dc0dee3","added_by":"auto","created_at":"2025-12-10 15:38:36","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":224046,"visible":true,"origin":"","legend":"\u003cp\u003eMIC and MBC values β-LP against different microbes. \u003cstrong\u003e(A)\u003c/strong\u003e MIC and MBC of β-LP against MRSA, Pseudomonas aeruginosa, S. aureus, and M. smegmatis. \u003cstrong\u003e(B)\u003c/strong\u003e MIC and MBC of β-LP against Candida albicans.\u003c/p\u003e","description":"","filename":"Figure5.CFU.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7982878/v1/513a90074279518cd344833a.jpg"},{"id":97822618,"identity":"1cb254c8-b753-4300-9b6f-749bbb8fd197","added_by":"auto","created_at":"2025-12-09 18:50:55","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":91010,"visible":true,"origin":"","legend":"\u003cp\u003eCFU assay showing the effect of (β-LP) on microbial viability. Upper panels (A–E) represent untreated controls, and lower panels (F–J) show β-LP-treated samples. MRSA (A vs. F), Pseudomonas aeruginosa (B vs. G), Staphylococcus aureus (C vs. H), Mycobacterium smegmatis (D vs. I), and Candida albicans (E vs. J).\u003c/p\u003e","description":"","filename":"Figure6.GROWTHOFINHIBITION.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7982878/v1/d613bebdbf50ab8777ededd5.jpg"},{"id":97822620,"identity":"dff7cea0-75b7-4338-ad6c-e05abb8f85cb","added_by":"auto","created_at":"2025-12-09 18:50:55","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":67854,"visible":true,"origin":"","legend":"\u003cp\u003eThe growth inhibition percentage of selected bacteria after treatment with β-lapachone.\u003c/p\u003e","description":"","filename":"Figure7.antibiofilmactivity.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7982878/v1/d715fb28d5a1862766c8fc83.jpg"},{"id":97897821,"identity":"cdf488d8-26d0-43e4-849b-7742934c46c0","added_by":"auto","created_at":"2025-12-10 15:38:18","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":79879,"visible":true,"origin":"","legend":"\u003cp\u003eThe percentage of biofilm inhibition of selected bacteria after treatment with β-lapachone.\u003c/p\u003e","description":"","filename":"Figure8.KINETICGROWTHOFBL.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7982878/v1/87c72d12330189a1d7434a39.jpg"},{"id":97896722,"identity":"121d4332-c7a7-4df8-a624-f1b4cf1553a7","added_by":"auto","created_at":"2025-12-10 15:36:56","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":78246,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of β-LP on microbes growth kinetics. Representative bacterial strains of \u003cstrong\u003e(A)\u003c/strong\u003e MRSA, \u003cstrong\u003e(B)\u003c/strong\u003e Pseudomonas aeruginosa, \u003cstrong\u003e(C)\u003c/strong\u003e Staphylococcus aureus, \u003cstrong\u003e(D)\u003c/strong\u003e Mycobacterium smegmatis, and \u003cstrong\u003e(E)\u003c/strong\u003e Candida albicans. were treated with different concentrations (MIC x 0.5, MIC x 1 and MIC x 2) of β-LP. Growth cycle of untreated organisms served as growth control. Optical density at 600 nm was measured at regular time intervals of 2 hours.\u003c/p\u003e","description":"","filename":"Figure9.HeightoftheairofBL.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7982878/v1/8aae874267af1018384a6fc1.jpg"},{"id":97896658,"identity":"f825d3c3-2057-423c-a760-e5fcf417087e","added_by":"auto","created_at":"2025-12-10 15:36:52","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":81687,"visible":true,"origin":"","legend":"\u003cp\u003eCatalase activity in both untreated and treated bacteria with β-LP. Black bars represent control (untreated) samples, while gray bars represent treated samples. The height of the air bubble column (mm) represents the catalase activity levels in each strain.\u003c/p\u003e","description":"","filename":"Figure10.cytoplasmicleakageassayofBL.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7982878/v1/29368fe18bfba3f32dfc8d2c.jpg"},{"id":97822623,"identity":"454cc609-067b-4703-acc9-468ded82edc3","added_by":"auto","created_at":"2025-12-09 18:50:55","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":174002,"visible":true,"origin":"","legend":"\u003cp\u003eCytoplasmic leakage assay for microbes treated with β-LP. The absorbance at 595 nm was used to measure intracellular leakage.\u003c/p\u003e","description":"","filename":"Figure11.MTT.png","url":"https://assets-eu.researchsquare.com/files/rs-7982878/v1/882927a3bf926b663854a5e0.png"},{"id":97898516,"identity":"106b63e6-d4c3-4cf7-9f4b-7b02055ae405","added_by":"auto","created_at":"2025-12-10 15:39:14","extension":"jpg","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":34132,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different concentrations of β-LP on cell viability as assessed by the MTT assay.\u003c/p\u003e","description":"","filename":"Figure12MTT.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7982878/v1/bfe8eaa9c472b6567f24f8e7.jpg"},{"id":97822630,"identity":"7535c5f9-7210-4011-850d-e4d488522166","added_by":"auto","created_at":"2025-12-09 18:50:55","extension":"jpg","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":87335,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different concentrations of β-LP on cell viability as assessed by the MTT assay. The bar graph shows a concentration-dependent decrease in cell viability (%), with the highest.\u003c/p\u003e","description":"","filename":"Figure13.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7982878/v1/ff8aa5cd2e416173969c0b95.jpg"},{"id":97896698,"identity":"a2f81255-1e59-497e-8f4a-2d00db06afd4","added_by":"auto","created_at":"2025-12-10 15:36:53","extension":"jpg","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":99433,"visible":true,"origin":"","legend":"\u003cp\u003eFlow cytometry quadrant plots illustrate the effect of β-lapachone at doses of 0 (Control), 2, 5, 10, and 20 µM. Annexin V-FITC and Propidium Iodide (PI) staining are used to identify viable, apoptotic, and necrotic populations.\u003c/p\u003e","description":"","filename":"Figure14..jpg","url":"https://assets-eu.researchsquare.com/files/rs-7982878/v1/bcd01ccc6aa194f8d96e8e91.jpg"},{"id":97822627,"identity":"1b18fce0-8431-47ef-8840-14bbbe230251","added_by":"auto","created_at":"2025-12-09 18:50:55","extension":"jpg","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":21439,"visible":true,"origin":"","legend":"\u003cp\u003eCell cycle analysis histograms demonstrate DNA content (PI intensity) of cells treated with increasing concentrations of β-lapachone (0 (Control), 2, 5, 10, and 20 µM) during 24 hours. The peaks indicate the distribution of G0/G1, S, and G2/M phases, as measured by flow cytometry.\u003c/p\u003e","description":"","filename":"Figure15.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7982878/v1/9bdcd5294d08ff7727a5389f.jpg"},{"id":97822632,"identity":"4c66a196-c5a1-4aad-b9a6-1a2d0954fcef","added_by":"auto","created_at":"2025-12-09 18:50:55","extension":"jpg","order_by":16,"title":"Figure 16","display":"","copyAsset":false,"role":"figure","size":18601,"visible":true,"origin":"","legend":"\u003cp\u003eBinding affinities and molecular interactions of Beta Lapachone with multiple target proteins of Candida albicans. (A) CYP51 with Beta Lapachone: Pi-Pi: PHE228; Pi-Alkyl: TYR118, LEU121, PHE233, LEU376, MET508. (B) DHFR with Beta Lapachone: H-bonds: ALA11; Pi-Alkyl/Sigma/Sulfur: MET25.\u003c/p\u003e","description":"","filename":"Figure16..jpg","url":"https://assets-eu.researchsquare.com/files/rs-7982878/v1/ae1c5ab437784e2d2787884c.jpg"},{"id":97822638,"identity":"7ba87cc6-2dd3-420f-a188-e7a0609fe41f","added_by":"auto","created_at":"2025-12-09 18:50:55","extension":"jpg","order_by":17,"title":"Figure 17","display":"","copyAsset":false,"role":"figure","size":20516,"visible":true,"origin":"","legend":"\u003cp\u003eBinding affinities and molecular interactions of Beta Lapachone with multiple target proteins of MRSA. (A) PBP2a with Beta Lapachone: H-bonds: TYR441, GLU602; Pi-Pi/Alkyl: TYR519, ALA601. (B) Sortase A with Beta Lapachone: H-bonds: ASN114, SER116, ARG197; Pi-Alkyl/Sigma: PRO163, VAL168, THR180, ILE199.\u003c/p\u003e","description":"","filename":"Figure17.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7982878/v1/2e250757d31c48d10e0e5eab.jpg"},{"id":97822633,"identity":"0dd5fee2-6f86-4e2b-be7b-a6dc47022e97","added_by":"auto","created_at":"2025-12-09 18:50:55","extension":"jpg","order_by":18,"title":"Figure 18","display":"","copyAsset":false,"role":"figure","size":22507,"visible":true,"origin":"","legend":"\u003cp\u003eBinding affinities and molecular interactions of Beta Lapachone with multiple target proteins of M. smegmatis. (A) Cargo Desulfurase with Beta Lapachone: H-bonds: ASN328, ARG532; Pi-Alkyl: ALA184, ARG512. (B) InhA with Beta Lapachone: H-bond: GLY14; Pi-Pi/Sigma/Alkyl: PHE41, VAL65, ILE16, ILE95, ILE122.\u003c/p\u003e","description":"","filename":"Figure18.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7982878/v1/84632facc22819af76012ee8.jpg"},{"id":97898211,"identity":"b5b1a881-f77b-4b70-abd7-85e117cd223b","added_by":"auto","created_at":"2025-12-10 15:38:48","extension":"jpg","order_by":19,"title":"Figure 19","display":"","copyAsset":false,"role":"figure","size":20169,"visible":true,"origin":"","legend":"\u003cp\u003eBinding affinities and molecular interactions of Beta Lapachone with multiple target proteins of P. aeruginosa. (A) LasR with Beta Lapachone: H-bonds: ARG61; Pi-Pi/Alkyl/Sigma/Anion: TYR64, ASP73, LEU36, ALA50, VAL76, ALA127. (B) OprF with Beta Lapachone: Pi-Pi/Alkyl: SER39, ILE74, ASP72, LYS130.\u003c/p\u003e","description":"","filename":"Figure19.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7982878/v1/51ccdf041f5d864e4379ac2a.jpg"},{"id":97822645,"identity":"0a8b9b21-bf48-4f00-8a83-cc0712eb9f02","added_by":"auto","created_at":"2025-12-09 18:50:55","extension":"png","order_by":20,"title":"Figure 20","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":"\u003cp\u003eBinding affinities and molecular interactions of Beta Lapachone with multiple target proteins of S. aureus. (A) AHL with Beta Lapachone: H-bonds: LYS21, GLU289. (B) TarO with Beta Lapachone: Pi-Pi: PHE154; Pi-Alkyl: VAL347, ALA155, LEU342; Interaction with ASP355.\u003c/p\u003e","description":"","filename":"placeholderimageCopy.png","url":"https://assets-eu.researchsquare.com/files/rs-7982878/v1/f846f8bfeee9d45a2c6efc5e.png"},{"id":99312721,"identity":"6d7ebef8-486d-4cda-a15b-6fbd5962cdba","added_by":"auto","created_at":"2025-12-31 16:19:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3775491,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7982878/v1/a94f9283-17cd-4b65-9304-86c175fe2844.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Deciphering the Therapeutic Promise of β-Lapachone against Pathogenic Multidrug Resistant Microbes and Breast Cancer Cell","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMultidrug-resistant (MDR) bacteria are a significant risk to public health because they can adapt to multiple antibiotic treatments. Resistance to at least one of three or more distinct classes of antibiotics, including ampicillin, sulfonamides, and chloramphenicol, is commonly used to identify MDR strains. On the other hand, strains that are extensively drug-resistant (XDR) are resistant to nearly every antibiotic that is currently on the market, meaning that only one or two effective treatment options remain. These consist of azithromycin, carbapenems, and piperacillin. XDR strains that are resistant to third-generation cephalosporins such as ceftazidime, cefuroxime, and ceftriaxone, as well as the fluoroquinolone ciprofloxacin, are noteworthy examples (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). When bacteria change in ways that make drugs used to treat infections less effective, this is known as bacterial antimicrobial resistance, or AMR (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). According to epidemiological studies acquiring extra-chromosomal genetic material through horizontal gene transfer, antibiotic use is directly associated with the development of resistant bacterial strains (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). In Gram-negative bacteria, reduced outer membrane permeability, efflux pump activity, break down antibiotics by the synthesis of enzymes, the formation of biofilms, and changes to antibiotic targets that allow them to avoid the effects of antibiotics (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). AMR is one of the most pressing public health issues of our time due to decrease antibiotic effect on multidrug-resistant (MDR) bacteria, which present a growing threat to global health (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMany antimicrobials have been developed and marketed over the years with the shared objective of treating and curing mild to severe infections (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Beyond the severe human impact, AMR has a significant financial cost that will only increase if resistance is not addressed. Failure to act will result in a massive loss of world production of \u003cspan\u003e$\u003c/span\u003e100 trillion by 2050 (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). The development of extremely virulent MDR bacteria highlights the urgent demand for substitute therapies in order to successfully stop MDR infections and their spread (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eA natural product is a chemical that is made by living things, such as microbes, plants, mushrooms, and animals. In addition to being used for centuries to treat serious illnesses, plants are still frequently employed to create novel therapeutic candidates (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Scientists are paying attention to natural chemicals because they may make useful models for creating new medication compounds (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMedicinal plants are regarded for their therapeutic characteristics and synergistic effects, which help with illness treatment and prevention resulting in greater manufacturing and a decrease in synthetic drug use (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe evolution of drug resistance in cancer cells, as well as the limits of current chemotherapeutic drugs, have highlighted the critical need to identify compounds with significant anticancer properties and novel modes of action. Natural compounds attract the interest of researchers as viable sources of new anticancer therapies due to their various biological features and ability to overcome resistance mechanisms. β-LP, has been extensively researched for its capacity to produce oxidative stress, impair DNA replication, and promote apoptosis in multiple cancer types. The purpose of this study is to evaluate the antimicrobial and anticancer potential of β-LP by using in vitro biological studies and in silico molecular docking approach against pathogenic and multidrug-resistant bacteria and cancer cell.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eMaterials\u003c/strong\u003e\u003cbr\u003eThis study included \u0026beta;-lapachone (10 mM) as natural antimicrobial agents was first dissolved in dimethyl sulfoxide (DMSO) to ensure optimum dilution and application. For culture and sensitivity testing, microbiological media and reagents included Mueller-Hinton agar (MHA) and Mueller-Hinton broth (MHB). Phosphate-buffered saline (PBS), crystal violet solution, catalase test reagents, and bacterial strains. Antimicrobial activity was determined with 96-well microdilution plates.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/h2\u003e\n\u003ch2\u003e\u003cstrong\u003e\u003cem\u003eIn-Vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;antimicrobial activity\u003c/strong\u003e\u003c/h2\u003e\n\u003ch3 id=\"_Toc198500275\"\u003e\u003cstrong\u003eBacterial Strains and Growth Conditions\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThis study included a variety of microbial strains, including multidrug-resistant (MDR) organisms such VRE, MRSA, \u003cem\u003eAcinetobacter baumannii, Pseudomonas aeruginosa, and Neisseria spp.\u003c/em\u003e, as well as other clinically relevant pathogens like \u003cem\u003eSalmonella spp., Escherichia coli, Staphylococcus aureus, Mycobacterium smegmatis, and Candida albicans\u003c/em\u003e. All strains were stored as glycerol stocks in the freezer at -80 \u0026deg;C. All strains were grown in Mueller-Hinton broth (MHB). Bacterial suspensions for experimental testing were prepared by diluting the cultures in MHB to an optical density (O.D.) of 0.05 at a wavelength of 610 nm. Following adjustment, the bacterial suspensions were incubated at 37\u0026deg;C for 20-25 minutes to stabilize the inoculum prior to testing.\u003c/p\u003e\n\u003ch3 id=\"_Toc198500276\"\u003e\u003cstrong\u003ePreparation of Compounds\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe \u0026beta;-lapachone was synthesized at a 10 mM stock concentration. For experimental purposes, \u0026beta;-lapachone was diluted to a working concentration of 2.5 mM with dimethyl sulfoxide (DMSO) as the solvent. The dilution was performed according to the formula \u003cstrong\u003eC1V1 = C2V2\u003c/strong\u003e, where C1 and C2 represents the beginning and final concentrations respectively, and V1 and V2 represent the beginning and final volumes.\u0026nbsp;\u003c/p\u003e\n\u003ch3 id=\"_Toc198500277\"\u003e\u003cstrong\u003eEvaluation of antibacterial efficacy by agar well diffusion\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe \u0026beta;-lapachone was tested for antibacterial activity using the agar well diffusion method (12). Mueller-Hinton agar (MHA) plates were prepared, and wells of 6 mm in diameter were made with a sterilized syringe cap. The surface of each plate was equally inoculated with sterile cotton swabs soaked in the prepared bacterial suspension. Following this, 20 \u0026mu;L of dimethyl sulfoxide (DMSO) was added to the control well, and 20 \u0026mu;L of \u0026beta;-lapachone was added to the corresponding wells. Plates were incubated aerobically at 37\u0026deg;C overnight. \u0026nbsp;After incubation, the diameter of the zone of inhibition (ZOI) along with well was measured in millimeters (mm) to determine antimicrobial activity.\u003c/p\u003e\n\u003ch3 id=\"_Toc198500278\"\u003e\u003cstrong\u003eDetermination of MICs and MBC\u003c/strong\u003e\u003cstrong\u003es\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eMinimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) were determined for \u0026beta;-lapachone against selected microbial strains. \u0026beta;-lapachone was tested against MRSA, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eMycobacterium smegmatis\u003c/em\u003e, \u003cem\u003eCandida albicans\u003c/em\u003e, and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e. \u0026beta;-lapachone was serially diluted two-fold from a starting concentration of 2.5 mM. Microbial cultures were cultured in Mueller-Hinton broth (MHB) and adjusted to have an optical density of 0.05. The adjusted suspension (180 \u0026mu;L) was added to each well of a 96-well microdilution plate, followed by 20 \u0026mu;L of \u0026beta;-lapachone. The control wells received 180 \u0026mu;L of microbial solution and 20 \u0026mu;L of MHB. Plates were\u0026nbsp;incubated at 37\u0026deg;C for overnight. The MIC was defined as the lowest concentration that produced no observable growth. To determine MBC, 10 \u0026mu;L of clear wells were subcultured on Mueller-Hinton agar and incubated overnight at 37\u0026deg;C. The MBC had the lowest concentration with no colony formation (13).\u003c/p\u003e\n\u003ch3 id=\"_Toc198500279\"\u003e\u003cstrong\u003eDetermination Colony forming unit\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eTo assess bacterial viability after treatment, colony forming units (CFUs) were performed using ten-fold serial dilutions from the MIC wells (14). From each MIC well, 62.5 \u0026mu;L was transferred into 562.5 \u0026mu;L of Mueller-Hinton broth (MHB), and the dilutions were plated onto Mueller-Hinton agar plates (MHA). For control samples, 62.5 \u0026mu;L of bacterial culture and 562.5 \u0026mu;L of MHB were similarly plated. Plates were incubated overnight at 37\u0026deg;C to compare bacterial growth between treated and control samples.\u003c/p\u003e\n\u003ch3 id=\"_Toc198500280\"\u003e\u003cstrong\u003eEvaluation of growth inhibition\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe growth inhibition assay assessed the antibacterial activity of \u0026beta;-lapachone against certain microorganisms. In a 96-well microtiter plate, 20 \u0026mu;L of \u0026beta;-lapachone at MIC concentration was added to 180 \u0026mu;L of bacterial culture. Control wells held 180 \u0026mu;L of bacterial suspension and 20 \u0026mu;L of Mueller-Hinton broth (MHB). The plate was incubated at 37\u0026deg;C, and absorbance at 600 nm was monitored after 24 hrs using a FLUOstar Omega plate reader (BMG LABTECH, Germany) (15). Growth inhibition was estimated by comparing the absorbance values of treatment and control wells using the following formula:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInhibition (%) = ((\u0026mu;C \u0026ndash; \u0026mu;T) / \u0026mu;C) \u0026times; 100\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhere \u0026mu;C is Mean absorbance of control wells (O.D. 600) and \u0026mu;T is Mean absorbance of treated wells (O.D.600)\u003c/p\u003e\n\u003ch3 id=\"_Toc198500281\"\u003e\u003cstrong\u003eAntibiofilm activity of \u0026beta;-lapachone\u0026nbsp;\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eSelected microbial strains were used to test the prevention of biofilm development. In 96-well polystyrene microplates, 180 \u0026mu;L of bacterial suspension (OD 600 = 0.05) was added to each well, followed by 20 \u0026mu;L of \u0026beta;-lapachone at their respective MIC values. The strains studied were \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, MRSA, \u003cem\u003eMycobacterium smegmatis, Pseudomonas aeruginosa\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Candida albicans\u003c/em\u003e. Plates were incubated at 37\u0026deg;C without shaking for 24 hours to generate biofilms. Control wells contained 180 \u0026mu;L of bacterial suspension and 20 \u0026mu;L of Mueller-Hinton broth (MHB). Plates were incubated overnight and then put in the oven at 60\u0026deg;C for 30-45 minutes. Next, 50 \u0026mu;L of 1% crystal violet solution was added to each well (16). Biofilm formation was measured at 610 nm with a BMG LABTECH FLUOstar Omega plate reader (BMG LABTECH, Allmendgr\u0026uuml;n, Ortenberg, Germany).\u003c/p\u003e\n\u003ch3 id=\"_Toc198500282\"\u003e\u003cstrong\u003eKinetic assay for time killing performance\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eA kinetic assay was performed to assess the antibacterial activity of \u0026beta;-lapachone against several pathogens, including \u003cem\u003eStaphylococcus aureus,\u003c/em\u003e MRSA, \u003cem\u003eMycobacterium smegmatis, Pseudomonas aeruginosa\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Candida albicans\u003c/em\u003e. In a 96-well microplate, 180 \u0026mu;L of bacterial suspension (adjusted to 0.05 OD at 610 nm in Mueller-Hinton broth) was combined with 20 \u0026mu;L of \u0026beta;-lapachone at three concentrations: MIC x 0.5, MIC x 1, and MIC x 2. Control wells contained 180 \u0026mu;L of bacterial culture and 20 \u0026mu;L of MHB.\u003cbr\u003e\u0026nbsp;The plates were incubated at 37\u0026deg;C and absorbance was measured at every 2 hours with the FLUOstar Omega plate reader (BMG LABTECH, Allmendgr\u0026uuml;n, Ortenberg, Germany). By comparing absorbance measurements between treated and control wells, growth kinetics were tracked over the incubation period in order to identify the compounds\u0026apos; time-dependent bactericidal or fungicidal effects (17).\u003c/p\u003e\n\u003ch3 id=\"_Toc198500283\"\u003e\u003cstrong\u003eCatalase Activity Assay\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eBacterial cultures treated with \u0026beta;-lapachone at their respective MIC values were incubated overnight at 37\u0026deg;C in a shaking incubator at 200 RPM in order to measure catalase activity. The same conditions were used to incubate untreated bacterial cultures that were produced in the medium alone as a control. Following incubation, each culture received a direct addition of 100 \u0026mu;L of hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), and the mixture was promptly checked to produce bubbles. To assess catalase activity, the height of the ensuing air bubble column was measured with a ruler.\u003c/p\u003e\n\u003ch3 id=\"_Toc198500284\"\u003e\u003cstrong\u003eCytoplasmic leakage Assay\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe cytoplasmic leakage assay\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ewas used to evaluate membrane integrity after compound treatment. A 24 hour bacterial suspension of each examined organism was centrifuged at 3500 rpm for 15 minutes. To eliminate any remaining media, the bacterial pellets were rinsed with phosphate-buffered saline (PBS, pH 7.0). After adding 1mL of PBS, the pellets were shaken and incubated at 37\u0026deg;C for 24 hours. After incubation, 50 \u0026mu;L of \u0026beta;-LP at a final concentration of 2.5 mM was added to the corresponding samples and kept for 30 minutes at room temperature. The treated and the control suspensions were centrifuged at 3500 rpm for an additional 15 minutes. The supernatant was collected and examined spectrophotometrically at 595 nm with the FLUOstar Omega plate reader (BMG LABTECH, Allmendgrun, Ortenberg, Germany) to quantify the level of DNA released by the cells (18).\u003c/p\u003e\n\u003ch2 id=\"_Toc198500285\"\u003e\u003cstrong\u003e\u003cem\u003eIn-Vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Anti-Cancer Activity\u003c/strong\u003e\u003c/h2\u003e\n\u003ch3 id=\"_Toc198500286\"\u003e\u003cstrong\u003eCell Culture\u003c/strong\u003e\u003cstrong\u003e\u003cspan dir=\"RTL\"\u003e \u003c/span\u003e\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe human breast cancer cell line BT-474 from ATCC was cultured in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium: Nutrient Mix F-12 (D-MEM/F-12 1:1) medium supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin and 100 mg/ml streptomycin in a 37\u0026deg;C incubator with 5% CO2. The cells were passaged by harvesting with trypsin/ EDTA and seeding into flask.\u0026nbsp;\u003c/p\u003e\n\u003ch3 id=\"_Toc198500287\"\u003e\u003cstrong\u003eMTT assay\u003c/strong\u003e\u003cstrong\u003e\u003cspan dir=\"RTL\"\u003e \u003c/span\u003e\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe cytotoxicity of the test samples was evaluated using the MTT assay (19). Briefly, cells were seeded in a 96-well plate at a density of 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well and allowed to adhere overnight under standard culture conditions. After incubation, the cells were treated with various concentrations of the \u0026beta;-LP and incubated for an additional 24 hours. Following treatment, 20 \u0026micro;L of MTT solution (5 mg/mL in PBS) was added to each well, and the plates were incubated for 4 hours at 37\u0026deg;C. Subsequently, the culture medium was carefully aspirated, and 150 \u0026micro;L of dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals. The absorbance was measured at 570 nm using a microplate reader. Cell viability was expressed as a percentage relative to untreated control cells, and all experiments were performed in triplicate to ensure reproducibility.\u003c/p\u003e\n\u003ch3 id=\"_Toc198500288\"\u003e\u003cstrong\u003eFlow cytometry\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eFlow cytometry was used to assess apoptosis and necrosis after \u0026beta;-lapachone treatment. Cells were seeded in 6-well plates at a density of approximately 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well and allowed to adhere overnight at 37\u0026deg;C in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e. Cells were then treated with various concentrations of \u0026beta;-LP (0, 2, 5, 10, and 20 \u0026micro;M) for 24 hours, with untreated cells serving as the control. After treatment, both adherent and floating cells were collected, washed twice with cold phosphate-buffered saline (PBS), and resuspended in 100 \u0026micro;L of binding buffer. Cells were stained by adding 5 \u0026micro;L of Annexin V-FITC and 5 \u0026micro;L of Propidium Iodide (PI) and incubated for 15 minutes at room temperature in the dark. After staining, 400 \u0026micro;L of binding buffer was added to each sample, and flow cytometric analysis was performed immediately using a BD FACSCalibur flow cytometer. Annexin V-FITC and PI signals were detected in FL1 (green) and FL2 (red) channels, respectively, with proper compensation settings to minimize signal overlap. A minimum of 10,000 events were acquired per sample. The cell population was analyzed into four quadrants: viable cells (Annexin V\u003csup\u003e-\u003c/sup\u003e/PI\u003csup\u003e-\u003c/sup\u003e), early apoptotic cells (Annexin V\u003csup\u003e+\u003c/sup\u003e/PI\u003csup\u003e-\u003c/sup\u003e), late apoptotic/secondary necrotic cells (Annexin V\u003csup\u003e+\u003c/sup\u003e/PI\u003csup\u003e+\u003c/sup\u003e), and necrotic cells (Annexin V\u003csup\u003e-\u003c/sup\u003e/PI\u003csup\u003e+\u003c/sup\u003e). Data were analyzed using FlowJo software, and results were expressed as mean \u0026plusmn; standard deviation from three independent experiments.\u003c/p\u003e\n\u003ch3 id=\"_Toc198500289\"\u003e\u003cstrong\u003eCell Cycle arrest\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eFor cell cycle analysis, cells were seeded in 6-well plates at a density of approximately 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well and incubated overnight at 37\u0026deg;C in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e. After adhesion, cells were treated with various concentrations of \u0026beta;-LP (0, 2, 5, 10, and 20 \u0026micro;g/mL) for 24 hours, while untreated cells served as the control. Following treatment, both floating and adherent cells were harvested by trypsinization, washed twice with cold phosphate-buffered saline (PBS), and centrifuged at 300 \u0026times; g for 5 minutes at 4\u0026deg;C. The resulting pellets were fixed in ice-cold 70% ethanol, added dropwise while gently vortexing, and incubated at -20\u0026deg;C for at least 2 hours. After fixation, cells were washed with PBS, and resuspended in a staining solution containing 50 \u0026micro;g/mL propidium iodide (PI) and 100 \u0026micro;g/mL RNase A. The samples were incubated at room temperature in the dark for 30 minutes to ensure complete staining of DNA. Flow cytometry was performed using a BD FACSCalibur system, with excitation at 488 nm and emission detected in the FL2 channel. For each sample, 10,000\u0026ndash;20,000 events were collected. Doublets were excluded by appropriate gating strategies. DNA content histograms were generated, and the distribution of cells across the G0/G1, S, and G2/M phases was analyzed using FlowJo software (20). The results were expressed as the percentage of cells in each phase and reported as mean \u0026plusmn; standard deviation (SD) from three independent experiments.\u003c/p\u003e\n\u003ch2 id=\"_Toc198500291\"\u003e\u003cstrong\u003e\u003cem\u003eIn-Silico\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;study\u003c/strong\u003e\u003c/h2\u003e\n\u003ch3 id=\"_Toc198500292\"\u003e\u003cstrong\u003eLigand and Target Selection\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eBeta-Lapachone (PubChem CID: 3885) was selected based on their pharmacological relevance and compliance with Lipinski\u0026rsquo;s Rule of Five. Their 3D structures were retrieved from PubChem in SDF format, converted to PDB using Open Babel, and energy-minimized using the MMFF94 force field.\u003c/p\u003e\n\u003cp\u003eTen protein targets from five pathogenic microorganisms were selected based on their roles in microbial survival, virulence, and antibiotic resistance. These include:\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003e\u003cem\u003eCandida albicans\u003c/em\u003e: CYP51 (PDB: 5TZ1), DHFR (PDB: 1AI9),\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMRSA: PBP2a (PDB: 1VQQ), Sortase A (PDB: 1T2P),\u0026nbsp;\u003c/li\u003e\n \u003cli\u003e\u003cem\u003eMycobacterium smegmatis\u003c/em\u003e: InhA (PDB: 2NV6), Cysteine Desulfurase (PDB: 8KG1),\u003c/li\u003e\n \u003cli\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e: LasR (PDB: 3IX3), OprF (PDB: 4RLC),\u003c/li\u003e\n \u003cli\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e: Alpha-Hemolysin (PDB: 7AHL), TarO (PDB: 5EZM).\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eProtein structures were obtained from the RCSB PDB.\u003c/p\u003e\n\u003ch3 id=\"_Toc198500293\"\u003e\u003cstrong\u003ePreparation of Proteins and Ligands\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eProtein structures were preprocessed using AutoDock Tools by removing water molecules and heteroatoms, adding polar hydrogens, and assigning Gasteiger charges. Ligands were prepared and converted into PDBQT format using AutoDock 4.2. Potential binding pockets were predicted using DogSiteScorer for grid box configuration.\u003c/p\u003e\n\u003ch3 id=\"_Toc198500294\"\u003e\u003cstrong\u003eMolecular Docking and Analysis\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eMolecular docking was performed using AutoDock Vina v1.2.x. Binding affinities (in kcal/mol) were recorded for all ligand\u0026ndash;protein complexes. Top-ranked complexes were further analyzed for molecular interactions using Biovia Discovery Studio.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eAll statistical analyses and graphical representations were conducted using GraphPad Prism (version 10.4.2). Statistical analyses were performed using SPSS software (version 20) and the value of p \u0026lt; 0.05 was taken as statistically noteworthy.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2 style=\"text-align: left;\"\u003e\u003cstrong\u003e\u003cem\u003eIn-Vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Antimicrobial Activity\u003c/strong\u003e\u003c/h2\u003e\n\u003ch3 id=\"_Toc198500297\" style=\"text-align: left;\"\u003e\u003cstrong\u003eEvaluation of antibacterial efficacy by zone of inhibition\u003c/strong\u003e\u003c/h3\u003e\n\u003cp style=\"text-align: left;\"\u003eThe antimicrobial activity of \u0026beta;-lapachone was investigated against MDR as shown in\u003cstrong\u003e\u0026nbsp;Figure 1.\u003c/strong\u003e The zone of inhibition (ZOI) is against MDR bacteria using the well diffusion method. The bacteria studied were \u003cstrong\u003e(A)\u003c/strong\u003e MRSA, \u003cstrong\u003e(B)\u003c/strong\u003e Neisseria spp., \u003cstrong\u003e(C)\u003c/strong\u003e VRE, \u003cstrong\u003e(D)\u003c/strong\u003e Acinetobacter baumannii, and \u003cstrong\u003e(E)\u003c/strong\u003e Pseudomonas aeruginosa. Well number 2 consisted of \u0026beta;-lapachone and common pathogenic microbial strains shown in \u003cstrong\u003eFigure 2.\u003c/strong\u003e The zone of inhibition (ZOI) is against pathogenic microbes using the well diffusion method. The bacteria studied were \u003cstrong\u003e(A)\u003c/strong\u003e Salmonella spp., \u003cstrong\u003e(B)\u003c/strong\u003e Pseudomonas aeruginosa, \u003cstrong\u003e(C)\u003c/strong\u003e Escherichia coli, \u003cstrong\u003e(D)\u003c/strong\u003e Staphylococcus aureus, \u003cstrong\u003e(E)\u003c/strong\u003e Mycobacterium smegmatis, and \u003cstrong\u003e(F)\u003c/strong\u003e Candida albicans. Well number 2 consisted of \u0026beta;-lapachone. using the agar well diffusion method. \u0026beta;-LP demonstrated measurable activity against MRSA, with inhibition zones of 19 mm Figure 8. Among the remaining MDR strains, such as Neisseria, VRE, Acinetobacter, and Pseudomonas, none of the compounds showed significant antimicrobial activity.\u003c/p\u003e\n\u003cp style=\"text-align: left;\"\u003eAt the same time, \u0026beta;-LP showed the strongest inhibitory effect against common pathogenic microorganisms in\u003cstrong\u003e\u0026nbsp;Figure 3.\u003c/strong\u003e Effects of \u0026beta;-lapachone on microbial strains.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eIncluded MRSA, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eMycobacterium smegmatis\u003c/em\u003e, and \u003cem\u003eCandida albicans\u003c/em\u003e. It produced distinct zones of inhibition (ZOI) of 19 mm against \u003cem\u003ePseudomonas\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e, 20 mm against \u003cem\u003eM. smegmatis\u003c/em\u003e, and a strong antimicrobial effect with ZOI of 38 mm against \u003cem\u003eCandida albicans\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003ch3 id=\"_Toc198500298\" style=\"text-align: left;\"\u003e\u003cstrong\u003eDetermination of MICs and MBCs\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/h3\u003e\n\u003cp style=\"text-align: left;\"\u003e\u0026beta;-lapachone shown strong antimicrobial effect against Gram-positive bacteria and Candida albicans. The MIC values for MRSA, S. aureus, and M. smegmatis were all 0.156 mM, while P. aeruginosa had a slightly higher MIC of 0.312 mM. \u0026nbsp;Accordingly, MBC values for MRSA, S. aureus, M. smegmatis, and P. aeruginosa were 1.25 mM, 0.312 mM, and 0.625 mM respectively, indicating efficient bactericidal activity shown in \u003cstrong\u003eFigure 4.\u003c/strong\u003e MIC and MBC values \u0026beta;-LP against different microbes. \u003cstrong\u003e(A)\u003c/strong\u003e For \u0026beta;-LP: MRSA, Pseudomonas aeruginosa, S. aureus, and M. smegmatis. \u003cstrong\u003e(B)\u003c/strong\u003e The MIC and MBC of \u0026beta;-LP against Candida albicans. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eC.\u0026nbsp;albicans\u0026nbsp;showed remarkable sensitivity to \u0026beta;-LP, with MIC of 0.004 mM and MBC of 0.009 mM, indicating promising antifungal characteristics (\u003cstrong\u003eFigure 4).\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ch3 id=\"_Toc198500299\" style=\"text-align: left;\"\u003e\u003cstrong\u003eDetermination Colony forming unit\u003c/strong\u003e\u003c/h3\u003e\n\u003cp style=\"text-align: left;\"\u003eTreatment with \u0026beta;-lapachone (\u0026beta;-LP) significantly reduced colony-forming units (CFUs) in all examined microbes compared to untreated controls\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eas showen in\u003cstrong\u003e\u0026nbsp;Figure 5.\u0026nbsp;\u003c/strong\u003eCFU assay showing the effect of (\u0026beta;-LP) on microbial viability. Upper panels (A\u0026ndash;E) represent untreated controls, and lower panels (F\u0026ndash;J) show \u0026beta;-LP-treated samples. MRSA (A vs. F), \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (B vs. G), \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (C vs. H), \u003cem\u003eMycobacterium smegmatis\u003c/em\u003e (D vs. I), and \u003cem\u003eCandida albicans\u003c/em\u003e (E vs. J). \u0026nbsp;In MRSA, the control plate contained a high density of colonies, whereas the treated sample had a significant drop in CFUs. \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e developed densely and widely in the control plate, but the treated plate had fewer and more distributed colonies. Similarly, \u0026beta;-LP therapy significantly reduced colony count in \u003cem\u003eStaphylococcus aureus\u003c/em\u003e compared to the control group. \u003cem\u003eMycobacterium smegmatis\u003c/em\u003e exhibited pigmented growth in the control, but treatment with \u0026beta;-LP significantly reduced colony density and distribution. The most pronounced effect was seen in \u003cem\u003eCandida albicans\u003c/em\u003e, where the treated plate showed a significant drop in CFU count compared to the heavy growth in untreated control. \u0026beta;-LP exhibits substantial antibacterial and antifungal activity, reducing viable microbial populations across many species.\u0026nbsp;\u003c/p\u003e\n\u003ch3 id=\"_Toc198500300\" style=\"text-align: left;\"\u003e\u003cstrong\u003eEvaluation percentage of growth inhibition\u003c/strong\u003e\u003c/h3\u003e\n\u003cp style=\"text-align: left;\"\u003eThe percentage of growth inhibition evaluates the antibacterial and antifungal capacity of \u0026beta;-LP by inhibiting the growth of specific bacterial and fungal strains. The experiment was designed to determine the proportion of growth inhibition caused by every compound under standardized conditions\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eas shown in\u003cstrong\u003e\u0026nbsp;Figure 6.\u003c/strong\u003e The growth inhibition percentage of selected bacteria after treatment with \u0026beta;-lapachone. All the organisms tested showed measurable growth suppression after being treated with \u0026beta;-LP. \u0026beta;-LP specifically inhibited MRSA by 42.21%, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e by 57.20%, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e by 50.96%, \u003cem\u003eMycobacterium smegmatis\u003c/em\u003e by 34.05%, and \u003cem\u003eCandida albicans\u003c/em\u003e by 58.3%. These findings suggest that \u0026beta;-LP has moderate antibacterial and antifungal action, with the strongest inhibition found against \u003cem\u003eP. aeruginosa\u0026nbsp;\u003c/em\u003eand \u003cem\u003eC. albicans.\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch3 id=\"_Toc198500301\" style=\"text-align: left;\"\u003e\u003cstrong\u003ePercentage of antibiofilm activity of \u0026beta;-lapachone\u0026nbsp;\u003c/strong\u003e\u003c/h3\u003e\n\u003cp style=\"text-align: left;\"\u003eThis experiment evaluated the effectiveness of \u0026beta;-lapachone in inhibiting biofilm formation in for medicinal purposes relevant microbes. Biofilm production is a crucial role in microbes persistence and resistance, making it an important target for antimicrobial drugs.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eAs illustrated in\u003cstrong\u003e\u0026nbsp;Figure 7.\u0026nbsp;\u003c/strong\u003eThe percentage of biofilm inhibition of selected bacteria after treatment with formed by \u0026beta;-lapachone. \u0026beta;-LP treatment resulted in essential levels of biofilm inhibition across all tested strains. The inhibition percentages for MRSA were around 91.8%, 83.7% for \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, 76.3% for \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, 82.5% for \u003cem\u003eMycobacterium smegmatis\u003c/em\u003e, and 85.7% for \u003cem\u003eCandida albicans\u003c/em\u003e. These findings suggest that \u0026beta;-LP has excellent antibiofilm action.\u003c/p\u003e\n\u003ch3 id=\"_Toc198500302\" style=\"text-align: left;\"\u003e\u003cstrong\u003eGrowth kinetic assay for time killing performance\u003c/strong\u003e\u003c/h3\u003e\n\u003cp style=\"text-align: left;\"\u003eIn order to assess the antibacterial and antifungal activities of \u0026beta;-LP over a 20-hour period, a kinetic growth experiment was conducted. In this experiment, growth was tracked using absorbance measurements to evaluate the inhibitory effect of \u0026beta;-LP on microbial proliferation at various concentrations (MIC x 0.5, MIC x 1 and MIC x 2). Regarding \u0026beta;-LP, each microbe that was examined, including MRSA, \u003cem\u003ePseudomonas aeruginosa, Staphylococcus aureus, Mycobacterium smegmatis, and Candida albicans\u003c/em\u003e growth were recorded at MIC x 0.5, MIC x 1 and MIC x 2 concentrations, except for \u003cem\u003eC. albicans\u003c/em\u003e, where only MIC x 1 and MIC x 2\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eas shown in\u003cstrong\u003e\u0026nbsp;Figure 8.\u003c/strong\u003e Effect of \u0026beta;-LP on microbes growth kinetics. Representative bacterial strains of \u003cstrong\u003e(A)\u003c/strong\u003e MRSA, \u003cstrong\u003e(B)\u003c/strong\u003e \u003cem\u003ePseudomonas aeruginosa,\u003c/em\u003e \u003cstrong\u003e(C)\u003c/strong\u003e \u003cem\u003eStaphylococcus aureus,\u003c/em\u003e \u003cstrong\u003e(D)\u003c/strong\u003e \u003cem\u003eMycobacterium smegmatis,\u0026nbsp;\u003c/em\u003eand \u003cstrong\u003e(E)\u003c/strong\u003e \u003cem\u003eCandida albicans\u003c/em\u003e were treated with different concentrations (MIC x 0.5, MIC x 1 and MIC x 2) of \u0026beta;-LP. Growth cycle of untreated organisms served as growth control. Optical density at 600 nm was measured at regular time intervals of 2 hours. All untreated control groups showed normal development progression over time. In contrast, \u0026beta;-LP treatment caused a concentration-dependent decrease in growth across all strains. At MIC x 0.5, considerable inhibition was detected, whereas MIC x 1 induced severe suppression and MIC x 2 led in near-complete inhibition of growth. \u0026nbsp;These results demonstrate that \u0026beta;-LP, efficiently suppresses microbial growth in all strains studied in a way that is dependent on both time and dose.\u003c/p\u003e\n\u003ch3 id=\"_Toc198500303\" style=\"text-align: left;\"\u003e\u003cstrong\u003eCatalase Activity Assay\u003c/strong\u003e\u003c/h3\u003e\n\u003cp style=\"text-align: left;\"\u003eThe purpose of the catalase activity experiment was to measure the catalase enzyme activity and assess the effects of \u0026beta;-LP therapy on the oxidative stress response of different microbes as shown in \u003cstrong\u003eFigure 9.\u003c/strong\u003e Catalase activity in both untreated and treated bacteria. Treatment with \u0026beta;-LP Black bars represents control (untreated) samples, while gray bars represent treated samples. The height of the air bubble column (mm) represents the catalase activity levels in each strain. Catalase activity was determined by measuring the height of the oxygen bubble column that developed upon exposure to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. In this assay, \u0026beta;-LP treatment resulted in a widespread decrease of catalase activity across the majority of tested strains. MRSA showed a moderate reduction (5 mm bubble height), \u003cem\u003eStaphylococcus aureus\u003c/em\u003e showed limited activity (1 mm), while \u003cem\u003eP. aeruginosa, M. smegmatis\u003c/em\u003e, and \u003cem\u003eC. albicans\u003c/em\u003e produced no bubbles at all. Either the substance directly inhibiting the enzyme or severe oxidative stress that destroys the enzyme itself can cause a decrease in catalase activity. These data show that \u0026beta;-LP limit microbial catalase activity, either directly or by causing oxidative stress, and that bubble height is a functional indicator of how bacteria respond to compounds-induced oxidative imbalances.\u003c/p\u003e\n\u003ch3 id=\"_Toc198500304\" style=\"text-align: left;\"\u003e\u003cstrong\u003eCytoplasmic leakage Assay\u003c/strong\u003e\u003c/h3\u003e\n\u003cp style=\"text-align: left;\"\u003eThe cytoplasmic leakage assay, which measures the release of intracellular contents as indicated by absorbance at 595 nm, was used to assess the membrane-disruptive effects of \u0026beta;-LP on different microbes. Greater cytoplasmic leakage and an impaired membrane are indicated by an increase in absorbance. In the \u0026beta;-LP-treated group shown in\u003cstrong\u003e\u0026nbsp;Figure 10.\u0026nbsp;\u003c/strong\u003eCytoplasmic leakage assay for microbes treated with \u0026beta;-LP. The absorbance at 595 nm was used to measure intracellular leakage. Displays the results for \u0026beta;-LP -treated cells. Black bars represent untreated controls, while gray bars represent treated samples., all tested strains (MRSA, \u003cem\u003ePseudomonas aeruginosa, Staphylococcus aureus, Mycobacterium smegmatis\u003c/em\u003e, and \u003cem\u003eCandida albicans\u003c/em\u003e) showed slightly higher absorbance than their respective untreated controls, suggesting slight membrane disruption. \u003cem\u003eM. smegmatis\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e showed the most marked increases.\u003c/p\u003e\n\u003ch2 id=\"_Toc198500305\" style=\"text-align: left;\"\u003e\u003cstrong\u003e\u003cem\u003eIn-Vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Anti-Cancer Activity\u003c/strong\u003e\u003c/h2\u003e\n\u003ch3 id=\"_Toc198500306\" style=\"text-align: left;\"\u003e\u003cstrong\u003eMTT Assay\u003c/strong\u003e\u003cstrong\u003e\u003cspan dir=\"RTL\"\u003e \u003c/span\u003e\u003c/strong\u003e\u003c/h3\u003e\n\u003cp style=\"text-align: left;\"\u003eThe microscopic images\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eshown in\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFigure\u003cspan dir=\"RTL\"\u003e \u003c/span\u003e11.\u003c/strong\u003e Effect of different concentrations of \u0026beta;-LP on cell viability as assessed by the MTT assay. illustrate the morphological changes in cells after treatment with varying concentrations of \u0026beta;-LP during the MTT assay. In the control group, the cells appeared healthy, densely populated, and exhibited typical spindle-shaped morphology with good adherence to the culture surface, indicating normal cell viability and proliferation. Upon treatment with 125 \u0026micro;g/mL, the cells largely retained their structure, although slight morphological alterations such as mild rounding and a slight reduction in cell density were observed, suggesting early signs of cytotoxic stress. At 250 ug/mL, more pronounced changes were evident; cells exhibited partial rounding, shrinkage, and decreased density compared to the control, indicating moderate cytotoxic effects. Exposure to 500 \u0026micro;g/mL resulted in significant cytotoxicity, characterized by severe cell rounding, detachment from the substrate, and reduced cellular spread. At the highest concentration of 1000 \u0026micro;g/mL, the cells were highly compromised, with extensive rounding, loss of normal structure, and a marked reduction in cell number, demonstrating near-complete cytotoxicity. Overall, these observations confirm a dose-dependent decrease in cell viability and health, with increasing concentrations of \u0026beta;-LP leading to progressive and severe morphological damage, consistent with mitochondrial dysfunction detected in the MTT assay. The MTT assay results demonstrate a clear concentration-dependent decrease in cell viability upon treatment with \u0026beta;-LP as shown in \u003cstrong\u003eFigure 12\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e Effect of different concentrations of \u0026beta;-LP on cell viability as assessed by the MTT assay. The bar graph shows a concentration-dependent decrease in cell viability (%), with the highest. The untreated cell control exhibited 100% viability, confirming normal cellular health in the absence of any toxic exposure. As the concentration of the test compound increased, a progressive decline in cell viability was observed. At 125 \u0026micro;g/mL, the mean cell viability dropped to 29.84%, indicating early signs of cytotoxicity. Further reduction was noted at 250 \u0026micro;g/mL with 22.56% viability, and a sharper decline at 500 \u0026micro;g/mL, where viability reduced to 14.37%. The most substantial cytotoxic effect was recorded at the highest concentration of 1000 \u0026micro;g/mL, where mean cell viability plummeted to just 5.23%. These results strongly indicate that \u0026beta;-LP exerts dose-dependent cytotoxic effects on the treated cells, with higher concentrations causing significant impairment of cell metabolic activity and survival, as reflected by the decreased formazan production measured in the MTT assay.\u003c/p\u003e\n\u003ch3 id=\"_Toc198500307\" style=\"text-align: left;\"\u003e\u003cstrong\u003eFlow cytometry\u003c/strong\u003e\u003c/h3\u003e\n\u003cp style=\"text-align: left;\"\u003eThe effect of \u0026beta;-lapachone on inducing apoptosis and necrosis was evaluated using flow cytometry, and the results in \u003cstrong\u003eTable 1\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e Effect of \u0026beta;-lapachone on early apoptosis, late apoptosis, necrosis, and total cell death percentages as determined by flow cytometry analysis. Cells were treated with increasing concentrations of \u0026beta;-lapachone (0\u0026ndash;20 \u0026micro;M) for 24 hours. Data are presented as mean \u0026plusmn; standard deviation (SD) from three independent experiments.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003edemonstrated a clear dose-dependent increase in cell death. In the control group (0 \u0026micro;M), minimal cell death was observed, with early apoptosis at 2.1%, late apoptosis at 1.2%, necrosis at 1.0%, and a total cell death of only 4.3%, confirming the baseline health of untreated cells. Upon treatment with 2 \u0026micro;M \u0026beta;-lapachone, early apoptosis increased significantly to 15.5%, while late apoptosis and necrosis rose to 5.4% and 2.5%, respectively, leading to a total cell death of 23.4%. At 5 \u0026micro;M, the early apoptotic population expanded to 32.8%, accompanied by 10.1% late apoptosis and 3.0% necrosis, resulting in a total of 45.9% cell death. Increasing the concentration to 10 \u0026micro;M further elevated early apoptosis to 48.6% and late apoptosis to 18.7%, with a slight rise in necrosis (4.5%), producing a total cell death of 71.8%. At the highest tested concentration (20 \u0026micro;M), \u0026beta;-lapachone induced a profound cytotoxic effect, with early apoptosis reaching 61.2%, late apoptosis 28.5%, and necrosis 5.8%, culminating in a near-complete total cell death of 95.5%. These results clearly demonstrate that \u0026beta;-lapachone triggers apoptosis predominantly, with necrosis contributing minimally, and that its cytotoxic effect on cells is both concentration-dependent and primarily apoptotic in nature. The flow cytometry quadrant plots display the effect of \u0026beta;-lapachone treatment on cell viability, apoptosis, and necrosis, as measured by Annexin V-FITC and Propidium Iodide (PI) staining shown in \u003cstrong\u003eFigure 13\u003c/strong\u003e\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eFlow cytometry quadrant plots illustrate the effect of \u0026beta;-lapachone at doses of 0 (Control), 2, 5, 10, and 20 \u0026micro;M. Annexin V-FITC and Propidium Iodide (PI) staining are used to identify viable, apoptotic, and necrotic populations.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eEach quadrant represents a distinct cell population: Lower left (Annexin V\u003csup\u003e-\u003c/sup\u003e/PI\u003csup\u003e-\u003c/sup\u003e): Viable (live) cells, Lower right (Annexin V\u003csup\u003e+\u003c/sup\u003e/PI\u003csup\u003e-\u003c/sup\u003e): Early apoptotic cells, Upper right (Annexin V\u003csup\u003e+\u003c/sup\u003e/PI\u003csup\u003e+\u003c/sup\u003e): Late apoptotic or secondary necrotic cells and Upper left (Annexin V\u003csup\u003e-\u003c/sup\u003e/PI\u003csup\u003e+\u003c/sup\u003e): Necrotic cells.\u003c/p\u003e\n\u003cp style=\"text-align: left;\"\u003eIn the control group (0 \u0026micro;M \u0026beta;-lapachone), the vast majority of cells (95.7%) remained viable, with only minimal early apoptosis (2.1%), late apoptosis (1.2%), and necrosis (1.0%), indicating excellent baseline cell health. Upon treatment with 2 \u0026micro;M \u0026beta;-lapachone, there was a noticeable increase in early apoptosis (15.5%) and late apoptosis (5.4%), with viability dropping to 76.6%. This early shift toward apoptosis shows the initial cytotoxic effect of the compound at a low concentration. At 5 \u0026micro;M, apoptosis became more pronounced, with early apoptosis rising to 32.8% and late apoptosis to 10.1%, reducing viable cells to 54.1%. A minor increase in necrosis (3.0%) was also observed. Treatment with 10 \u0026micro;M \u0026beta;-lapachone caused a dramatic shift: only 28.2% of cells remained viable, while early apoptosis reached 48.6% and late apoptosis 18.7%. Necrosis slightly increased to 4.5%, indicating that \u0026beta;-lapachone at this concentration predominantly induces apoptosis rather than direct necrotic death. At the highest concentration, 20 \u0026micro;M, the majority of cells underwent apoptosis: early apoptosis was 61.2%, late apoptosis was 28.5%, and necrosis was slightly elevated at 5.8%. Only 4.5% of cells remained viable, indicating almost complete cell death primarily through the apoptotic pathway. Overall, these results clearly demonstrate a dose-dependent increase in apoptosis, with \u0026beta;-lapachone inducing both early and late apoptosis efficiently at higher concentrations, while necrosis remains relatively low even at 20 \u0026micro;M. This highlights \u0026beta;-lapachone\u0026rsquo;s strong apoptotic-inducing potential with minimal necrotic effects.\u003c/p\u003e\n\u003cp style=\"text-align: left;\"\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003cstrong\u003e: Effect of \u0026beta;-lapachone on early apoptosis, late apoptosis, necrosis, and total cell death percentages as determined by flow cytometry analysis.\u003c/strong\u003e Cells were treated with increasing concentrations of \u0026beta;-lapachone (0\u0026ndash;20 \u0026micro;M) for 24 hours. Data are presented as mean \u0026plusmn; standard deviation (SD) from three independent experiments.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cstrong\u003e\u003cspan dir=\"LTR\"\u003eConcentration of \u0026beta;-lapachone (\u0026micro;M)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cstrong\u003e\u003cspan dir=\"LTR\"\u003eEarly Apoptosis (%)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cstrong\u003e\u003cspan dir=\"LTR\"\u003eLate Apoptosis (%)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cstrong\u003e\u003cspan dir=\"LTR\"\u003eNecrosis (%)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cstrong\u003e\u003cspan dir=\"LTR\"\u003eTotal Cell Death (%)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e0 (Control)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e2.1 \u0026plusmn; 0.3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e1.2 \u0026plusmn; 0.2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e1.0 \u0026plusmn; 0.2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e4.3 \u0026plusmn; 0.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e15.5 \u0026plusmn; 1.2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e5.4 \u0026plusmn; 0.8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e2.5 \u0026plusmn; 0.6\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e23.4 \u0026plusmn; 1.8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e32.8 \u0026plusmn; 2.0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e10.1 \u0026plusmn; 1.1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e3.0 \u0026plusmn; 0.7\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e45.9 \u0026plusmn; 2.4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e10\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e48.6 \u0026plusmn; 2.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e18.7 \u0026plusmn; 1.8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e4.5 \u0026plusmn; 0.8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e71.8 \u0026plusmn; 3.2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e20\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e61.2 \u0026plusmn; 3.1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e28.5 \u0026plusmn; 2.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e5.8 \u0026plusmn; 0.9\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e95.5 \u0026plusmn; 4.0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003ch3 id=\"_Toc198500308\" style=\"text-align: left;\"\u003e\u003cstrong\u003eCell Cycle Arrest\u003c/strong\u003e\u003c/h3\u003e\n\u003cp style=\"text-align: left;\"\u003eFlow cytometry analysis based on DNA content (propidium iodide staining) revealed that \u0026beta;-lapachone induces a dose-dependent S-phase arrest in treated cells. On the X-axis, DNA content is displayed, with 2N DNA content corresponding to the G0/G1 phase (resting or initial growth phase), DNA content between 2N and 4N representing the S-phase (where DNA synthesis and replication occur), and 4N DNA content corresponding to the G2/M phase (mitotic preparation). The Y-axis represents the number of cells detected at each DNA content level. In the control group, cells exhibited a normal distribution, characterized by a strong G0/G1 peak around 2N, a moderate S-phase spread, and a clear G2/M peak around 4N. Upon treatment with 2 \u0026micro;M \u0026beta;-lapachone, a slight reduction in the G0/G1 peak was observed along with a minor increase in the S-phase shoulder, indicating early signs of S-phase accumulation. At 5 \u0026micro;M, the G0/G1 peak further decreased, and the S-phase region broadened, suggesting more cells were arrested during DNA synthesis. With 10 \u0026micro;M \u0026beta;-lapachone, a significant S-phase accumulation was evident around 3.5 PI intensity, accompanied by a notable reduction in the G0/G1 population. The effect was most pronounced at 20 \u0026micro;M, where the S-phase peak became broad and dominant, and the G0/G1 peak was minimal, indicating that the majority of the cells were blocked in S-phase. Throughout the increasing concentrations, the G2/M peak remained relatively small and stable, confirming that cells were not progressing into mitosis \u003cstrong\u003eTable 2\u003c/strong\u003e. Flow cytometry was used to analyze the cell cycle distribution after 24 hours of treatment with \u0026beta;-LP at various concentrations (0 (Control), 2, 5, 10, and 20 \u0026micro;M) using propidium iodide (PI). Based on DNA content, cells were divided into three phases: G0/G1, S, and G2/M. and \u003cstrong\u003eFigure 14\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e Cell cycle analysis histograms demonstrate DNA content (PI intensity) of cells treated with increasing concentrations of \u0026beta;-lapachone (0 (Control), 2, 5, 10, and 20 \u0026micro;M) during 24 hours. The peaks indicate the distribution of G0/G1, S, and G2/M phases, as measured by flow cytometry. These observations suggest that \u0026beta;-lapachone blocks cell cycle progression by arresting cells in the S-phase, likely through inhibition of DNA replication or replication fork stalling mechanisms. Overall, the study demonstrated that \u0026beta;-lapachone treatment led to a progressive and concentration-dependent S-phase arrest, with the S-phase population increasing from approximately 29.1% in the control to 50.1% at 20 \u0026micro;M, while the G0/G1 population decreased correspondingly.\u003c/p\u003e\n\u003cp style=\"text-align: left;\"\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e2:\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFlow cytometry was used to analyze the cell cycle distribution after 24 hours of treatment with \u0026beta;-LP at various concentrations (0 (Control), 2, 5, 10, and 20 \u0026micro;M) using propidium iodide (PI).\u0026nbsp;\u003c/strong\u003eBased on DNA content, cells were divided into three phases: G0/G1, S, and G2/M.\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"519\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cstrong\u003e\u003cspan dir=\"LTR\"\u003eConcentration\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cstrong\u003e\u003cspan dir=\"LTR\"\u003eG0/G1 Phase (%)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cstrong\u003e\u003cspan dir=\"LTR\"\u003eS Phase (%)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cstrong\u003e\u003cspan dir=\"LTR\"\u003eG2/M Phase (%)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003eControl\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e53.5 \u0026plusmn; 2.0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e29.1 \u0026plusmn; 1.7\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e17.4 \u0026plusmn; 1.3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e2 \u0026micro;M\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e50.2 \u0026plusmn; 2.1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e32.8 \u0026plusmn; 1.8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e17.0 \u0026plusmn; 1.4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e5 \u0026micro;M\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e45.8 \u0026plusmn; 2.4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e37.5 \u0026plusmn; 2.1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e16.7 \u0026plusmn; 1.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e10 \u0026micro;M\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e38.2 \u0026plusmn; 2.6\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e44.0 \u0026plusmn; 2.3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e17.8 \u0026plusmn; 1.6\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e20 \u0026micro;M\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e32.4 \u0026plusmn; 2.8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e50.1 \u0026plusmn; 2.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003e17.5 \u0026plusmn; 1.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp style=\"text-align: left;\"\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch2 id=\"_Toc198500309\" style=\"text-align: left;\"\u003e\u003cstrong\u003e\u003cem\u003eIn-Silico\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Study\u003c/strong\u003e\u003c/h2\u003e\n\u003cp style=\"text-align: left;\"\u003eThe docking study evaluated the binding affinities and molecular interactions of Beta Lapachone with multiple target proteins from various organisms. Docking scores and detailed molecular interactions are presented in Table 3 along with corresponding figures.\u003c/p\u003e\n\u003cp style=\"text-align: left;\"\u003e\u003cstrong\u003eTable 3: Docking Score of Beta Lapachone with multiple target proteins\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"text-align: left;\"\u003e\u0026nbsp;\u003cimg src=\"https://myfiles.space/user_files/58895_8739fc6c57c1c19a/58895_custom_files/img1765287156.jpg\" alt=\"image\" width=\"570\" height=\"238\"\u003e\u003c/p\u003e\n\u003cp style=\"text-align: left;\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp style=\"text-align: left;\"\u003e\u003cstrong\u003eInteraction Analysis:\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eCandida albicans\u003c/em\u003e\u003c/strong\u003e (\u003cstrong\u003eCYP51 \u0026amp; DHFR) and Beta-Lapachone Complex\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"text-align: left;\"\u003eDocking analysis of Beta-Lapachone with \u003cem\u003eCandida albicans\u0026nbsp;\u003c/em\u003eCYP51 (PDB ID: 5TZ1) revealed multiple hydrophobic interactions stabilizing the ligand within the active site. A key \u0026pi;\u0026ndash;\u0026pi; T-shaped interaction was observed between the aromatic ring of Beta-Lapachone and PHE228 (4.92 \u0026Aring;), enhancing binding via aromatic stacking. Additional pi\u0026ndash;alkyl contacts with TYR118 (4.59 \u0026Aring;), LEU121 (3.97 \u0026Aring;), PHE233 (4.96 \u0026Aring;), LEU376 (5.38 \u0026Aring;), and MET508 (5.06 \u0026Aring;) contribute to a non-polar binding pocket, facilitating van der Waals stabilization. These interactions underscore the role of hydrophobic complementarity in ligand accommodation and support Beta-Lapachone\u0026rsquo;s potential as a CYP51 inhibitor targeting ergosterol biosynthesis \u003cstrong\u003e(Figure 15A\u003c/strong\u003e).\u003c/p\u003e\n\u003cp style=\"text-align: left;\"\u003eDocking of Beta-Lapachone with \u003cem\u003eCandida albicans\u003c/em\u003e DHFR (PDB ID: 1AI9) revealed a robust interaction network involving both polar and hydrophobic contacts. ALA11 formed two strong hydrogen bonds (1.82 \u0026Aring; and 2.74 \u0026Aring;), alongside a pi\u0026ndash;alkyl interaction (5.30 \u0026Aring;), anchoring the ligand within the active site. MET25 contributed additional stabilization via two pi\u0026ndash;alkyl contacts (4.60 \u0026Aring; and 5.30 \u0026Aring;), a pi\u0026ndash;sigma interaction (3.69 \u0026Aring;), and a unique pi\u0026ndash;sulfur interaction (3.83 \u0026Aring;), reflecting diverse van der Waals and soft acid\u0026ndash;base interactions. This combination of directional hydrogen bonds and multipoint hydrophobic contacts underscores Beta-Lapachone\u0026rsquo;s high binding affinity and supports its potential to inhibit DHFR-mediated folate biosynthesis in \u003cem\u003eC. albicans\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e(Figure 15B).\u003c/strong\u003e\u003c/p\u003e\n\u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cstrong\u003e\u003cspan dir=\"LTR\"\u003eInteraction Analysis: \u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan dir=\"LTR\"\u003eMRSA (PBP2a \u0026amp; Sortase A) and \u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan dir=\"LTR\"\u003eBeta-Lapachone Complex\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003eDocking of Beta-Lapachone with \u003cem\u003eMRSA\u003c/em\u003e PBP2a (PDB ID: 1VQQ) revealed a strong interaction network comprising both polar and hydrophobic contacts. TYR441 formed two conventional hydrogen bonds (1.96 \u0026Aring; and 2.25 \u0026Aring;), and GLU602 contributed an additional hydrogen bond (2.28 \u0026Aring;), anchoring the ligand via strong electrostatic interactions. TYR519 engaged in a \u0026pi;\u0026ndash;\u0026pi; T-shaped interaction (5.36 \u0026Aring;) and a \u0026pi;\u0026ndash;alkyl contact (5.54 \u0026Aring;), while ALA601 formed three \u0026pi;\u0026ndash;alkyl interactions (3.95 \u0026Aring;, 4.43 \u0026Aring;, and 5.45 \u0026Aring;), reflecting extensive hydrophobic complementarity. This diverse interaction profile supports the stable accommodation of Beta-Lapachone within the PBP2a active site and highlights its potential as an effective inhibitor against \u0026beta;-lactam resistance in MRSA \u003cstrong\u003e(Figure 16A)\u003c/strong\u003e.\u003c/span\u003e\u003c/p\u003e\n\u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003eDocking of Beta-Lapachone with \u003cem\u003eMRSA\u003c/em\u003e Sortase A (PDB ID: 1T2P) revealed a robust interaction network involving both polar and hydrophobic contacts. Conventional hydrogen bonds were formed with ASN114 (1.78 \u0026Aring;), SER116 (2.53 \u0026Aring;), and ARG197 (2.76 \u0026Aring; and 2.88 \u0026Aring;), anchoring the ligand via strong polar interactions. Hydrophobic stabilization was provided by \u0026pi;\u0026ndash;alkyl interactions with PRO163 (5.05 \u0026Aring;) and ILE199 (4.95 \u0026Aring;), along with \u0026pi;\u0026ndash;sigma interactions involving THR180 (4.00 \u0026Aring;) and ILE199 (3.94 \u0026Aring;). A \u0026pi;\u0026ndash;donor hydrogen bond with VAL168 (2.91 \u0026Aring;) further contributed to binding specificity. This diverse interaction profile suggests that Beta-Lapachone is well-accommodated within the SrtA active site and may effectively inhibit its function, highlighting its potential as an anti-virulence therapeutic against MRSA \u003cstrong\u003e(Figure 16B)\u003c/strong\u003e.\u003c/span\u003e\u003cspan dir=\"LTR\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cstrong\u003e\u003cspan dir=\"LTR\"\u003eInteraction Analysis: \u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u003cspan dir=\"LTR\"\u003eMycobacterium smegmatis\u003c/span\u003e\u003c/em\u003e\u003c/strong\u003e\u003cspan dir=\"LTR\"\u003e\u0026nbsp;(\u003cstrong\u003eCysteine Desulfurase \u0026amp; InhA) and Beta-Lapachone Complex\u003c/strong\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003eDocking of Beta-Lapachone with \u003cem\u003eMycobacterium smegmatis\u003c/em\u003e cysteine desulfurase (PDB ID: 8KG1) revealed a robust interaction profile comprising both polar and hydrophobic contacts. ARG532 forms two strong hydrogen bonds (1.89 \u0026Aring; and 2.55 \u0026Aring;), and ASN328 contributes an additional hydrogen bond (2.42 \u0026Aring;), anchoring the ligand through directional electrostatic interactions. Hydrophobic stabilization is driven by ALA184, which engages in four \u0026pi;\u0026ndash;alkyl interactions (4.01 \u0026Aring;, 4.19 \u0026Aring;, 4.71 \u0026Aring;, and 4.87 \u0026Aring;), and ARG512, which forms a \u0026pi;\u0026ndash;alkyl contact at 4.57 \u0026Aring;. This combination of hydrogen bonding and extensive van der Waals forces supports a stable and high-affinity binding conformation for Beta-Lapachone, suggesting its potential to inhibit cysteine desulfurase activity and interfere with sulfur metabolism in \u003cem\u003eM. smegmatis\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e(Figure 17A).\u003c/strong\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003eMolecular docking of Beta-Lapachone with \u003cem\u003eMycobacterium smegmatis\u003c/em\u003e enoyl-acyl carrier protein reductase (InhA; PDB ID: 2NV6) revealed a highly stabilized binding conformation supported by both polar and hydrophobic interactions. A strong conventional hydrogen bond with GLY14 (2.46 \u0026Aring;) provides directional polar anchoring. Aromatic stabilization is mediated by two \u0026pi;\u0026ndash;\u0026pi; stacking interactions with PHE41 (3.89 \u0026Aring; and 4.82 \u0026Aring;), while \u0026pi;\u0026ndash;alkyl contacts with VAL65, ILE16, and ILE95 (4.39\u0026ndash;5.30 \u0026Aring;) enhance hydrophobic compatibility. Additionally, \u0026pi;\u0026ndash;sigma interactions with ILE95 (3.71 \u0026Aring;, 3.92 \u0026Aring;) and ILE122 (3.93 \u0026Aring;) contribute further orbital overlap and van der Waals stabilization. This diverse interaction profile suggests that Beta-Lapachone effectively occupies the InhA active site, with the potential to inhibit mycolic acid biosynthesis and exert antimycobacterial activity against \u003cem\u003eM. smegmatis\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e(Figure 17B)\u003c/strong\u003e.\u003c/span\u003e\u003c/p\u003e\n\u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cstrong\u003e\u003cspan dir=\"LTR\"\u003eInteraction Analysis: \u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u003cspan dir=\"LTR\"\u003ePseudomonas aeruginosa\u003c/span\u003e\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan dir=\"LTR\"\u003e (LasR \u0026amp; OprF) and \u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan dir=\"LTR\"\u003eBeta-Lapachone Complex\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003eDocking of Beta-Lapachone with the LasR transcriptional regulator of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (PDB ID: 3IX3) reveals a robust and multifaceted interaction network. Two strong conventional hydrogen bonds with ARG61 (1.99 \u0026Aring;, 2.77 \u0026Aring;) provide electrostatic anchoring. A \u0026pi;\u0026ndash;anion interaction with ASP73 (3.94 \u0026Aring;) contributes additional charge-based stabilization. Aromatic interactions include two \u0026pi;\u0026ndash;\u0026pi; stacking contacts with TYR64 (4.91 \u0026Aring;, 5.52 \u0026Aring;), reinforcing ligand orientation.\u003c/span\u003e\u003c/p\u003e\n\u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003eExtensive hydrophobic interactions further stabilize the complex: LEU36 forms both \u0026pi;\u0026ndash;alkyl (4.67 \u0026Aring;) and \u0026pi;\u0026ndash;sigma (3.87 \u0026Aring;) interactions; TYR47 (5.25 \u0026Aring;), ALA50 (5.32 \u0026Aring;), and VAL76 (3.96\u0026ndash;5.33 \u0026Aring;) contribute additional \u0026pi;\u0026ndash;alkyl contacts; and ALA127 engages in four \u0026pi;\u0026ndash;alkyl interactions (3.77\u0026ndash;5.04 \u0026Aring;), supporting van der Waals compatibility. This rich interaction profile positions Beta-Lapachone securely within the LasR binding pocket, suggesting its potential to inhibit quorum sensing, impair virulence factor expression, and reduce biofilm formation, thus making it a promising antivirulence agent against \u003cem\u003eP. aeruginosa\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e(Figure 18A).\u003c/strong\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003eDocking of Beta-Lapachone with the outer membrane protein OprF of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (PDB ID: 4RLC) reveals a robust interaction network involving electrostatic, aromatic, hydrophobic, and hydrogen bonding contacts. Central to the stabilization are multiple \u0026pi;\u0026ndash;anion interactions: LYS13 (3.28 \u0026Aring;, 3.59 \u0026Aring;), ASP72 (3.43 \u0026Aring;), and ASP134 (4.63 \u0026Aring;), anchoring the ligand via charge\u0026ndash;\u0026pi; electron attraction. A \u0026pi;\u0026ndash;\u0026pi; T-shaped interaction with PHE11 (5.49 \u0026Aring;) adds aromatic complementarity.\u003c/span\u003e\u003c/p\u003e\n\u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003eHydrophobic stabilization is achieved through alkyl contacts with ALA44 (3.01 \u0026Aring;, 5.15 \u0026Aring;) and ILE74 (5.18 \u0026Aring;, 5.27 \u0026Aring;), enhancing van der Waals compatibility. Two strong hydrogen bonds with SER46 (2.24 \u0026Aring;) and SER89 (2.59 \u0026Aring;) provide directional polar stabilization and aid in precise ligand positioning.\u003c/span\u003e\u003c/p\u003e\n\u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003eThis diverse interaction profile positions Beta-Lapachone securely within the OprF binding site, suggesting potential to disrupt porin-mediated transport or membrane integrity and highlighting its promise as an antimicrobial agent targeting \u003cem\u003eP. aeruginosa\u0026nbsp;\u003c/em\u003e(\u003cstrong\u003e(Figure 18B\u003c/strong\u003e).\u003c/span\u003e\u003c/p\u003e\n\u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cstrong\u003e\u003cspan dir=\"LTR\"\u003eInteraction Analysis: \u003cem\u003eStaphylococcus aureus\u0026nbsp;\u003c/em\u003e\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan dir=\"LTR\"\u003e(\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan dir=\"LTR\"\u003e\u0026alpha;-hemolysin\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan dir=\"LTR\"\u003e\u0026nbsp;\u0026amp; TarO) and\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan dir=\"LTR\"\u003eBeta-Lapachone Complex\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003eDocking analysis of Beta-Lapachone with \u0026alpha;-hemolysin (PDB ID: 7AHL) from \u003cem\u003eStaphylococcus aureus\u003c/em\u003e reveals a triad of stabilizing interactions that contribute to a strong and specific ligand fit. A key conventional hydrogen bond is formed with LYS21 (2.51 \u0026Aring;), providing directional polar anchoring. LYS21 also engages in a carbon\u0026ndash;hydrogen bond (3.35 \u0026Aring;), adding complementary electrostatic stabilization. Additionally, a \u0026pi;\u0026ndash;anion interaction with GLU289 (3.82 \u0026Aring;) reinforces ligand binding through attraction between the aromatic \u0026pi;-system and the negatively charged carboxylate side chain. Together, these interactions suggest a robust and energetically favorable binding mode for Beta-Lapachone within the AHL cavity, highlighting its potential to inhibit the pore-forming activity of \u0026alpha;-hemolysin and function as an antivirulence agent against \u003cem\u003eS. aureus\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e(Figure 19A)\u003c/strong\u003e.\u003c/span\u003e\u003c/p\u003e\n\u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003eMolecular docking of Beta-Lapachone with the TarO protein of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (PDB ID: 5EZM) reveals a highly stabilized binding mode dominated by \u0026pi;-system-mediated interactions. Two key \u0026pi;\u0026ndash;anion interactions with ASP55 (3.65 \u0026Aring;, 4.53 \u0026Aring;) anchor the ligand through electrostatic attraction between the aspartate\u0026rsquo;s carboxylate group and the aromatic \u0026pi;-electrons of the ligand.\u003c/span\u003e\u003c/p\u003e\n\u003cp dir=\"RTL\" style=\"text-align: left;\"\u003e\u003cspan dir=\"LTR\"\u003eA dense hydrophobic network reinforces binding affinity through multiple \u0026pi;\u0026ndash;alkyl interactions involving ALA151 (4.12 \u0026Aring;, 4.59 \u0026Aring;), PHE154 (4.88 \u0026Aring;), LEU342 (5.31 \u0026Aring;), PRO343 (4.13 \u0026Aring;, 4.86 \u0026Aring;), and VAL347 (4.31 \u0026Aring;, 4.94 \u0026Aring;). Notably, PHE154 also engages in a \u0026pi;\u0026ndash;\u0026pi; stacking interaction (4.83 \u0026Aring;), enhancing aromatic complementarity within the cavity.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"text-align: left;\"\u003eThis rich profile of electrostatic and hydrophobic contacts supports a robust and energetically favorable conformation for Beta-Lapachone, suggesting its potential to inhibit TarO and disrupt wall teichoic acid biosynthesis, a critical pathway in \u003cem\u003eS. aureus\u003c/em\u003e cell wall integrity and virulence \u003cstrong\u003e(Figure 19B).\u003c/strong\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe prevalence of multidrug-resistant (MDR) microorganisms has become a major worldwide health concern, decreasing the efficacy of many traditional antibiotics and requiring the urgent development of alternative therapeutic options. This study evaluated the antimicrobial and antifungal effects of β-LP against clinically important MDR organisms. A range of microbiological and biochemical studies were conducted to examine their antimicrobial efficacy and the underlying mechanisms of action. The results showed that β-LP had consistent antimicrobial action across many experiments. β-LP demonstrated broad-spectrum antibacterial and antifungal action, with large inhibition zones and low MIC/MBC values, especially against MRSA, \u003cem\u003eS. aureus\u003c/em\u003e, and \u003cem\u003eC. albicans\u003c/em\u003e. Previous research has shown that β-LP causes intracellular reactive oxygen species (ROS), resulting in redox disorder, membrane damage, and reduced cellular metabolism (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). In CFU assays, β-LP significantly decreased viable bacterial and fungal populations, with growth almost eliminated in numerous treatment groups. Growth kinetic assay confirmed these findings by demonstrating dose-dependent inhibition of growth over 20 hour, emphasizing the compounds\u0026rsquo; concentration- and time-dependent death properties. These findings were similar with prior research Mir et al., 2023 (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), indicating that they had effective bactericidal and fungicidal capabilities. β-LP demonstrated antibiofilm action in all strains studied, with β-LP reducing biofilm formation by more than 70% in most species. This is a significant feature since biofilm-associated cells are known to have increased resistance to antimicrobial treatments.\u003c/p\u003e\u003cp\u003eβ-LP also significantly inhibited catalase function in treated bacteria and fungus, indicating oxidative stress as a key mechanism of action. Catalase inhibition by β-LP is consistent with previous work demonstrating its potential to interfere with antioxidant defenses, therefore enhancing ROS the formation and cell death (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Cytoplasmic leakage experiments provided additional confirmation of membrane damage. As a result of this data, it seems that β-LP promotes intracellular oxidative collapse. β-LP has powerful anticancer effects on breast cancer cells in a dose-dependent manner, as demonstrated by MTT viability assays and flow cytometry. β-LP drastically reduced cell viability while inducing apoptosis with minimal necrosis, indicating that apoptosis is the predominant mechanism of cytotoxicity. Flow cytometry analysis showed a gradual increase in early and late apoptotic populations with increasing concentrations of β-LP, reaching 95.5% and 89.4% total cell death, respectively, at the maximum tested dose (20 \u0026micro;M). Calahorra et al. (2024) Click or tap here to enter text.found that β-LP improved apoptotic activity in triple-negative breast cancer (TNBC) cell lines, especially when combined with hydroxytyrosol (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Although our study did not include combination therapy, β-LP 's individual efficacy is consistent with their findings, showing its pro-apoptotic capabilities in breast cancer models. Furthermore, β-lapachone significantly disrupted the cell cycle. Our study found that β-lapachone administration resulted in a concentration-dependent S-phase arrest. The S-phase population increased from 29.1% in the control group to 50.1% at 20 \u0026micro;M, whereas the G0/G1 population decreased. Previous research on colon and breast cancer found that β-lapachone impaired DNA replication (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Comparative literature backs up our findings. Recent research with oxaliplatin-resistant colorectal cancer cells (HCT116-OxPt-R) revealed that β-lapachone induces more cytotoxicity in resistant cells than in non-resistant cells. These findings highlight β-lapachone's potential for treating sensitive cancer types and overcoming chemoresistance by inducing multi-targeted apoptosis (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). These findings support the therapeutic benefits of β-lapachone in cancer treatment. Their capacity to cause apoptosis at different cell cycle arrest points complementary mechanisms that could be investigated in combination treatments. Future studies should optimize the use of β-lapachone in resistant or aggressive cancer types by analyzing synergistic combinations and biological pathways. β-LP consistently exhibited lower docking scores, indicating stronger interactions with target proteins. Notably, β-LP showed exceptional binding to dihydrofolate reductase of \u003cem\u003eCandida albicans\u003c/em\u003e and PBP2a of MRSA, both critical targets for antifungal and antibacterial treatments.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe study emphasizes the therapeutic potential of β-lapachone (β-LP) as multiple-function compound having antimicrobial and anticancer properties. The compound was highly effective in inhibiting multidrug-resistant microbes, disrupting biofilms, and inducing oxidative stress in vitro. These findings are especially important in light of developing antimicrobial resistance, which remains one of the most serious global health challenges. It is noteworthy that anticancer evaluations of β-LP against breast cancer cells have shown promising results. The in-silico docking research confirmed these findings by demonstrating the compounds' high binding affinities to important microbial and cellular targets, confirming their proposed modes of action.\u003c/p\u003e\u003cp\u003eNevertheless, it is imperative to assess the potential toxicity, pharmacokinetic properties, and side effects of the compounds. The study results suggest that β-LP could be used as future candidate drugs for the treatment of microbial diseases and breast cancer and might be recommended to biomedical and pharmaceutical applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors express their gratitude to the Deanship of Research and Graduate Studies at King Khalid University for financial support.\u0026nbsp;\u003c/p\u003e\n\u003cp dir=\"\"\u003e\u003cstrong\u003e\u003cspan dir=\"\"\u003eAuthor Contributions\u003c/span\u003e\u003c/strong\u003e\u003cspan dir=\"\"\u003e:\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp dir=\"\"\u003e\u003cspan dir=\"\"\u003eRAA, IA, YA: Concept, Data Collection, Writing Manuscript\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eIA, SOR, AD, MSA: Data Analysis\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eRAA, AKM, MS: Writing Manuscript\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eMAA, MYQ, MMAS, MS: Data Curation, Project Management\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eIA, AD, YA, MAA: Concept\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eIA: Funding. All authors reviewed the manuscript.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors express their gratitude to the Deanship of Research and Graduate Studies at King Khalid University for funding this work through the Large Research Group Project under grant number RGP.02/503/46 and the authors would like to express their thanks to Researchers supporting project number (ORF-2025_1326) King Saud University, Riyadh, Saudi Arabia.\u003c/p\u003e\n\u003cp dir=\"\"\u003e\u003cstrong\u003e\u003cspan dir=\"\"\u003eCompeting Interest:\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp dir=\"\"\u003e\u003cspan dir=\"\"\u003eThe authors declare no competing interests.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp dir=\"\"\u003e\u003cstrong\u003e\u003cspan dir=\"\"\u003eData Availability:\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp dir=\"\"\u003e\u003cspan dir=\"\"\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAlmutairy B. Extensively and multidrug-resistant bacterial strains: case studies of antibiotics resistance. Vol. 15, Frontiers in Microbiology. Frontiers Media SA; 2024.\u003c/li\u003e\n \u003cli\u003eMurray CJ, Ikuta KS, Sharara F, Swetschinski L, Robles Aguilar G, Gray A, et al. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. The Lancet. 2022 Feb 12;399(10325):629\u0026ndash;55.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eLee Ventola C. The Antibiotic Resistance Crisis Part 1: Causes and Threats. Vol. 40. 2015.\u003c/li\u003e\n \u003cli\u003eElmaidomy AH, Shady NH, Abdeljawad KM, Elzamkan MB, Helmy HH, Tarshan EA, et al. Antimicrobial potentials of natural products against multidrug resistance pathogens: a comprehensive review. Vol. 12, RSC Advances. Royal Society of Chemistry; 2022. p. 29078\u0026ndash;102.\u003c/li\u003e\n \u003cli\u003eMurugaiyan J, Anand Kumar P, Rao GS, Iskandar K, Hawser S, Hays JP, et al. Progress in Alternative Strategies to Combat Antimicrobial Resistance: Focus on Antibiotics. Vol. 11, Antibiotics. MDPI; 2022.\u003c/li\u003e\n \u003cli\u003eTackling Drug-Resistant Infections Globally: Final Report And Recommendations The Review On Antimicrobial Resistance Chaired By Jim O\u0026rsquo;neill. 2016.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eLiu X, Zhang Z, Tomczak N, Yang C, Li C, Liu R, et al. A bioinspired silica nanocomposite for enhanced multidrug-resistant bacteria treatment and wash-free imaging. Nanoscale Adv. 2023 Feb 20;5(6):1631\u0026ndash;5.\u003c/li\u003e\n \u003cli\u003eOrhan IE. Pharmacognosy: Science of natural products in drug discovery. BioImpacts. 2014;4(3):109\u0026ndash;10.\u003c/li\u003e\n \u003cli\u003eJamshidi-Kia F, Lorigooini Z, Amini-Khoei H. Medicinal plants: Past history and future perspective. Vol. 7, Journal of HerbMed Pharmacology. Nickan Research Institute; 2018. p. 1\u0026ndash;7.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eTerreni M, Taccani M, Pregnolato M. New antibiotics for multidrug-resistant bacterial strains: Latest research developments and future perspectives. Vol. 26, Molecules. MDPI AG; 2021.\u003c/li\u003e\n \u003cli\u003eBorges A, Abreu AC, Dias C, Saavedra MJ, Borges F, Sim\u0026otilde;es M. New perspectives on the use of phytochemicals as an emergent strategy to control bacterial infections including biofilms. Vol. 21, Molecules. MDPI AG; 2016.\u003c/li\u003e\n \u003cli\u003eMagaldi S, S. Mata-Essayag, C. Hartung de Capriles, C. Perez, M. T. Colella, Carolina Olaizola, et al.\u0026nbsp;Well diffusion for antifungal susceptibility testing. \u003cem\u003eInt J Infect Dis\u0026nbsp;\u003c/em\u003e2004; 8: 39-45.\u003c/li\u003e\n \u003cli\u003eFazly A, Charu Jain, Amie C. Dehner, Luca Issi, Elizabeth A. Lilly, Akbar Ali, et al. Chemical screening identifies filastatin, a small molecule inhibitor of candida albicans adhesion, morphogenesis, and pathogenesis. \u003cem\u003eProc Natl Acad Sci U S A\u0026nbsp;\u003c/em\u003e2013; 110: 13594-13599.\u003c/li\u003e\n \u003cli\u003eRaut JS, Ravikumar B. Shinde, Nitin M. Chauhan and S. Mohan Karuppayil. Terpenoids of plant origin inhibit morphogenesis, adhesion, and biofilm formation by candida albicans. \u003cem\u003eBiofouling\u0026nbsp;\u003c/em\u003e2013; 29: 87-96.\u003c/li\u003e\n \u003cli\u003eJin Y, Lakshman P. Samaranayake, Yuthika Samaranayake and Hak Kong Yip. Biofilm formation of candida albicans is variably affected by saliva and dietary sugars. \u003cem\u003eArch Oral Biol\u0026nbsp;\u003c/em\u003e2004; 49: 789-798.\u003c/li\u003e\n \u003cli\u003eLeekha S, Christine L. Terrell and Randall S. Edson. General principles of antimicrobial therapy. \u003cem\u003eMayo Clin Proc\u0026nbsp;\u003c/em\u003e2011; 86: 156-167.\u003c/li\u003e\n \u003cli\u003eSultan Z. Alasmari, Mohammed H. Makkawi, Irfan Ahmad, Abdulrahim R. Hakami, Abdulrahman A. Almehizia, Adel S. El-Azab, Alaa A.-M. Abdel-Aziz, Mohammed Ghazwani.\u003c/li\u003e\n \u003cli\u003eAntibacterial evaluation of 2-(6-Chloro-2-p-tolylquinazolin-4-ylthio) acetonitrile against pathogenic bacterial isolates with special reference to biofilm formation inhibition and anti-adherence properties. Journal of King Saud University-Science. 2024;36(8):103316.\u003c/li\u003e\n \u003cli\u003eShalini V, Shanmugam R, Manigandan P. Cytoplasmic Leakage and Protein Leakage Analysis of \u003cem\u003eOcimum Gratissimum\u003c/em\u003e Stem Extract-Mediated Silver Nanoparticles Against Wound Pathogens. J Pharm Bioallied Sci. 2024 Apr;16(Suppl 2):S1354-S1359.\u003c/li\u003e\n \u003cli\u003eGoel A, Prasad AK, Parmar VS, Ghosh B and Saini N: Apoptogenic effect of 7,8-diacetoxy-4-methylcoumarin and 7,8- diacetoxy-4-methylthiocoumarin in human lung adenocarcinoma cell line: Role of nf-kappab, akt, ros and map kinase pathway. Chem Biol Interact 179(2-3): 363-374, 2009.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eRaha P, Thomas S, Thurn KT, Park J and Munster PN: Combined histone deacetylase inhibition and tamoxifen induces apoptosis in tamoxifen-resistant breast cancer models, by reversing bcl-2 overexpression. Breast Cancer Res 17(26): 015-0533, 2015. PMID: 25848915.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMoraes DC, Curvelo JAR, Anjos CA, Moura KCG, Pinto MCFR, Portela MB, et al. \u0026beta;-lapachone and \u0026alpha;-nor-lapachone modulate Candida albicans viability and virulence factors. J Mycol Med. 2018 Jun 1;28(2):314\u0026ndash;9.\u003c/li\u003e\n \u003cli\u003eFernandes, A.W.C.; Santos, V.L.D.A.; Ara\u0026uacute;jo, C.R.M.; de Oliveira, H.P.; da Costa, M.M. Anti-biofilm Effect of \u0026beta;-Lapachone and Lapachol Oxime Against Isolates of Staphylococcus aureus. Curr. Microbiol. 2020, 77, 204\u0026ndash;209.\u003c/li\u003e\n \u003cli\u003eZhao W, Jiang L, Fang T, Fang F, Liu Y, Zhao Y, et al. \u0026beta;-Lapachone Selectively Kills Hepatocellular Carcinoma Cells by Targeting NQO1 to Induce Extensive DNA Damage and PARP1 Hyperactivation. Front Oncol. 2021 Oct 5;11.\u003c/li\u003e\n \u003cli\u003eHuang L, Pardee AB. \u0026beta;-Lapachone induces cell cycle arrest and apoptosis in human colon cancer cells. Mol Med. 1999;5(11):711\u0026ndash;720.\u003c/li\u003e\n \u003cli\u003eJung EJ, Kim HJ, Shin SC, Kim GS, Jung JM, Hong SC, et al. \u0026beta;-Lapachone exerts anticancer effects by downregulating p53, Lys-acetylated proteins, TrkA, p38 MAPK, SOD1, caspase-2, CD44 and NPM in oxaliplatin-resistant HCT116 colorectal cancer cells. Int J Mol Sci. 2023;24(12):9867. doi:10.3390/ijms24129867.\u003c/li\u003e\n \u003cli\u003eMacedo, L.; Fernandes, T.; Silveira, L.; Mesquita, A.; Franchitti, A.; Ximenes, E. \u0026beta;-Lapachone activity in synergy with conventional antimicrobials against methicillin resistant Staphylococcus aureus strains. Phytomedicine 2013, 21, 25\u0026ndash;29.\u003c/li\u003e\n \u003cli\u003eLlor, C.; Bjerrum, L. Antimicrobial resistance: Risk associated with antibiotic overuse and initiatives to reduce the problem. Ther. Adv. Drug Saf. 2014, 5, 229\u0026ndash;241.\u0026nbsp;\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":"Beta lapachone, Antimicrobial, Anticancer, MDR pathogens, Breast cancer","lastPublishedDoi":"10.21203/rs.3.rs-7982878/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7982878/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe growing rise of multidrug-resistant (MDR) infections, as well as the worldwide prevalence of cancer, one of the major causes of mortality, are two of the most serious and significant issues facing modern medicine. This study evaluated the antimicrobial and anticancer properties of beta lapachone in vitro and in silico. Antimicrobial activity was assessed using established assays such as diffusion methods, MIC and MBC determinations, CFU reduction, biofilm inhibition, oxidative stress analysis, and membrane integrity disruption. The beta lapachone investigated showed strong antimicrobial properties, significantly decreasing microbial viability, preventing biofilm formation, and generating membrane damage and oxidative stress.\u003c/p\u003e\u003cp\u003eAnticancer activity was tested on breast cancer cell lines using MTT viability assays, Annexin V/PI flow cytometry, and cell cycle analysis. The beta lapachone had dose-dependent cytotoxic effects, with triggering S-phase arrest, which led to increase apoptosis.\u003c/p\u003e\u003cp\u003eMolecular docking investigations showed their affinity for important microbial and cellular targets, proving the hypothesized mechanisms of action. These findings emphasize the beta lapachone therapeutic promise as dual-function medicines capable of treating both MDR infections and malignant tumors, thereby tackling two of the most serious dangers to world health.\u003c/p\u003e","manuscriptTitle":"Deciphering the Therapeutic Promise of β-Lapachone against Pathogenic Multidrug Resistant Microbes and Breast Cancer Cell","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-09 18:50:50","doi":"10.21203/rs.3.rs-7982878/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":"5be07593-ae7d-4152-a0dc-43acaf775a6d","owner":[],"postedDate":"December 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":59221519,"name":"Biological sciences/Cancer"},{"id":59221520,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":59221521,"name":"Biological sciences/Drug discovery"},{"id":59221522,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2025-12-26T01:23:39+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-09 18:50:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7982878","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7982878","identity":"rs-7982878","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}