Synergistic Potential of Bis-Benzidinedioxime Palladium(II) and Benzidinedioxime as Enhancers of Gentamicin Efficacy Against Resistant Bacteria: Insights from Molecular Docking and Antimicrobial Studies

Synergistic Potential of Bis-Benzidinedioxime Palladium(II) and Benzidinedioxime as Enhancers of Gentamicin Efficacy Against Resistant Bacteria: Insights from Molecular Docking and Antimicrobial Studies | 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 Synergistic Potential of Bis-Benzidinedioxime Palladium(II) and Benzidinedioxime as Enhancers of Gentamicin Efficacy Against Resistant Bacteria: Insights from Molecular Docking and Antimicrobial Studies Reem M. A. Ebrahim, Nooh Mohamed Hajhamed, Abdallah Elssir Ahmed, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7216024/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 rising threat of antibiotic resistance necessitates innovative strategies to enhance the effectiveness of traditional antibiotics, such as aminoglycosides. This research explores the potential of bis-benzidinedioxime palladium(II) (Pd) and benzidinedioxime (L) as adjuvants to boost Gentamicin (G) activity against resistant strains of Staphylococcus aureus and Salmonella. typhi. Molecular docking using MOE2024 showed that Pd has stronger binding affinities (docking scores: -10.54 to -7.48 kcal/mol) across all aminoglycoside-modifying enzyme targets (1V0C, 3HAM, and 1KNY), especially with 1V0C, which exhibits the strongest interactions due to electrostatic contacts with aspartate and glutamate residues. Benzidinedioxime and Gentamicin had moderate affinities (-7.06 to -6.41 kcal/mol and − 8.33 to -7.6 kcal/mol, respectively). Antimicrobial tests using the cup plate method showed that Pd and L alone had no significant activity (inhibition zones of 0–14 mm), while Gentamicin was ineffective against both strains. However, combinations of G + L and G + Pd showed strong synergy, with inhibition zones of 23–30 mm and 18–24 mm, respectively, especially against S. typhimurium. These findings suggest that benzidinedioxime and bis-benzidinedioxime palladium(II) enhance gentamicin’s effectiveness by strongly inhibiting Aminoglycoside-N-Acetyltransferase-(6')-Ib [AAC(6')-Ib] (PDB: 1V0C) and Aminoglycoside-O-Phosphotransferase-(2'')-IIa [APH(2'')-IIa] (PDB ID: 3HAM). Additionally, bis-benzidinedioxime palladium(II) blocks Aminoglycoside-O-Phosphotransferase-(2'')-IIa [APH(2'')-IIa] without changing the structure of the palladium complex. These results highlight the potential of dinuclear palladium complexes and their ligands as adjuvants to fight against aminoglycoside resistance, laying the groundwork for further studies and the development of new therapies. Biological sciences/Biochemistry Biological sciences/Drug discovery Biological sciences/Microbiology Antibiotic resistance bis-benzidinedioxime palladium (II) benzidinedioxime Gentamicin molecular docking antimicrobial synergy Staphylococcus aureus Salmonella. typhi Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The global surge in bacterial infections caused by antimicrobial-resistant strains is one of the most significant public health phenomena endangering lives worldwide 1 – 4 . Compounding this crisis is the diminishing effectiveness of most essential antibiotics, such as aminoglycosides, which are commonly used to combat serious infections caused by both Gram-negative and Gram-positive pathogens. This growing vulnerability underscores the urgency to explore novel strategies to restore or enhance antibiotic efficacy of our existing antibiotics armory 4 – 11 . One promising approach involves using metal-based compounds, such as dinuclear complexes, as adjuvants to antibiotic therapy 12 – 15 . Dinuclear metal complexes consist of two metallic centers connected by bridging ligands, often exhibiting unique structural and chemical properties that make them potential antimicrobial agents or enhancers of antibiotic activity 16 – 20 . The synergistic effects between dinuclear complexes and aminoglycosides have been attributed to several mechanisms, including increased bacterial membrane permeability, enhanced binding to bacterial ribosomal targets, and inhibition of resistance enzymes that deactivate aminoglycosides 21 , 22 . Furthermore, the binding properties of specific dinuclear complexes allow them to target bacterial ribosomal sites synergistically with aminoglycosides. Aminoglycosides function by binding to the 30S ribosomal subunit, leading to errors in protein synthesis and bactericidal effects. Dinuclear complexes may stabilize these interactions or introduce additional structural distortions in the ribosomal architecture, thereby enhancing the antibiotic's effectiveness 23 . Another critical mechanism involves inhibiting aminoglycoside-modifying enzymes, such as acetyltransferases, phosphotransferases, and nucleotidyltransferases 24 – 26 . These enzymes are often responsible for bacterial resistance by chemically modifying aminoglycosides at specific positions and rendering them inactive 24 , 26 , 27 . Dinuclear complexes may interact directly with these resistance enzymes, inhibiting their function and preserving the antibiotic's activity 28 , 29 . Additionally, some complexes generate oxidative stress through the production of reactive oxygen species (ROS), which damages bacterial DNA, proteins, and membranes, thereby complementing the action of aminoglycosides 29 – 32 . Platinum-based dinuclear compounds like [Pt₂(phen)₄Cl₂] (phen = 1,10-phenanthroline) destabilize bacterial membranes and disrupt nucleic acid synthesis, exhibiting synergistic effects with aminoglycosides attributed to increased antibiotic uptake and ROS-mediated damage 33 , 34 . Similarly, copper dinuclear complexes coordinated with Schiff base ligands (e.g., Cu₂(bpy)₂(OH) ₂, bpy = 2,2′-bipyridine) demonstrate potent antimicrobial properties by disrupting membranes and promoting oxidative stress, thereby enhancing the effectiveness of aminoglycosides against resistant strains 35 . Additionally, zinc-based dinuclear complexes, particularly those with imidazole or carboxylate ligands, enhance membrane permeability and disrupt biofilm formation, making them ideal for combination with aminoglycosides to target persistent infections 36 . Likewise, palladium-based dinuclear complexes have attracted attention due to their versatile coordination chemistry, strong antimicrobial properties, high stability, and ability to bind biomolecules, making them practical for disrupting bacterial processes 20 , 37 , 38 . The palladium complex reacted with the aminoglycoside enzyme through different synergistic mechanisms. First, membrane disruption 37 , 39 : The planar structure of palladium complexes allows intercalation into bacterial membranes, increasing permeability and aminoglycoside uptake 39 . Second, DNA binding: Palladium complexes bind bacterial DNA, disrupting transcription, replication, and amplifying the bactericidal effects of aminoglycosides 37 . Lastly, oxidative stress induction: ROS generated by these complexes damage bacterial components, further weakening their defenses 37 . These findings highlight the importance of thoroughly evaluating the interactions between metal complexes and antibiotics. The combined use does not universally result in enhanced antimicrobial effects and may, depending on the specific agents involved, lead to reduced efficacy 40 This paper examines the potential of dinuclear complexes to enhance the efficacy of aminoglycoside antibiotics, focusing on their mechanisms of action and the implications for combating antibiotic resistance. Understanding these interactions can pave the way for innovative therapeutic strategies to address the growing threat of drug-resistant bacterial infections. Material and Methods Material : Gentamicin (gentamicin 80 mg/2 mL injections, Amipharma, Sudan), Mueller-Hinton media (Sigma-Aldrich, Germany), and dimethyl sulfoxide (99.9%, Alpha Chemika, India) were used as received., Benzidinedioxime and bis-benzidinedioxime palladium(II) were synthesized and characterized in the previous study 20 Antimicrobial activity The antimicrobial potency of 10 mg mL − 1 of the synthesized compounds, Gentamicin, and a combination of synthetic compounds and Gentamicin was evaluated using the cup plate method, as described by Seeley et al. (1975). Using resistant isolated microorganisms, Gram-positive bacteria: Staphylococcus aureus, and Gram-negative bacteria, Salmonella. typhi. All strains were provided by the National Research Centre (NRC) in Khartoum, Sudan. Molecular docking Ligand Preparation Using the MOE environment, ligands (Gentamicin, Benzidinedioxime and bis-benzidinedioxime palladium(II) ) were prepared. The Builder module in MOE was used to create the initial 3D structures, which were then energy-minimized to achieve a stable conformation. The minimizations employed the MMFF94x force field, with partial charges automatically assigned based on the force field parameters. This process continued until a root-mean-square deviation (RMSD) gradient of 0.01 kcal mol⁻¹ Å⁻¹ was reached, ensuring an optimized ligand geometry for docking simulations. Protein Structure Preparation Three aminoglycoside-modifying enzyme crystal structures were obtained from the Protein Data Bank (PDB) for use as docking targets: Aminoglycoside-N-Acetyltransferase-(6')-Ib [AAC(6')-Ib] (PDB: 1V0C), Aminoglycoside-O-phosphotransferase-(2'')-IIa [APH(2'')-IIa] (PDB ID: 3HAM), and Aminoglycoside-O-nucleotidyltransferase-(4') [ANT(4')] (PDB ID: 1KNY). Each protein structure was prepared using the Structure Preparation module in MOE. Missing hydrogen atoms and structural irregularities, such as missing side chains or loops, were added, and protonation states were assigned using the Protonate3D tool at a physiological pH of 7.4. Water molecules and non-essential ligands, such as crystallization additives, were removed from the PDB files to focus on protein-ligand interactions. The structures were then optimized using the MMFF94x force field, with a tethering constraint applied to heavy atoms to maintain their experimental conformation while refining hydrogen positions. Docking Protocol Using MOE's Dock module, docking simulations were performed to identify the binding sites for each protein. The Site Finder tool in MOE was used to locate potential binding pockets by analyzing their geometric and chemical properties. The Triangle Matcher algorithm generated initial poses by aligning ligand triplets with receptor site points, placing the ligands in the binding site. Each pose was refined using the Rigid Receptor protocol, where the protein structure remained fixed while the ligand optimized its interactions within the binding pocket. To evaluate molecular interactions, including van der Waals, electrostatic, and hydrogen-bonding effects, we used the MMFF94x force field. The refined poses were ranked based on the London DG scoring function, which estimates the free energy of binding by considering entropic, desolvation, and interaction energy terms. Pose Analysis and Validation The top-ranked docking poses were visually analyzed using MOE’s visualization tools to assess their conformational plausibility and interactions with key binding pocket residues. Meaningful interactions, such as hydrogen bonds, hydrophobic contacts, π-π stacking, and cation-π interactions, were studied using the Ligand Interactions module. Distances and angles between ligand functional groups and protein residues were measured to evaluate the strength and specificity of the interactions. Data Analysis Binding affinities were reported as London dG scores (kcal mol⁻¹), with lower values indicating more favorable binding. Key interacting residues were identified and cross-checked with literature data to confirm their biological relevance. The docking results were further validated by comparing the predicted binding modes with experimental data from related aminoglycoside-modifying enzymes, ensuring consistency with known resistance mechanisms. Result and Discussion Antimicrobial activity : The antimicrobial activity of bis-benzidinedioxime palladium(II) (Pd), benzidinedioxime (L), Gentamicin (G), and their combinations was evaluated against resistant strains of Staphylococcus aureus and Salmonella. typhi using the cup plate method. The results, presented as inhibition zone diameters (mm), provide insights into the standalone and synergistic effects of these compounds, particularly in the context of combating antibiotic resistance. Gentamicin (G) alone exhibited no measurable antimicrobial activity against either S. aureus or S. typhimurium (inhibition zones of 0 mm), confirming the resistance of these bacterial strains to this aminoglycoside antibiotic. This lack of activity aligns with the growing challenge of aminoglycoside resistance, often mediated by aminoglycoside-modifying enzymes such as acetyltransferases, phosphotransferases, or nucleotidyltransferases, which chemically inactivate the antibiotic. Similarly, benzidinedioxime (L) alone showed no antimicrobial effect against either bacterial strain (0 mm inhibition zones), suggesting that the ligand itself lacks inherent antibacterial properties at the tested concentration of 10 mg mL⁻¹. This is consistent with the expectation that ligands without metal coordination often exhibit limited antimicrobial activity due to the absence of reactive or disruptive chemical moieties (Fig. 1 ). In contrast, bis-benzidinedioxime palladium(II) (Pd) demonstrated modest antimicrobial activity against S. typhimurium (14 mm inhibition zone) but no activity against S. aureus (0 mm). The selective activity against the Gram-negative S. typhimurium may be attributed to the planar geometry and coordination chemistry of the palladium(II) complex, which likely facilitates interactions with the bacterial outer membrane, increasing permeability or disrupting membrane integrity 37 . The lack of activity against S. aureus suggests that the thicker peptidoglycan layer of Gram-positive bacteria may hinder the complex’s ability to penetrate or interact effectively with critical cellular targets (Fig. 1 ). Table 1 Inhibition zone (mm) of Gentamicin, benzidinedioxime, and bis-benzidinedioxime palladium(II) and the combination of the benzidinedioxime and bis-benzidinedioxime palladium(II) with Gentamicin against resistant bacteria ( Staphylococcus aureus and Salmonella. typhi ). Compounds S. aureus S. Typhi Inhibition zone (mm) Inhibition zone (mm) Gentmicin (G) 0 0 Benzidinedioxime (L) 0 0 G + L 23 30 Bis-benzidinedioxime Palladium(II) (Pd) 0 14 G + Pd 18 24 The most striking results were observed with the combinations of Gentamicin with benzidinedioxime (G + L) and Gentamicin with bis-benzidinedioxime palladium(II) (G + Pd). The G + L combination produced significant inhibition zones of 23 mm against S. aureus and 30 mm against S. typhimurium . These results are unexpected, given the lack of standalone activity for both Gentamicin and benzidinedioxime, suggesting a synergistic interaction that restores or enhances Gentamicin’s antibacterial efficacy Table 1 . This synergy may arise from benzidinedioxime’s ability to interact with bacterial membranes or resistance enzymes, potentially increasing gentamicin uptake or inhibiting aminoglycoside-modifying enzymes. The larger inhibition zone against S. typhimurium compared to S. aureus indicates that the Gram-negative outer membrane may be more susceptible to disruption by benzidinedioxime, facilitating greater antibiotic penetration. The G + Pd combination also exhibited significant antimicrobial activity, with inhibition zones of 18 mm against S. aureus and 24 mm against S. typhimurium . These results are particularly notable given Gentamicin’s inactivity alone and the limited standalone activity of Pd against S. typhimurium (Table 1 ). The synergistic effect of G + Pd is likely driven by the palladium complex’s multifaceted mechanisms, including membrane disruption, DNA binding, and the generation of reactive oxygen species (ROS), as described in the introduction 41 . The palladium(II) complex’s planar structure may intercalate into bacterial membranes, enhancing gentamicin uptake. At the same time, its DNA-binding properties and ROS production could amplify the bactericidal effects of Gentamicin by disrupting bacterial replication and cellular integrity. The antimicrobial results complement the molecular docking data, which demonstrated that bis-benzidinedioxime palladium(II) exhibited the highest binding affinities across three protein targets (1V0C, 3HAM, and 1KNY), with docking scores ranging from − 10.54 kcal/mol (1V0C) to -7.48 kcal/mol (1KNY). These strong binding affinities, particularly with enzymes such as aminoglycoside 2-acetyltransferase and nucleotidyltransferase, suggest that Pd may target key bacterial metabolic pathways, potentially inhibiting folate synthesis and contributing to its standalone activity against S. typhimurium . The docking data also revealed extensive hydrogen bonding and electrostatic interactions with residues such as Asp and Glu, which may correlate with the complex’s ability to disrupt bacterial membranes or bind to resistance enzymes, thereby enhancing Gentamicin’s efficacy in the G + Pd combination. Benzidinedioxime, despite its weaker docking scores (-7.06 to -6.35 kcal/mol), showed remarkable synergy with Gentamicin in the antimicrobial assays (Table 1 ). This suggests that its role as an adjuvant may not rely solely on strong binding to protein targets but instead on subtler effects, such as membrane perturbation or weak interactions with resistance enzymes, which facilitate Gentamicin’s access to the bacterial ribosome. The docking results for Gentamicin (scores ranging from − 8.33 to -7.6 kcal/mol) indicate moderate binding to ribosomal or enzymatic targets Table 1 . Still, its inactivity alone underscores the dominance of resistance mechanisms in these strains. The synergy observed in G + L and G + Pd combinations highlights the potential of these adjuvants to overcome such resistance. The significant synergistic effects of G + L and G + Pd combinations underscore the potential of dinuclear palladium complexes and their ligands as adjuvants to restore the efficacy of aminoglycosides against bacteria that are resistant to them. The larger inhibition zones against S. typhimurium compared to S. aureus suggest that these combinations may be particularly effective against Gram-negative pathogens, which are often more challenging due to their outer membrane barrier. The mechanisms proposed in the introduction—membrane disruption, DNA binding, and ROS generation—are supported by the antimicrobial results and docking data, particularly for Pd, which likely enhances gentamicin uptake and amplifies its bactericidal effects through oxidative stress and metabolic disruption. The unexpected synergy of benzidinedioxime with Gentamicin suggests that even non-metalated ligands may play a role in overcoming resistance, possibly by modulating membrane permeability or inhibiting resistance enzymes. This finding warrants further investigation into the specific interactions between benzidinedioxime and bacterial targets, as well as its potential to enhance the efficacy of other classes of antibiotics. Molecular docking : Molecular docking is a powerful computational tool for predicting the binding affinity and interaction profiles of ligands with protein targets, providing insights into their potential as therapeutic agents. In this study, the docking performance of three compounds—bis-benzidinedioxime palladium(II), benzidinedioxime, and Gentamicin against three protein targets (1V0C, 3HAM, and 1KNY) was evaluated using MOE2024 software. The results, including binding energies, root-mean-square deviation (RMSD) refined values, and 2D interaction profiles, offer a comprehensive understanding of the molecular interactions and binding preferences of these compounds. Binding Energy, expressed as the docking score (S) in kcal/mol, reflects the strength of ligand-protein interactions, with more negative values indicating stronger binding. Across the three protein targets, bis-benzidinedioxime palladium(II) consistently exhibited the highest binding affinity, with docking scores ranging from − 10.54 kcal/mol (1V0C) to -7.48 kcal/mol (1KNY), Table 2 . This suggests that the palladium(II) complex forms more stable interactions with the protein targets compared to benzidinedioxime and Gentamicin. Benzidinedioxime displayed moderate binding affinities, with scores ranging from − 7.06 kcal/mol (1V0C) to -6.35 kcal/mol (3HAM), while Gentamicin showed the lowest affinities, with scores between − 8.33 kcal/mol (1V0C) and − 7.6 kcal/mol (1KNY), Table 2 . The superior binding affinity of bis-benzidinedioxime palladium(II) may be attributed to the presence of the palladium ion, which likely enhances coordination interactions with key residues in the protein binding pockets. Out of the protein targets, 1V0C showed the strongest binding interactions with all three compounds, with bis-benzidinedioxime palladium(II) having the highest docking score (-10.54 kcal/mol) (Table 2 ). This suggests that 1V0C may have a binding pocket shape and chemical environment that's especially well-suited for these ligands. On the other hand, 1KNY generally had low binding affinities, especially for the bis-benzidinedioxime palladium(II) and gentamicin complexes, suggesting possible steric or electronic mismatches in these protein-ligand interactions. Table 2 Binding energy and RMSD refined value of docking of bis-benzidinedioxime palladium(II), benzidinedioxime, and Gentamicin with Aminoglycoside-N-Acetyltransferase-(6')-Ib [AAC(6')-Ib] (PDB: 1V0C), Aminoglycoside-O-phosphotransferase-(2'')-IIa [APH(2'')-IIa] (PDB ID: 3HAM), and Aminoglycoside-O-nucleotidyltransferase-(4') [ANT (4')] (PDB ID: 1KNY) by using MOE2024 software compounds 1V0C 3HAM 1KNY S Rmsd refind S Rmsd refind S Rmsd refind bis-benzidinedioxime palladium(II) -10.54 1.65 -8.804 1.32 -7.48 1.13 Benzidinedioxime -7.06 1.24 -6.35 1.1 -6.68 1.34 gentamicin -8.33 .952 -7.77 1.75 -7.6 1.68 The RMSD refined values clarify the accuracy and reliability of the docked poses, with lower values indicating better alignment with the reference structure. Bis-benzidinedioxime palladium(II) showed RMSD values ranging from 1.13 Å (1KNY) to 1.65 Å (1V0C), suggesting reasonable pose reliability across all targets. Benzidinedioxime exhibited the lowest RMSD values, ranging from 1.1 Å (3HAM) to 1.34 Å (1KNY), indicating highly accurate docking poses, particularly for 3HAM. Gentamicin displayed a broader range of RMSD values (0.952 Å for 1V0C to 1.75 Å for 3HAM), suggesting variability in docking reliability, with the best performance observed for 1V0C. The low RMSD values for benzidinedioxime, particularly with 1V0C (1.24 Å) (Table 2 ), indicate that its docked poses closely match the reference structure, potentially due to its smaller molecular size and simpler structure, which may allow for better accommodation within the binding pocket. The 2D interaction diagrams, Figs. 2 – 4 , provide detailed insights into the specific residue interactions and binding modes of the three compounds with each protein target. The key interactions for each protein-ligand complex, focusing on hydrogen bonds, hydrophobic interactions, and other non-covalent contacts, as well as their implications for binding stability. Bis-benzidinedioxime palladium(II) forms extensive interactions with 1V0C, including hydrogen bonds with residues such as Asp152, Asp119, Trp102, and Trp49, with interaction distances ranging from 3.18 Å to 3.84 Å and energies from − 0.9 to -3.4 kcal/mol Fig. 2 . The presence of multiple hydrogen bonds, particularly with negatively charged aspartate residues, suggests strong electrostatic interactions facilitated by the palladium(II) ion. Benzidinedioxime interacts with a smaller subset of residues, including Trp102 and Asp100, with interaction energies (-1.4 kcal/mol), reflecting its lower binding affinity. Gentamicin forms hydrogen bonds with residues such as Trp164, Asp115, and Asp152; however, its interactions are less extensive, consistent with its moderate docking score of -8.33 kcal/mol (Fig. 1 ). Previous studies have elucidated the functional importance of specific residues for gentamicin binding and catalysis. Asp115 and Asp152 form critical, buried ionic interactions with amino groups on rings I and II, with Asp115 acting as a proton acceptor (general base). Ser98 binds the 3′′-NH3 group, while Asp100 interacts with both the 3′′-NH3 and 4′′-OH groups 42 . Furthermore, the tryptophan residues Trp49 and Trp102 contribute significantly via stacking interactions with rings I and II of the aminoglycoside. These binding studies provide a structural rationale for inhibition. The potent binding of Bis-Benzidinedioxime Palladium(II) stems from its ability to exploit the enzyme's intrinsic negative electrostatic potential, forming a dense network of hydrogen bonds, particularly with key catalytic aspartates (Asp115, Asp152). By occupying these critical residues and mimicking aspects of aminoglycoside binding (e.g., interactions with Trp residues), this complex likely acts as a competitive inhibitor, physically blocking the active site and preventing the natural substrate (like Gentamicin) from binding and undergoing acetylation. The weaker binding of benzidinedioxime alone underscores the importance of the palladium ion in enhancing these critical electrostatic and hydrogen-bonding interactions necessary for effective inhibition. Bis-benzidinedioxime palladium(II) interacts with 3HAM through hydrogen bonds with Glu58, Asp232, Asp29, Asp192, and Gly212, with interaction energies ranging from − 0.7 to -4.1 kcal/mol. Benzidinedioxime forms weaker interactions with residue Asp213, with energies around − 0.9 kcal/mol, reflecting its lower docking score (-6.35 kcal/mol). Gentamicin interacts with Asp192, exhibiting lower interaction energies of -0.6 kcal/mol, which suggests lower binding stability (Fig. 3 ). Critically, structural analysis shows Gentamicin is positioned for catalysis: the 2''-OH group on its ring C (the phosphorylation target) is poised ~ 3.0 Å from the oxygen atom of Asp192. APH (2'') phosphorylates Gentamicin at the 2''-OH group. Our data supports the established mechanism where Asp192 acts as the essential catalytic base; It precisely orients the 2''-OH group for optimal nucleophilic attack on the gamma-phosphate of ATP and It likely acts as a "proton relay station", accepting the proton from the 2''-OH during the reaction (acting as a general base) 43 . The potent inhibition by Bis-Benzidinedioxime Palladium(II) stems from its ability to exploit the Catalytic Architecture by forming extensive, energetically favorable interactions across the active site, including a strong interaction with the critical Asp192, also block the Catalytic Base; by occupying Asp192 with high affinity (-4.1 kcal/mol interaction possible), the Pd complex directly obstructs this residue's essential functions – orienting the substrate's OH group and accepting its proton. Furthermore, it physically occludes the active site by forming a broad network of interactions (involving Glu58, Asp232, Asp29, and Gly212), effectively filling the active site cavity. This combination suggests the Pd complex acts as a highly effective competitive inhibitor. It outcompetes Gentamicin for binding, particularly by sequestering Asp192, thereby preventing the precise substrate positioning and proton transfer essential for phosphorylation. The weaker binding of benzidinedioxime alone underscores the critical role of the palladium ion in achieving this potent, mechanism-targeted inhibition and potentially stabilizing intermediates later in the process ("proton trap"). For 1KNY, bis-benzidinedioxime palladium(II) forms hydrogen bonds with Glu63, Glu67, Glu76, and Ser188, with energies − .05 to -5.4 kcal/mol. Benzidinedioxime interacts with Trp37 and Ser39, with weaker energies (-0.7 to -1.3 kcal/mol). Gentamicin interacts with Glu52, Glu76, Ser39, Ser49, and Mg, with energies ranging from − 0.5 to -3.1 kcal/mol, consistent with its docking score of -7.6 kcal/mol (Fig. 4 ). Crucially, specific glutamates (Glu67 with ring A, Glu76 with ring A, Glu142 with ring C) form strong hydrogen bonds critical for positioning the aminoglycoside. ANT (4') confers resistance by transferring an adenylyl group from Mg-ATP to the 4'-OH of aminoglycosides like Gentamicin and kanamycin, forming O-adenylylated product and MgPPi 44 . The results of molecular docking support the catalytic mechanism, where Glu145 serves as the primary catalytic base. Analysis indicates that the nucleophilic 4'-O atom of the aminoglycoside is positioned for activation (deprotonation). While O2α of ATP or Glu76 were potential activators, the distance data strongly suggest Glu145 (OE2) is responsible for activating the 4'-OH in the wild-type enzyme, acting as the primary proton acceptor. On the other hand, Glu76 acts as a backup base; if Glu145 is absent or disabled, Glu76 can potentially take over this essential proton-accepting role, highlighting functional redundancy in the catalytic machinery. Both Glu145 and Glu76 are critical "proton relay partners" for the reaction. The potent binding of the Pd complex suggests a highly effective inhibition strategy, as it sequesters the catalytic machinery by directly engaging and occupying multiple key glutamates involved in substrate binding (Glu67, Glu142) and catalysis (Glu76, potentially Glu145 by proximity). Its strong interaction with Glu76 (-5.4 kcal/mol possible) is particularly significant, given Glu76's dual role in substrate binding and its potential as a backup catalytic base. In addition, it blocks the Active Site; the network of interactions with Glu63, Glu67, Glu76, and Ser188 physically occludes the binding cleft, where both the aminoglycoside (such as Gentamicin) and the MgATP cofactor must bind and interact. Moreover, the Pd complex disrupts electrostatic steering & catalysis through binding tightly to the critical glutamates. The Pd complex disrupts the precise electrostatic environment needed to position the aminoglycoside's 4'-OH for nucleophilic attack and interferes with the proton transfer essential for activating the nucleophile (whether by Glu145 or Glu76). Additionally, it has an extensive binding network and a higher affinity (compared to benzidinedioxime), allowing it to effectively outcompete Gentamicin for the active site. In conclusion, Bis-Benzidinedioxime Palladium(II) acts as a potent competitive inhibitor of ANT(4') by exploiting the enzyme's catalytic architecture. It achieves this by forming a high-affinity complex that blocks key glutamates (Glu67, Glu76, Glu142), which are crucial for substrate binding, and occupies the catalytic base (Glu145 or Glu76), thereby preventing both substrate positioning and the essential proton transfer step required for adenylylation. The weaker binding of benzidinedioxime underscores the critical role of the palladium ion in enabling this potent, mechanism-targeted inhibition The docking results reveal that bis-benzidinedioxime palladium(II) consistently outperforms benzidinedioxime and Gentamicin in terms of binding affinity across all protein targets. This is likely due to the palladium(II) ion, which enhances coordination and electrostatic interactions with negatively charged residues such as aspartate and glutamate, as observed in the 2D interaction profiles. The presence of multiple hydrogen bonds contributes to the stability of the palladium complex in the binding pocket. Benzidinedioxime, lacking the palladium ion, exhibits weaker binding affinities and fewer interactions, suggesting that its simpler structure limits its ability to form strong contacts with the protein targets. Gentamicin, despite its larger and more complex structure, forms moderate interactions, likely due to its multiple hydroxyl and amine groups, which facilitate hydrogen bonding but may introduce steric constraints in certain binding pockets (e.g., 3HAM). The shape of the binding pocket and the type of amino acid residues of the protein play an essential role in the value of binding affinities of the compound with the protein. 1V0C appears to be the most accommodating target to benzidinedioxime and Bis-benzidinedioximme palladium(II), likely due to a favorable arrangement of polar and charged residues that match the ligands' functional groups. In contrast, 1KNY offers more difficult binding environments, possibly due to steric constraints or weak electrostatic interactions. Previous research found that benzidinedioxime had a medium inhibition zone against isolated S. aureus and S. typhimurium (14.3 ± 1.6 mm and 12.7 ± 1.8 mm, respectively). In contrast, Gentamicin showed a stronger inhibition zone (26 mm and 25 mm, respectively) (Ebrahim et al., 2023). This new result suggests that these bacteria have developed resistance to benzidinedioxime through at least one aminoglycoside enzyme. The molecular docking study of benzidinedioxime and bis-benzidinedioxime in Fig. 4 showed that the hydrogen atom on the hydroxyl group doesn't interact with Glu 142 or Glu 76, resulting in a weak nucleophilic attack of the oxygen atom on the phosphate group. This suggests that nucleotidyltransferase enzymes don't contribute to bacterial resistance. The 2D interaction of benzidinedioxime and bis-benzidinedioxime with Aminoglycoside-O-phosphotransferase-(2'')-IIa [APH(2'')-IIa] (PDB ID: 3HAM) in Fig. 3 shows a hydrogen bond formed between benzidinedioxime and Asp 213, increasing its nucleophilic attack properties on the phosphate group in the enzyme. When benzidinedioxime is linked to the protein and ADP by a hydroxyl group, an unstable transition state forms according to the SN2 mechanism. This unstable state leads to the breakdown of the hydrogen-oxygen bond and the formation of an oxygen-ADP bond, facilitating benzidinedioxime phosphorylation, as shown in (Fig. 5 ). Despite the bis-benzidinedioxime palladium(II) establishing a strong hydrogen bond with Glu 56 in Aminoglycoside-O-phosphotransferase-(2'')-IIa [APH(2'')-IIa] (PDB ID: 3HAM) through its hydroxyl group, the enzyme did not modify it due to the significant distance of 10.81 °A between the hydroxyl group and ADP, as illustrated in (Fig. 6 ). The findings from the antimicrobial test corroborated this observation, as bis-benzidinedioxime palladium(II) exhibited antimicrobial effects against S. Typhi (14 mm), as summarized in Table 1 . This outcome suggests that the resistance of S. Typhi to aminoglycosides was not attributed to Aminoglycoside-O-phosphotransferase-(2'')-IIa [APH(2'')-IIa]. Figure 2 illustrates the resistance mechanism of Aminoglycoside-N-Acetyltransferase-(6')-Ib [AAC(6')-Ib] (PDB: 1V0C) to Benzidinedioxime and bis-benzidinedioxime palladium(II). Bis-benzidinedioxime palladium(II) forms two ionic bonds with Asp 152 and Asp 115, creating a strong nucleophilic attack on COA due to the negative charge on the imine group. This property enables a strong nucleophilic attack on the acetyl group of COA. Benzidinedioxime is modified by COA through a nucleophilic interaction of the nitrogen atom's lone pair of electrons with the acetyl group in COA. The study concluded that S. Aureus is resistant to both compounds and Gentamicin due to Aminoglycoside-N-Acetyltransferase-(6')-Ib [AAC(6')-Ib]. Molecular docking and antimicrobial activity results demonstrated the synergistic potential of benzidinedioxime and bis-benzidinedioxime palladium(II) to enhance the efficacy of Gentamicin against resistant bacteria (Fig. 7 ). Conclusion This study demonstrates the significant potential of bis-benzidinedioxime palladium(II) (Pd) and its ligand, benzidinedioxime (L), as adjuvants to overcome gentamicin resistance in Staphylococcus aureus and Salmonella. typhi. Molecular docking revealed that Pd exhibits superior binding affinity, particularly against the aminoglycoside-modifying enzymes AAC(6')-Ib (1V0C) and APH(2'')-IIa (3HAM), driven by strong electrostatic interactions with key Asp/Glu residues. The palladium complex's strong electrostatic interactions, especially with aspartate and glutamate residues, highlight its potential as a lead compound for further drug development. Antimicrobial assays further demonstrate that bis-benzidinedioxime palladium(II) (Pd) and benzidinedioxime (L) significantly enhance the efficacy of gentamicin (G) against resistant Staphylococcus aureus and Salmonella. Typhi, yielding inhibition zones of 18–24 mm and 23–30 mm, respectively. This potent synergy suggests that both Pd and L function by effectively inhibiting key resistance enzymes, AAC(6')-Ib and APH(2'')-IIa, thereby protecting gentamicin from inactivation. Furthermore, the docking analysis indicates that Pd achieves inhibition of APH(2'')-IIa while evading altering the structure of the palladium complex. These findings establish dinuclear palladium complexes and their ligand as promising candidates for the development of resistance-breaking adjuvants to revitalize aminoglycoside therapy, paving the way for mechanistic studies and structural optimization to develop innovative treatments for antibiotic-resistant bacterial infections. Declarations Author contributions Reem M. A. Ebrahim investigation, data analysis, writing original draft, visualization, Nooh Mohamed Hajhamed, molecular docking, writing, Abdallah Elssir Ahmed and Ayman Azhary, data analysis, visualization, writing, review and editing, Yousif Sulfab, and Elmugdad A. Ali supervision, writing, review and editing. Data Availability Statement All data generated or analyzed during this study are included in this published article. Conflicts of interest The authors declare that they have no conflict of interest. Funding The authors received no funding for this work. References Isernia, S. et al. The key role of depression and supramarginal gyrus in frailty: a cross-sectional study. Frontiers in Aging Neuroscience 15 , 1292417 (2023). Mancuso, G., Midiri, A., Gerace, E. & Biondo, C. Bacterial antibiotic resistance: the most critical pathogens. Pathogens 10 , 1310 (2021). Read, A. F. & Woods, R. J. Antibiotic resistance management. 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S. & Tolmasky, M. E. Aminoglycoside modifying enzymes. Drug resistance updates 13 , 151-171 (2010). Jana, S. & Deb, J. Molecular understanding of aminoglycoside action and resistance. Applied microbiology and biotechnology 70 , 140-150 (2006). Serio, A. W., Magalhães, M. L., Blanchard, J. S. & Connolly, L. E. in Antimicrobial Drug Resistance: Mechanisms of Drug Resistance, Volume 1 213-229 (Springer, 2017). Ayipo, Y. O., Osunniran, W. A., Babamale, H. F., Ayinde, M. O. & Mordi, M. N. Metalloenzyme mimicry and modulation strategies to conquer antimicrobial resistance: Metal-ligand coordination perspectives. Coordination Chemistry Reviews 453 , 214317 (2022). Viganor, L., Howe, O., McCarron, P., McCann, M. & Devereux, M. The antibacterial activity of metal complexes containing 1, 10-phenanthroline: potential as alternative therapeutics in the era of antibiotic resistance. Current topics in medicinal chemistry 17 , 1280-1302 (2017). Guillouzo, A. & Guguen-Guillouzo, C. Antibiotics-induced oxidative stress. Current Opinion in Toxicology 20 , 23-28 (2020). Vatansever, F. et al. Antimicrobial strategies centered around reactive oxygen species–bactericidal antibiotics, photodynamic therapy, and beyond. FEMS microbiology reviews 37 , 955-989 (2013). Van Acker, H. & Coenye, T. The role of reactive oxygen species in antibiotic-mediated killing of bacteria. Trends in microbiology 25 , 456-466 (2017). Peega, T. Synthesis, characterization and investigation of the mode of action in the anticancer activity of novel platinum complexes , University of the Witwatersrand, Johannesburg, (2024). Navarro, M., Justo, R. M., Delgado, G. Y. S. & Visbal, G. Metallodrugs for the treatment of trypanosomatid diseases: Recent advances and new insights. Current Pharmaceutical Design 27 , 1763-1789 (2021). Etsana, K. Synthesis, Structural Investigation and Assessment of Antibacterial Activities of New Cu (II) Complexes Containing 2, 2’-Bipyridine and 1, 10-phenanthroline , (2017). Falcone, E. R. Investigating the Antiproliferative Activity of Synthetic Troponoids . (University of Connecticut, 2016). Olesya, S. & Alexander, P. Antimicrobial activity of mono-and polynuclear platinum and palladium complexes. Foods and Raw materials 8 , 298-311 (2020). Waziri, I. et al. New palladium (II) complexes from halogen substituted Schiff base ligands: Synthesis, spectroscopic, biological activity, density functional theory, and molecular docking investigations. Inorganica Chimica Acta 552 , 121505 (2023). Shaikh, S. et al. Mechanistic insights into the antimicrobial actions of metallic nanoparticles and their implications for multidrug resistance. International journal of molecular sciences 20 , 2468 (2019). Ventola, C. L. The antibiotic resistance crisis: part 1: causes and threats. Pharmacy and therapeutics 40 , 277 (2015). Claudel, M., Schwarte, J. V. & Fromm, K. M. New antimicrobial strategies based on metal complexes. Chemistry 2 , 849-899 (2020). Vetting, M. W. et al. Mechanistic and structural analysis of aminoglycoside N-acetyltransferase AAC (6′)-Ib and its bifunctional, fluoroquinolone-active AAC (6′)-Ib-cr variant. Biochemistry 47 , 9825-9835 (2008). Young, P. G. et al. The crystal structures of substrate and nucleotide complexes of Enterococcus faecium aminoglycoside-2′′-phosphotransferase-IIa [APH (2′′)-IIa] provide insights into substrate selectivity in the APH (2′′) subfamily. Journal of bacteriology 191 , 4133-4143 (2009). Martí, S., Bastida, A. & Świderek, K. Theoretical studies on mechanism of inactivation of kanamycin A by 4′-O-Nucleotidyltransferase. Frontiers in chemistry 6 , 660 (2019). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7216024","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":494860823,"identity":"6595b52b-5cc7-41c0-88d9-0f7c475937f2","order_by":0,"name":"Reem M. A. 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A.","lastName":"Ebrahim","suffix":""},{"id":494860824,"identity":"8ccb8d36-ce2e-4284-85c1-9e8c4ada355c","order_by":1,"name":"Nooh Mohamed Hajhamed","email":"","orcid":"","institution":"Sirius Training and Research Center","correspondingAuthor":false,"prefix":"","firstName":"Nooh","middleName":"Mohamed","lastName":"Hajhamed","suffix":""},{"id":494860825,"identity":"1ffca242-0b74-4260-bc7f-19c9c83a74eb","order_by":2,"name":"Abdallah Elssir Ahmed","email":"","orcid":"","institution":"Africa City of Technology","correspondingAuthor":false,"prefix":"","firstName":"Abdallah","middleName":"Elssir","lastName":"Ahmed","suffix":""},{"id":494860826,"identity":"2e521e65-e0a5-445f-830c-6f0729d8c2c9","order_by":3,"name":"Ayman Azhary","email":"","orcid":"","institution":"Sirius Training and Research Center","correspondingAuthor":false,"prefix":"","firstName":"Ayman","middleName":"","lastName":"Azhary","suffix":""},{"id":494860827,"identity":"007d6cd7-783b-4667-826b-3554c9bd454f","order_by":4,"name":"Yousif Sulfab","email":"","orcid":"","institution":"Al-Neelain University","correspondingAuthor":false,"prefix":"","firstName":"Yousif","middleName":"","lastName":"Sulfab","suffix":""},{"id":494860828,"identity":"8520d319-0c10-44ca-89a2-3134892429aa","order_by":5,"name":"Elmugdad A. Ali","email":"","orcid":"","institution":"Sudan University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Elmugdad","middleName":"A.","lastName":"Ali","suffix":""}],"badges":[],"createdAt":"2025-07-25 16:23:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7216024/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7216024/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88352171,"identity":"69e7aaac-ee43-4ce1-ad47-6c57db993931","added_by":"auto","created_at":"2025-08-05 14:29:18","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":73259,"visible":true,"origin":"","legend":"\u003cp\u003eRepresents the resistance of (Staphylococcus aureus and Salmonella. typhi) to Gentamicin and the synergistic effect of the combination of the benzidinedioxime and bis-benzidinedioxime palladium(II) with Gentamicin against resistant bacteria (Staphylococcus aureus and Salmonella. typhi )\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7216024/v1/d6734c839f713f3b70596067.jpg"},{"id":88352702,"identity":"737cf794-a09b-41aa-8c28-7aa426dd267f","added_by":"auto","created_at":"2025-08-05 14:37:18","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":138232,"visible":true,"origin":"","legend":"\u003cp\u003e2D interaction of docking of bis-benzidinedioxime palladium (II). Benzidinedioxime and gentamicin, respectively, with Aminoglycoside-N-Acetyltransferase-(6')-Ib [AAC(6')-Ib] (PDB: 1V0C)\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7216024/v1/68b7643812d85cc4417fb58a.jpg"},{"id":88352700,"identity":"34a49afc-85a8-4f15-881e-9cdbd262869f","added_by":"auto","created_at":"2025-08-05 14:37:18","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":126926,"visible":true,"origin":"","legend":"\u003cp\u003e2D interaction of docking of bis-benzidinedioxime palladium (II). Benzidinedioxime and gentamicin, respectively, with Aminoglycoside-O-phosphotransferase-(2'')-IIa [APH(2'')-IIa] (PDB ID: 3HAM)\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7216024/v1/2687d485593ff8e7c49ff80f.jpg"},{"id":88352703,"identity":"57e9afc2-cfee-4a70-b945-705135341d17","added_by":"auto","created_at":"2025-08-05 14:37:18","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":130170,"visible":true,"origin":"","legend":"\u003cp\u003e2D interaction of docking of bis-benzidinedioxime palladium (II). Benzidinedioxime and Gentamicin, respectively, with Aminoglycoside-O-nucleotidyltransferase-(4') [ANT (4')] (PDB ID: 1KNY)\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7216024/v1/873df355da156d2151200715.jpg"},{"id":88352175,"identity":"7fdecfe4-8fce-4bfc-8539-61b5b53aa190","added_by":"auto","created_at":"2025-08-05 14:29:18","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":120751,"visible":true,"origin":"","legend":"\u003cp\u003eRepresent modification mechanism of benzidinedioxime by Aminoglycoside-O-phosphotransferase-(2'')-IIa [APH(2'')-IIa] (PDB ID: 3HAM)\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7216024/v1/37d53f01f50c9bea6e3f3843.jpg"},{"id":88352177,"identity":"4eb0f53d-a8d0-43eb-b09b-831c08199f65","added_by":"auto","created_at":"2025-08-05 14:29:18","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":68183,"visible":true,"origin":"","legend":"\u003cp\u003eRepresents the mechanism of interaction and unmodification of bis-benzidinedioxime palladium(II) with Aminoglycoside-O-phosphotransferase-(2'')-IIa [APH(2'')-IIa] (PDB ID: 3HAM).\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7216024/v1/a0b8c3fa72a6a331255c4bb3.jpg"},{"id":88353558,"identity":"a82091e8-1d0b-410c-8874-e658dabf04d6","added_by":"auto","created_at":"2025-08-05 14:45:18","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":137524,"visible":true,"origin":"","legend":"\u003cp\u003eMechanism of synergistic potential of benzidinedioxime and bis-benzidinedioxime palladium(II) as enhancers of gentamicin efficacy against resistant bacteria. The figure was created using BioRender.com (accessed June 19, 2025).\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7216024/v1/95e11f4ae8c232017a117f10.jpg"},{"id":92390315,"identity":"e753ebe3-8af9-40aa-927e-bf270808c18a","added_by":"auto","created_at":"2025-09-29 08:24:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1538860,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7216024/v1/481c0b84-0bce-40ce-ab6d-a52f2cd308c0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synergistic Potential of Bis-Benzidinedioxime Palladium(II) and Benzidinedioxime as Enhancers of Gentamicin Efficacy Against Resistant Bacteria: Insights from Molecular Docking and Antimicrobial Studies","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe global surge in bacterial infections caused by antimicrobial-resistant strains is one of the most significant public health phenomena endangering lives worldwide \u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Compounding this crisis is the diminishing effectiveness of most essential antibiotics, such as aminoglycosides, which are commonly used to combat serious infections caused by both Gram-negative and Gram-positive pathogens. This growing vulnerability underscores the urgency to explore novel strategies to restore or enhance antibiotic efficacy of our existing antibiotics armory \u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6 CR7 CR8 CR9 CR10\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. One promising approach involves using metal-based compounds, such as dinuclear complexes, as adjuvants to antibiotic therapy \u003csup\u003e\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDinuclear metal complexes consist of two metallic centers connected by bridging ligands, often exhibiting unique structural and chemical properties that make them potential antimicrobial agents or enhancers of antibiotic activity \u003csup\u003e\u003cspan additionalcitationids=\"CR17 CR18 CR19\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The synergistic effects between dinuclear complexes and aminoglycosides have been attributed to several mechanisms, including increased bacterial membrane permeability, enhanced binding to bacterial ribosomal targets, and inhibition of resistance enzymes that deactivate aminoglycosides \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Furthermore, the binding properties of specific dinuclear complexes allow them to target bacterial ribosomal sites synergistically with aminoglycosides. Aminoglycosides function by binding to the 30S ribosomal subunit, leading to errors in protein synthesis and bactericidal effects. Dinuclear complexes may stabilize these interactions or introduce additional structural distortions in the ribosomal architecture, thereby enhancing the antibiotic's effectiveness \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAnother critical mechanism involves inhibiting aminoglycoside-modifying enzymes, such as acetyltransferases, phosphotransferases, and nucleotidyltransferases \u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. These enzymes are often responsible for bacterial resistance by chemically modifying aminoglycosides at specific positions and rendering them inactive \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Dinuclear complexes may interact directly with these resistance enzymes, inhibiting their function and preserving the antibiotic's activity \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Additionally, some complexes generate oxidative stress through the production of reactive oxygen species (ROS), which damages bacterial DNA, proteins, and membranes, thereby complementing the action of aminoglycosides \u003csup\u003e\u003cspan additionalcitationids=\"CR30 CR31\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePlatinum-based dinuclear compounds like [Pt₂(phen)₄Cl₂] (phen\u0026thinsp;=\u0026thinsp;1,10-phenanthroline) destabilize bacterial membranes and disrupt nucleic acid synthesis, exhibiting synergistic effects with aminoglycosides attributed to increased antibiotic uptake and ROS-mediated damage \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Similarly, copper dinuclear complexes coordinated with Schiff base ligands (e.g., Cu₂(bpy)₂(OH) ₂, bpy\u0026thinsp;=\u0026thinsp;2,2\u0026prime;-bipyridine) demonstrate potent antimicrobial properties by disrupting membranes and promoting oxidative stress, thereby enhancing the effectiveness of aminoglycosides against resistant strains \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Additionally, zinc-based dinuclear complexes, particularly those with imidazole or carboxylate ligands, enhance membrane permeability and disrupt biofilm formation, making them ideal for combination with aminoglycosides to target persistent infections \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Likewise, palladium-based dinuclear complexes have attracted attention due to their versatile coordination chemistry, strong antimicrobial properties, high stability, and ability to bind biomolecules, making them practical for disrupting bacterial processes \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe palladium complex reacted with the aminoglycoside enzyme through different synergistic mechanisms. First, membrane disruption \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e: The planar structure of palladium complexes allows intercalation into bacterial membranes, increasing permeability and aminoglycoside uptake \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Second, DNA binding: Palladium complexes bind bacterial DNA, disrupting transcription, replication, and amplifying the bactericidal effects of aminoglycosides \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Lastly, oxidative stress induction: ROS generated by these complexes damage bacterial components, further weakening their defenses \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThese findings highlight the importance of thoroughly evaluating the interactions between metal complexes and antibiotics. The combined use does not universally result in enhanced antimicrobial effects and may, depending on the specific agents involved, lead to reduced efficacy \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eThis paper examines the potential of dinuclear complexes to enhance the efficacy of aminoglycoside antibiotics, focusing on their mechanisms of action and the implications for combating antibiotic resistance. Understanding these interactions can pave the way for innovative therapeutic strategies to address the growing threat of drug-resistant bacterial infections.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003e\u003cb\u003eMaterial\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eGentamicin (gentamicin 80 mg/2 mL injections, Amipharma, Sudan), Mueller-Hinton media (Sigma-Aldrich, Germany), and dimethyl sulfoxide (99.9%, Alpha Chemika, India) were used as received., Benzidinedioxime and bis-benzidinedioxime palladium(II) were synthesized and characterized in the previous study \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAntimicrobial activity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe antimicrobial potency of 10 mg mL\u003csup\u003e− 1\u003c/sup\u003e of the synthesized compounds, Gentamicin, and a combination of synthetic compounds and Gentamicin was evaluated using the cup plate method, as described by Seeley et al. (1975). Using resistant isolated microorganisms, Gram-positive bacteria: Staphylococcus aureus, and Gram-negative bacteria, Salmonella. typhi. All strains were provided by the National Research Centre (NRC) in Khartoum, Sudan.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMolecular docking\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eLigand Preparation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eUsing the MOE environment, ligands (Gentamicin, Benzidinedioxime and bis-benzidinedioxime palladium(II) ) were prepared. The Builder module in MOE was used to create the initial 3D structures, which were then energy-minimized to achieve a stable conformation. The minimizations employed the MMFF94x force field, with partial charges automatically assigned based on the force field parameters. This process continued until a root-mean-square deviation (RMSD) gradient of 0.01 kcal mol⁻¹ Å⁻¹ was reached, ensuring an optimized ligand geometry for docking simulations.\u003c/p\u003e\u003cp\u003e\u003cb\u003eProtein Structure Preparation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThree aminoglycoside-modifying enzyme crystal structures were obtained from the Protein Data Bank (PDB) for use as docking targets: Aminoglycoside-N-Acetyltransferase-(6')-Ib [AAC(6')-Ib] (PDB: 1V0C), Aminoglycoside-O-phosphotransferase-(2'')-IIa [APH(2'')-IIa] (PDB ID: 3HAM), and Aminoglycoside-O-nucleotidyltransferase-(4') [ANT(4')] (PDB ID: 1KNY). Each protein structure was prepared using the Structure Preparation module in MOE. Missing hydrogen atoms and structural irregularities, such as missing side chains or loops, were added, and protonation states were assigned using the Protonate3D tool at a physiological pH of 7.4. Water molecules and non-essential ligands, such as crystallization additives, were removed from the PDB files to focus on protein-ligand interactions. The structures were then optimized using the MMFF94x force field, with a tethering constraint applied to heavy atoms to maintain their experimental conformation while refining hydrogen positions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDocking Protocol\u003c/b\u003e\u003c/p\u003e\u003cp\u003eUsing MOE's Dock module, docking simulations were performed to identify the binding sites for each protein. The Site Finder tool in MOE was used to locate potential binding pockets by analyzing their geometric and chemical properties. The Triangle Matcher algorithm generated initial poses by aligning ligand triplets with receptor site points, placing the ligands in the binding site. Each pose was refined using the Rigid Receptor protocol, where the protein structure remained fixed while the ligand optimized its interactions within the binding pocket. To evaluate molecular interactions, including van der Waals, electrostatic, and hydrogen-bonding effects, we used the MMFF94x force field. The refined poses were ranked based on the London DG scoring function, which estimates the free energy of binding by considering entropic, desolvation, and interaction energy terms.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePose Analysis and Validation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe top-ranked docking poses were visually analyzed using MOE’s visualization tools to assess their conformational plausibility and interactions with key binding pocket residues. Meaningful interactions, such as hydrogen bonds, hydrophobic contacts, π-π stacking, and cation-π interactions, were studied using the Ligand Interactions module. Distances and angles between ligand functional groups and protein residues were measured to evaluate the strength and specificity of the interactions.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eData Analysis\u003c/h2\u003e\u003cp\u003eBinding affinities were reported as London dG scores (kcal mol⁻¹), with lower values indicating more favorable binding. Key interacting residues were identified and cross-checked with literature data to confirm their biological relevance. The docking results were further validated by comparing the predicted binding modes with experimental data from related aminoglycoside-modifying enzymes, ensuring consistency with known resistance mechanisms.\u003c/p\u003e"},{"header":"Result and Discussion","content":"\u003cp\u003e\u003cb\u003eAntimicrobial activity\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eThe antimicrobial activity of bis-benzidinedioxime palladium(II) (Pd), benzidinedioxime (L), Gentamicin (G), and their combinations was evaluated against resistant strains of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eSalmonella. typhi\u003c/em\u003e using the cup plate method. The results, presented as inhibition zone diameters (mm), provide insights into the standalone and synergistic effects of these compounds, particularly in the context of combating antibiotic resistance.\u003c/p\u003e\u003cp\u003eGentamicin (G) alone exhibited no measurable antimicrobial activity against either \u003cem\u003eS. aureus\u003c/em\u003e or \u003cem\u003eS. typhimurium\u003c/em\u003e (inhibition zones of 0 mm), confirming the resistance of these bacterial strains to this aminoglycoside antibiotic. This lack of activity aligns with the growing challenge of aminoglycoside resistance, often mediated by aminoglycoside-modifying enzymes such as acetyltransferases, phosphotransferases, or nucleotidyltransferases, which chemically inactivate the antibiotic. Similarly, benzidinedioxime (L) alone showed no antimicrobial effect against either bacterial strain (0 mm inhibition zones), suggesting that the ligand itself lacks inherent antibacterial properties at the tested concentration of 10 mg mL⁻¹. This is consistent with the expectation that ligands without metal coordination often exhibit limited antimicrobial activity due to the absence of reactive or disruptive chemical moieties (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn contrast, bis-benzidinedioxime palladium(II) (Pd) demonstrated modest antimicrobial activity against \u003cem\u003eS. typhimurium\u003c/em\u003e (14 mm inhibition zone) but no activity against \u003cem\u003eS. aureus\u003c/em\u003e (0 mm). The selective activity against the Gram-negative \u003cem\u003eS. typhimurium\u003c/em\u003e may be attributed to the planar geometry and coordination chemistry of the palladium(II) complex, which likely facilitates interactions with the bacterial outer membrane, increasing permeability or disrupting membrane integrity \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. The lack of activity against \u003cem\u003eS. aureus\u003c/em\u003e suggests that the thicker peptidoglycan layer of Gram-positive bacteria may hinder the complex’s ability to penetrate or interact effectively with critical cellular targets (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eInhibition zone (mm) of Gentamicin, benzidinedioxime, and bis-benzidinedioxime palladium(II) and the combination of the benzidinedioxime and bis-benzidinedioxime palladium(II) with Gentamicin against resistant bacteria (\u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eSalmonella. typhi\u003c/em\u003e ).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCompounds\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eS. aureus\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eS. Typhi\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eInhibition zone (mm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eInhibition zone (mm)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGentmicin (G)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBenzidinedioxime (L)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eG + L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBis-benzidinedioxime Palladium(II) (Pd)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eG + Pd\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003eThe most striking results were observed with the combinations of Gentamicin with benzidinedioxime (G + L) and Gentamicin with bis-benzidinedioxime palladium(II) (G + Pd). The G + L combination produced significant inhibition zones of 23 mm against \u003cem\u003eS. aureus\u003c/em\u003e and 30 mm against \u003cem\u003eS. typhimurium\u003c/em\u003e. These results are unexpected, given the lack of standalone activity for both Gentamicin and benzidinedioxime, suggesting a synergistic interaction that restores or enhances Gentamicin’s antibacterial efficacy Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. This synergy may arise from benzidinedioxime’s ability to interact with bacterial membranes or resistance enzymes, potentially increasing gentamicin uptake or inhibiting aminoglycoside-modifying enzymes. The larger inhibition zone against \u003cem\u003eS. typhimurium\u003c/em\u003e compared to \u003cem\u003eS. aureus\u003c/em\u003e indicates that the Gram-negative outer membrane may be more susceptible to disruption by benzidinedioxime, facilitating greater antibiotic penetration.\u003c/p\u003e\u003cp\u003eThe G + Pd combination also exhibited significant antimicrobial activity, with inhibition zones of 18 mm against \u003cem\u003eS. aureus\u003c/em\u003e and 24 mm against \u003cem\u003eS. typhimurium\u003c/em\u003e. These results are particularly notable given Gentamicin’s inactivity alone and the limited standalone activity of Pd against \u003cem\u003eS. typhimurium\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The synergistic effect of G + Pd is likely driven by the palladium complex’s multifaceted mechanisms, including membrane disruption, DNA binding, and the generation of reactive oxygen species (ROS), as described in the introduction \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. The palladium(II) complex’s planar structure may intercalate into bacterial membranes, enhancing gentamicin uptake. At the same time, its DNA-binding properties and ROS production could amplify the bactericidal effects of Gentamicin by disrupting bacterial replication and cellular integrity.\u003c/p\u003e\u003cp\u003eThe antimicrobial results complement the molecular docking data, which demonstrated that bis-benzidinedioxime palladium(II) exhibited the highest binding affinities across three protein targets (1V0C, 3HAM, and 1KNY), with docking scores ranging from − 10.54 kcal/mol (1V0C) to -7.48 kcal/mol (1KNY). These strong binding affinities, particularly with enzymes such as aminoglycoside 2-acetyltransferase and nucleotidyltransferase, suggest that Pd may target key bacterial metabolic pathways, potentially inhibiting folate synthesis and contributing to its standalone activity against \u003cem\u003eS. typhimurium\u003c/em\u003e. The docking data also revealed extensive hydrogen bonding and electrostatic interactions with residues such as Asp and Glu, which may correlate with the complex’s ability to disrupt bacterial membranes or bind to resistance enzymes, thereby enhancing Gentamicin’s efficacy in the G + Pd combination.\u003c/p\u003e\u003cp\u003eBenzidinedioxime, despite its weaker docking scores (-7.06 to -6.35 kcal/mol), showed remarkable synergy with Gentamicin in the antimicrobial assays (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This suggests that its role as an adjuvant may not rely solely on strong binding to protein targets but instead on subtler effects, such as membrane perturbation or weak interactions with resistance enzymes, which facilitate Gentamicin’s access to the bacterial ribosome. The docking results for Gentamicin (scores ranging from − 8.33 to -7.6 kcal/mol) indicate moderate binding to ribosomal or enzymatic targets Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Still, its inactivity alone underscores the dominance of resistance mechanisms in these strains. The synergy observed in G + L and G + Pd combinations highlights the potential of these adjuvants to overcome such resistance.\u003c/p\u003e\u003cp\u003eThe significant synergistic effects of G + L and G + Pd combinations underscore the potential of dinuclear palladium complexes and their ligands as adjuvants to restore the efficacy of aminoglycosides against bacteria that are resistant to them. The larger inhibition zones against \u003cem\u003eS. typhimurium\u003c/em\u003e compared to \u003cem\u003eS. aureus\u003c/em\u003e suggest that these combinations may be particularly effective against Gram-negative pathogens, which are often more challenging due to their outer membrane barrier. The mechanisms proposed in the introduction—membrane disruption, DNA binding, and ROS generation—are supported by the antimicrobial results and docking data, particularly for Pd, which likely enhances gentamicin uptake and amplifies its bactericidal effects through oxidative stress and metabolic disruption.\u003c/p\u003e\u003cp\u003eThe unexpected synergy of benzidinedioxime with Gentamicin suggests that even non-metalated ligands may play a role in overcoming resistance, possibly by modulating membrane permeability or inhibiting resistance enzymes. This finding warrants further investigation into the specific interactions between benzidinedioxime and bacterial targets, as well as its potential to enhance the efficacy of other classes of antibiotics.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMolecular docking\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eMolecular docking is a powerful computational tool for predicting the binding affinity and interaction profiles of ligands with protein targets, providing insights into their potential as therapeutic agents. In this study, the docking performance of three compounds—bis-benzidinedioxime palladium(II), benzidinedioxime, and Gentamicin against three protein targets (1V0C, 3HAM, and 1KNY) was evaluated using MOE2024 software. The results, including binding energies, root-mean-square deviation (RMSD) refined values, and 2D interaction profiles, offer a comprehensive understanding of the molecular interactions and binding preferences of these compounds.\u003c/p\u003e\u003cp\u003eBinding Energy, expressed as the docking score (S) in kcal/mol, reflects the strength of ligand-protein interactions, with more negative values indicating stronger binding. Across the three protein targets, bis-benzidinedioxime palladium(II) consistently exhibited the highest binding affinity, with docking scores ranging from − 10.54 kcal/mol (1V0C) to -7.48 kcal/mol (1KNY), Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. This suggests that the palladium(II) complex forms more stable interactions with the protein targets compared to benzidinedioxime and Gentamicin. Benzidinedioxime displayed moderate binding affinities, with scores ranging from − 7.06 kcal/mol (1V0C) to -6.35 kcal/mol (3HAM), while Gentamicin showed the lowest affinities, with scores between − 8.33 kcal/mol (1V0C) and − 7.6 kcal/mol (1KNY), Table\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The superior binding affinity of bis-benzidinedioxime palladium(II) may be attributed to the presence of the palladium ion, which likely enhances coordination interactions with key residues in the protein binding pockets.\u003c/p\u003e\u003cp\u003eOut of the protein targets, 1V0C showed the strongest binding interactions with all three compounds, with bis-benzidinedioxime palladium(II) having the highest docking score (-10.54 kcal/mol) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This suggests that 1V0C may have a binding pocket shape and chemical environment that's especially well-suited for these ligands. On the other hand, 1KNY generally had low binding affinities, especially for the bis-benzidinedioxime palladium(II) and gentamicin complexes, suggesting possible steric or electronic mismatches in these protein-ligand interactions.\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBinding energy and RMSD refined value of docking of bis-benzidinedioxime palladium(II), benzidinedioxime, and Gentamicin with Aminoglycoside-N-Acetyltransferase-(6')-Ib [AAC(6')-Ib] (PDB: 1V0C), Aminoglycoside-O-phosphotransferase-(2'')-IIa [APH(2'')-IIa] (PDB ID: 3HAM), and Aminoglycoside-O-nucleotidyltransferase-(4') [ANT (4')] (PDB ID: 1KNY) by using MOE2024 software\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"10\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ecompounds\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e1V0C\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e\u003cp\u003e3HAM\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e\u003cp\u003e1KNY\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"1\" nameend=\"c10\" namest=\"c10\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eRmsd refind\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003eRmsd refind\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003eRmsd refind\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ebis-benzidinedioxime palladium(II)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-10.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e1.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-8.804\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e1.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-7.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e1.13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBenzidinedioxime\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-7.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e1.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-6.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e1.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-6.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e1.34\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003egentamicin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-8.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e.952\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-7.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e1.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-7.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e1.68\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003eThe RMSD refined values clarify the accuracy and reliability of the docked poses, with lower values indicating better alignment with the reference structure. Bis-benzidinedioxime palladium(II) showed RMSD values ranging from 1.13 Å (1KNY) to 1.65 Å (1V0C), suggesting reasonable pose reliability across all targets. Benzidinedioxime exhibited the lowest RMSD values, ranging from 1.1 Å (3HAM) to 1.34 Å (1KNY), indicating highly accurate docking poses, particularly for 3HAM. Gentamicin displayed a broader range of RMSD values (0.952 Å for 1V0C to 1.75 Å for 3HAM), suggesting variability in docking reliability, with the best performance observed for 1V0C. The low RMSD values for benzidinedioxime, particularly with 1V0C (1.24 Å) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), indicate that its docked poses closely match the reference structure, potentially due to its smaller molecular size and simpler structure, which may allow for better accommodation within the binding pocket.\u003c/p\u003e\u003cp\u003eThe 2D interaction diagrams, Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e–\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, provide detailed insights into the specific residue interactions and binding modes of the three compounds with each protein target. The key interactions for each protein-ligand complex, focusing on hydrogen bonds, hydrophobic interactions, and other non-covalent contacts, as well as their implications for binding stability. Bis-benzidinedioxime palladium(II) forms extensive interactions with 1V0C, including hydrogen bonds with residues such as Asp152, Asp119, Trp102, and Trp49, with interaction distances ranging from 3.18 Å to 3.84 Å and energies from − 0.9 to -3.4 kcal/mol Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The presence of multiple hydrogen bonds, particularly with negatively charged aspartate residues, suggests strong electrostatic interactions facilitated by the palladium(II) ion. Benzidinedioxime interacts with a smaller subset of residues, including Trp102 and Asp100, with interaction energies (-1.4 kcal/mol), reflecting its lower binding affinity. Gentamicin forms hydrogen bonds with residues such as Trp164, Asp115, and Asp152; however, its interactions are less extensive, consistent with its moderate docking score of -8.33 kcal/mol (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Previous studies have elucidated the functional importance of specific residues for gentamicin binding and catalysis. Asp115 and Asp152 form critical, buried ionic interactions with amino groups on rings I and II, with Asp115 acting as a proton acceptor (general base). Ser98 binds the 3′′-NH3 group, while Asp100 interacts with both the 3′′-NH3 and 4′′-OH groups \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Furthermore, the tryptophan residues Trp49 and Trp102 contribute significantly via stacking interactions with rings I and II of the aminoglycoside. These binding studies provide a structural rationale for inhibition. The potent binding of Bis-Benzidinedioxime Palladium(II) stems from its ability to exploit the enzyme's intrinsic negative electrostatic potential, forming a dense network of hydrogen bonds, particularly with key catalytic aspartates (Asp115, Asp152). By occupying these critical residues and mimicking aspects of aminoglycoside binding (e.g., interactions with Trp residues), this complex likely acts as a competitive inhibitor, physically blocking the active site and preventing the natural substrate (like Gentamicin) from binding and undergoing acetylation. The weaker binding of benzidinedioxime alone underscores the importance of the palladium ion in enhancing these critical electrostatic and hydrogen-bonding interactions necessary for effective inhibition.\u003c/p\u003e\u003cp\u003eBis-benzidinedioxime palladium(II) interacts with 3HAM through hydrogen bonds with Glu58, Asp232, Asp29, Asp192, and Gly212, with interaction energies ranging from − 0.7 to -4.1 kcal/mol. Benzidinedioxime forms weaker interactions with residue Asp213, with energies around − 0.9 kcal/mol, reflecting its lower docking score (-6.35 kcal/mol). Gentamicin interacts with Asp192, exhibiting lower interaction energies of -0.6 kcal/mol, which suggests lower binding stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCritically, structural analysis shows Gentamicin is positioned for catalysis: the 2''-OH group on its ring C (the phosphorylation target) is poised ~ 3.0 Å from the oxygen atom of Asp192. APH (2'') phosphorylates Gentamicin at the 2''-OH group. Our data supports the established mechanism where Asp192 acts as the essential catalytic base; It precisely orients the 2''-OH group for optimal nucleophilic attack on the gamma-phosphate of ATP and It likely acts as a \"proton relay station\", accepting the proton from the 2''-OH during the reaction (acting as a general base) \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe potent inhibition by Bis-Benzidinedioxime Palladium(II) stems from its ability to exploit the Catalytic Architecture by forming extensive, energetically favorable interactions across the active site, including a strong interaction with the critical Asp192, also block the Catalytic Base; by occupying Asp192 with high affinity (-4.1 kcal/mol interaction possible), the Pd complex directly obstructs this residue's essential functions – orienting the substrate's OH group and accepting its proton. Furthermore, it physically occludes the active site by forming a broad network of interactions (involving Glu58, Asp232, Asp29, and Gly212), effectively filling the active site cavity.\u003c/p\u003e\u003cp\u003eThis combination suggests the Pd complex acts as a highly effective competitive inhibitor. It outcompetes Gentamicin for binding, particularly by sequestering Asp192, thereby preventing the precise substrate positioning and proton transfer essential for phosphorylation. The weaker binding of benzidinedioxime alone underscores the critical role of the palladium ion in achieving this potent, mechanism-targeted inhibition and potentially stabilizing intermediates later in the process (\"proton trap\").\u003c/p\u003e\u003cp\u003eFor 1KNY, bis-benzidinedioxime palladium(II) forms hydrogen bonds with Glu63, Glu67, Glu76, and Ser188, with energies − .05 to -5.4 kcal/mol. Benzidinedioxime interacts with Trp37 and Ser39, with weaker energies (-0.7 to -1.3 kcal/mol). Gentamicin interacts with Glu52, Glu76, Ser39, Ser49, and Mg, with energies ranging from − 0.5 to -3.1 kcal/mol, consistent with its docking score of -7.6 kcal/mol (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCrucially, specific glutamates (Glu67 with ring A, Glu76 with ring A, Glu142 with ring C) form strong hydrogen bonds critical for positioning the aminoglycoside. ANT (4') confers resistance by transferring an adenylyl group from Mg-ATP to the 4'-OH of aminoglycosides like Gentamicin and kanamycin, forming O-adenylylated product and MgPPi \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. The results of molecular docking support the catalytic mechanism, where Glu145 serves as the primary catalytic base. Analysis indicates that the nucleophilic 4'-O atom of the aminoglycoside is positioned for activation (deprotonation). While O2α of ATP or Glu76 were potential activators, the distance data strongly suggest Glu145 (OE2) is responsible for activating the 4'-OH in the wild-type enzyme, acting as the primary proton acceptor. On the other hand, Glu76 acts as a backup base; if Glu145 is absent or disabled, Glu76 can potentially take over this essential proton-accepting role, highlighting functional redundancy in the catalytic machinery. Both Glu145 and Glu76 are critical \"proton relay partners\" for the reaction.\u003c/p\u003e\u003cp\u003eThe potent binding of the Pd complex suggests a highly effective inhibition strategy, as it sequesters the catalytic machinery by directly engaging and occupying multiple key glutamates involved in substrate binding (Glu67, Glu142) and catalysis (Glu76, potentially Glu145 by proximity). Its strong interaction with Glu76 (-5.4 kcal/mol possible) is particularly significant, given Glu76's dual role in substrate binding and its potential as a backup catalytic base. In addition, it blocks the Active Site; the network of interactions with Glu63, Glu67, Glu76, and Ser188 physically occludes the binding cleft, where both the aminoglycoside (such as Gentamicin) and the MgATP cofactor must bind and interact. Moreover, the Pd complex disrupts electrostatic steering \u0026amp; catalysis through binding tightly to the critical glutamates. The Pd complex disrupts the precise electrostatic environment needed to position the aminoglycoside's 4'-OH for nucleophilic attack and interferes with the proton transfer essential for activating the nucleophile (whether by Glu145 or Glu76). Additionally, it has an extensive binding network and a higher affinity (compared to benzidinedioxime), allowing it to effectively outcompete Gentamicin for the active site.\u003c/p\u003e\u003cp\u003eIn conclusion, Bis-Benzidinedioxime Palladium(II) acts as a potent competitive inhibitor of ANT(4') by exploiting the enzyme's catalytic architecture. It achieves this by forming a high-affinity complex that blocks key glutamates (Glu67, Glu76, Glu142), which are crucial for substrate binding, and occupies the catalytic base (Glu145 or Glu76), thereby preventing both substrate positioning and the essential proton transfer step required for adenylylation. The weaker binding of benzidinedioxime underscores the critical role of the palladium ion in enabling this potent, mechanism-targeted inhibition\u003c/p\u003e\u003cp\u003eThe docking results reveal that bis-benzidinedioxime palladium(II) consistently outperforms benzidinedioxime and Gentamicin in terms of binding affinity across all protein targets. This is likely due to the palladium(II) ion, which enhances coordination and electrostatic interactions with negatively charged residues such as aspartate and glutamate, as observed in the 2D interaction profiles. The presence of multiple hydrogen bonds contributes to the stability of the palladium complex in the binding pocket.\u003c/p\u003e\u003cp\u003eBenzidinedioxime, lacking the palladium ion, exhibits weaker binding affinities and fewer interactions, suggesting that its simpler structure limits its ability to form strong contacts with the protein targets. Gentamicin, despite its larger and more complex structure, forms moderate interactions, likely due to its multiple hydroxyl and amine groups, which facilitate hydrogen bonding but may introduce steric constraints in certain binding pockets (e.g., 3HAM).\u003c/p\u003e\u003cp\u003eThe shape of the binding pocket and the type of amino acid residues of the protein play an essential role in the value of binding affinities of the compound with the protein. 1V0C appears to be the most accommodating target to benzidinedioxime and Bis-benzidinedioximme palladium(II), likely due to a favorable arrangement of polar and charged residues that match the ligands' functional groups. In contrast, 1KNY offers more difficult binding environments, possibly due to steric constraints or weak electrostatic interactions.\u003c/p\u003e\u003cp\u003ePrevious research found that benzidinedioxime had a medium inhibition zone against isolated S. aureus and S. typhimurium (14.3 ± 1.6 mm and 12.7 ± 1.8 mm, respectively). In contrast, Gentamicin showed a stronger inhibition zone (26 mm and 25 mm, respectively) (Ebrahim et al., 2023). This new result suggests that these bacteria have developed resistance to benzidinedioxime through at least one aminoglycoside enzyme. The molecular docking study of benzidinedioxime and bis-benzidinedioxime in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e showed that the hydrogen atom on the hydroxyl group doesn't interact with Glu 142 or Glu 76, resulting in a weak nucleophilic attack of the oxygen atom on the phosphate group. This suggests that nucleotidyltransferase enzymes don't contribute to bacterial resistance. The 2D interaction of benzidinedioxime and bis-benzidinedioxime with Aminoglycoside-O-phosphotransferase-(2'')-IIa [APH(2'')-IIa] (PDB ID: 3HAM) in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows a hydrogen bond formed between benzidinedioxime and Asp 213, increasing its nucleophilic attack properties on the phosphate group in the enzyme. When benzidinedioxime is linked to the protein and ADP by a hydroxyl group, an unstable transition state forms according to the SN2 mechanism. This unstable state leads to the breakdown of the hydrogen-oxygen bond and the formation of an oxygen-ADP bond, facilitating benzidinedioxime phosphorylation, as shown in (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDespite the bis-benzidinedioxime palladium(II) establishing a strong hydrogen bond with Glu 56 in Aminoglycoside-O-phosphotransferase-(2'')-IIa [APH(2'')-IIa] (PDB ID: 3HAM) through its hydroxyl group, the enzyme did not modify it due to the significant distance of 10.81 °A between the hydroxyl group and ADP, as illustrated in (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The findings from the antimicrobial test corroborated this observation, as bis-benzidinedioxime palladium(II) exhibited antimicrobial effects against S. Typhi (14 mm), as summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. This outcome suggests that the resistance of S. Typhi to aminoglycosides was not attributed to Aminoglycoside-O-phosphotransferase-(2'')-IIa [APH(2'')-IIa].\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e illustrates the resistance mechanism of Aminoglycoside-N-Acetyltransferase-(6')-Ib [AAC(6')-Ib] (PDB: 1V0C) to Benzidinedioxime and bis-benzidinedioxime palladium(II). Bis-benzidinedioxime palladium(II) forms two ionic bonds with Asp 152 and Asp 115, creating a strong nucleophilic attack on COA due to the negative charge on the imine group. This property enables a strong nucleophilic attack on the acetyl group of COA. Benzidinedioxime is modified by COA through a nucleophilic interaction of the nitrogen atom's lone pair of electrons with the acetyl group in COA. The study concluded that S. Aureus is resistant to both compounds and Gentamicin due to Aminoglycoside-N-Acetyltransferase-(6')-Ib [AAC(6')-Ib]. Molecular docking and antimicrobial activity results demonstrated the synergistic potential of benzidinedioxime and bis-benzidinedioxime palladium(II) to enhance the efficacy of Gentamicin against resistant bacteria (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates the significant potential of bis-benzidinedioxime palladium(II) (Pd) and its ligand, benzidinedioxime (L), as adjuvants to overcome gentamicin resistance in Staphylococcus aureus and Salmonella. typhi. Molecular docking revealed that Pd exhibits superior binding affinity, particularly against the aminoglycoside-modifying enzymes AAC(6')-Ib (1V0C) and APH(2'')-IIa (3HAM), driven by strong electrostatic interactions with key Asp/Glu residues. The palladium complex's strong electrostatic interactions, especially with aspartate and glutamate residues, highlight its potential as a lead compound for further drug development. Antimicrobial assays further demonstrate that bis-benzidinedioxime palladium(II) (Pd) and benzidinedioxime (L) significantly enhance the efficacy of gentamicin (G) against resistant Staphylococcus aureus and Salmonella. Typhi, yielding inhibition zones of 18\u0026ndash;24 mm and 23\u0026ndash;30 mm, respectively. This potent synergy suggests that both Pd and L function by effectively inhibiting key resistance enzymes, AAC(6')-Ib and APH(2'')-IIa, thereby protecting gentamicin from inactivation. Furthermore, the docking analysis indicates that Pd achieves inhibition of APH(2'')-IIa while evading altering the structure of the palladium complex. These findings establish dinuclear palladium complexes and their ligand as promising candidates for the development of resistance-breaking adjuvants to revitalize aminoglycoside therapy, paving the way for mechanistic studies and structural optimization to develop innovative treatments for antibiotic-resistant bacterial infections.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eReem M. A. Ebrahim investigation, data analysis, writing original draft, visualization,\u0026nbsp;Nooh Mohamed Hajhamed, molecular docking, writing, Abdallah Elssir Ahmed\u003csup\u003e\u0026nbsp;\u003c/sup\u003eand Ayman Azhary,\u0026nbsp;data analysis,\u0026nbsp;visualization, writing, review\u0026nbsp;and editing, Yousif Sulfab, and Elmugdad A. Ali\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003esupervision,\u0026nbsp;writing, review and editing.\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors received no funding for this work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eIsernia, S.\u003cem\u003e et al.\u003c/em\u003e The key role of depression and supramarginal gyrus in frailty: a cross-sectional study. \u003cem\u003eFrontiers in Aging Neuroscience\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 1292417 (2023).\u003c/li\u003e\n\u003cli\u003eMancuso, G., Midiri, A., Gerace, E. \u0026amp; Biondo, C. 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Theoretical studies on mechanism of inactivation of kanamycin A by 4\u0026prime;-O-Nucleotidyltransferase. \u003cem\u003eFrontiers in chemistry\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 660 (2019).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Antibiotic resistance, bis-benzidinedioxime palladium (II), benzidinedioxime, Gentamicin, molecular docking, antimicrobial synergy, Staphylococcus aureus, Salmonella. typhi","lastPublishedDoi":"10.21203/rs.3.rs-7216024/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7216024/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe rising threat of antibiotic resistance necessitates innovative strategies to enhance the effectiveness of traditional antibiotics, such as aminoglycosides. This research explores the potential of bis-benzidinedioxime palladium(II) (Pd) and benzidinedioxime (L) as adjuvants to boost Gentamicin (G) activity against resistant strains of Staphylococcus aureus and Salmonella. typhi. Molecular docking using MOE2024 showed that Pd has stronger binding affinities (docking scores: -10.54 to -7.48 kcal/mol) across all aminoglycoside-modifying enzyme targets (1V0C, 3HAM, and 1KNY), especially with 1V0C, which exhibits the strongest interactions due to electrostatic contacts with aspartate and glutamate residues. Benzidinedioxime and Gentamicin had moderate affinities (-7.06 to -6.41 kcal/mol and \u0026minus;\u0026thinsp;8.33 to -7.6 kcal/mol, respectively). Antimicrobial tests using the cup plate method showed that Pd and L alone had no significant activity (inhibition zones of 0\u0026ndash;14 mm), while Gentamicin was ineffective against both strains. However, combinations of G\u0026thinsp;+\u0026thinsp;L and G\u0026thinsp;+\u0026thinsp;Pd showed strong synergy, with inhibition zones of 23\u0026ndash;30 mm and 18\u0026ndash;24 mm, respectively, especially against S. typhimurium. These findings suggest that benzidinedioxime and bis-benzidinedioxime palladium(II) enhance gentamicin\u0026rsquo;s effectiveness by strongly inhibiting Aminoglycoside-N-Acetyltransferase-(6')-Ib [AAC(6')-Ib] (PDB: 1V0C) and Aminoglycoside-O-Phosphotransferase-(2'')-IIa [APH(2'')-IIa] (PDB ID: 3HAM). Additionally, bis-benzidinedioxime palladium(II) blocks Aminoglycoside-O-Phosphotransferase-(2'')-IIa [APH(2'')-IIa] without changing the structure of the palladium complex. These results highlight the potential of dinuclear palladium complexes and their ligands as adjuvants to fight against aminoglycoside resistance, laying the groundwork for further studies and the development of new therapies.\u003c/p\u003e","manuscriptTitle":"Synergistic Potential of Bis-Benzidinedioxime Palladium(II) and Benzidinedioxime as Enhancers of Gentamicin Efficacy Against Resistant Bacteria: Insights from Molecular Docking and Antimicrobial Studies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-05 14:29:14","doi":"10.21203/rs.3.rs-7216024/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":"3a5637b8-57f5-4c4a-afce-ebccdc479dbf","owner":[],"postedDate":"August 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":52554750,"name":"Biological sciences/Biochemistry"},{"id":52554751,"name":"Biological sciences/Drug discovery"},{"id":52554752,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2025-09-29T08:24:21+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-05 14:29:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7216024","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7216024","identity":"rs-7216024","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}