Target-Mediated Fluoroquinolone Resistance in Neisseria gonorrhoeae: Actions of Ciprofloxacin against Gyrase and Topoisomerase IV.

Fluoroquinolones make up a critically important class of antibacterials administered worldwide to treat human infections. However, their clinical utility has been curtailed by target-mediated resistance, which is caused by mutations in the fluoroquinolone targets, gyrase and topoisomerase IV. An important pathogen that has been affected by this resistance is Neisseria gonorrhoeae, the causative agent of gonorrhea. Over 82 million new cases of this sexually transmitted infection were reported globally in 2020. Despite the impact of fluoroquinolone resistance on gonorrhea treatment, little is known about the interactions of this drug class with its targets in this bacterium. Therefore, we investigated the effects of the fluoroquinolone ciprofloxacin on the catalytic and DNA cleavage activities of wild-type gyrase and topoisomerase IV and the corresponding enzymes that harbor mutations associated with cellular and clinical resistance to fluoroquinolones. Results indicate that ciprofloxacin interacts with both gyrase (its primary target) and topoisomerase IV (its secondary target) through a water-metal ion bridge that has been described in other species. Moreover, mutations in amino acid residues that anchor this bridge diminish the susceptibility of the enzymes for the drug, leading to fluoroquinolone resistance. Results further suggest that ciprofloxacin primarily induces its cytotoxic effects by enhancing gyrase-mediated DNA cleavage as opposed to inhibiting the DNA supercoiling activity of the enzyme. In conclusion, this work links the effects of ciprofloxacin on wild-type and resistant gyrase to results reported for cellular and clinical studies and provides a mechanistic explanation for the targeting and resistance of fluoroquinolones in N. gonorrhoeae.

A CcdB toxin-derived peptide acts as a broad-spectrum antibacterial therapeutic in infected mice.

The bacterial toxin CcdB (Controller of Cell death or division B) targets DNA Gyrase, an essential bacterial topoisomerase, which is also the molecular target for fluoroquinolones. Here, we present a short cell-penetrating 24-mer peptide, CP1-WT, derived from the Gyrase-binding region of CcdB and examine its effect on growth of Escherichia coli, Salmonella Typhimurium, Staphylococcus aureus and a carbapenem- and tigecycline-resistant strain of Acinetobacter baumannii in both axenic cultures and mouse models of infection. The CP1-WT peptide shows significant improvement over ciprofloxacin in terms of its in vivo therapeutic efficacy in treating established infections of S. Typhimurium, S. aureus and A. baumannii. The molecular mechanism likely involves inhibition of Gyrase or Topoisomerase IV, depending on the strain used. The study validates the CcdB binding site on bacterial DNA Gyrase as a viable and alternative target to the fluoroquinolone binding site.

In silico evaluation of usnic acid derivatives to discover potential antibacterial drugs against DNA gyrase B and DNA topoisomerase IV.

Due to the rising increase in infectious diseases brought on by bacteria and anti-bacterial drug resistance, antibacterial therapy has become difficult. The majority of first-line antibiotics are no longer effective against numerous germs, posing a new hazard to global human health in the 21st century. Through the drug-likeness screening, 184 usnic acid derivatives were selected from an in-house database of 340 usnic acid compounds. The pharmacokinetics (ADMET) prediction produced fifteen hit compounds, of which the lead molecule was subsequently obtained through a molecular docking investigation. The lead compounds, labelled compound-277 and compound-276, respectively, with the substantial binding affinity towards the enzymes were obtained through further docking simulation on the DNA gyrase and DNA topoisomerase proteins. Additionally, molecular dynamic (MD) simulation was performed for 300 ns on the lead compounds in order to confirm the stability of the docked complexes and the binding pose discovered during docking tests. Due to their intriguing pharmacological characteristics, these substances may be promising therapeutic candidate for anti-bacterial medication.Communicated by Ramaswamy H. Sarma.

Role of the Water-Metal Ion Bridge in Quinolone Interactions with Escherichia coli Gyrase.

Fluoroquinolones are an important class of antibacterials, and rising levels of resistance threaten their clinical efficacy. Gaining a more full understanding of their mechanism of action against their target enzymes-the bacterial type II topoisomerases gyrase and topoisomerase IV-may allow us to rationally design quinolone-based drugs that overcome resistance. As a step toward this goal, we investigated whether the water-metal ion bridge that has been found to mediate the major point of interaction between Escherichia coli topoisomerase IV and Bacillus anthracis topoisomerase IV and gyrase, as well as Mycobacterium tuberculosis gyrase, exists in E. coli gyrase. This is the first investigation of the water-metal ion bridge and its function in a Gram-negative gyrase. Evidence suggests that the water-metal ion bridge does exist in quinolone interactions with this enzyme and, unlike the Gram-positive B. anthracis gyrase, does use both conserved residues (serine and acidic) as bridge anchors. Furthermore, this interaction appears to play a positioning role. These findings raise the possibility that the water-metal ion bridge is a universal point of interaction between quinolones and type II topoisomerases and that it functions primarily as a binding contact in Gram-positive species and primarily as a positioning interaction in Gram-negative species. Future studies will explore this possibility.

Development and radiosterilization of new hydrazono-quinoline hybrids as DNA gyrase and topoisomerase IV inhibitors: Antimicrobial and hemolytic activities against uropathogenic isolates with molecular docking study.

This study aimed to synthesize new potent quinoline derivatives based on hydrazone moieties and evaluate their antimicrobial activity. The newly synthesized hydrazono-quinoline derivatives 2, 5a, 9, and 10b showed the highest antimicrobial activity with MIC values ≤1.0 µg/ml against bacteria and ≤8.0 µg/ml against the fungi. Further, these derivatives exhibited bactericidal and fungicidal effects with MBC/MIC and MFC/MIC ratio ≤4. Surprisingly, the most active compounds displayed good inhibition to biofilm formation with MBEC values ranging between (40.0 ± 10.0 - 230.0 ± 31.0) and (67.0 ± 24.0 - 347.0 ± 15.0) µg/ml against Staphylococcus aureus and Pseudomonas aeruginosa, respectively. The hemolytic assays confirmed that the hydrazono-quinoline derivatives are non-toxic with low % lysis values ranging from 4.62% to 14.4% at a 1.0 mg/ml concentration. Besides, compound 5a exhibited the lowest hemolytic activity value of ~4.62%. Furthermore, the study suggests that the hydrazono-quinoline analogs exert their antibacterial activity as dual inhibitors for DNA gyrase and DNA topoisomerase IV enzymes with IC50 values ranging between (4.56 ± 0.3 - 21.67 ± 0.45) and (6.77 ± 0.4 - 20.41 ± 0.32) µM, respectively. Additionally, the recent work advocated that compound 5a showed the reference SAL at the É£-radiation dose of 10.0 kGy in the sterilization process without affecting its chemical structure. Finally, the in silico drug-likeness, toxicity properties, and molecular docking simulation were performed. Besides, the result exhibited good oral-bioavailability, lower toxicity prediction, and lower binding energy with good binding mode rather than the positive control.

Identification of secondary metabolites from Crescentia cujete as promising antibacterial therapeutics targeting type 2A topoisomerases through molecular dynamics simulation.

The potential of fluoroquinolones as remarkable antibacterial agents evolved from their ability to generate 'poison' complexes between type IIA topoisomerases [topo2As (DNA gyrases and topoisomerases IV)] and DNA. However, the overuse of fluoroquinolones coupled with chromosomal mutations in topo2As has increased incidence of resistance and consequently undermined the application of the currently available fluoroquinolones in clinical practice. In this study, the molecular mechanism of interaction between the secondary metabolites of Crescentia cujete (an underutilized plant with proven anti-bacterial activity) and topo2As was investigated using computational methods. Through molecular docking, the top five compounds with the best affinity for each topo2A were identified and subjected to molecular dynamics simulation over a period of 100 ns. The results revealed that the identified compounds had higher binding energy values than the reference standards against the topo2As except for topoisomerase IV ParC, and this was consistent with the results of the structural stability and compactness of the resulting complexes. Specifically, cistanoside D (-49.18 kcal/mol), chlorogenic acid (-55.55 kcal/mol), xylocaine (-33.08 kcal/mol), and naringenin (-35.48 kcal/mol) had the best affinity for DNA gyrase A, DNA gyrase B, topoisomerase IV ParC, and topoisomerase IV ParE, respectively. Of the constituents of C. cujete evaluated, only apigenin and luteolin had affinity for all the four targets. These observations are indicative of the identified compounds as potential inhibitors of topo2As as evidenced from the molecular interactions including hydrogen bonds established with the active site amino acids of the respective targets. This is the first in silico report on the antibacterial effect of C. cujete and the findings would guide structural modification of the identified compounds as novel inhibitors of topo2As for further in vitro and in vivo assessments.

Hydrazone analogues with promising antibacterial profiles: Synthesis, morphology, in vitro and in silico approaches.

The emergence of resistance to antibacterial drugs remains an important global threat that necessitates an urgent need for the discovery of alternative drugs. This study was undertaken to synthesize some novel nitroaryl/heteroaryl hydrazone derivatives as potential antibacterial agents. After synthesizing by a simple reaction between quinoline/quinazoline hydrazine and nitroaryl/heteroaryl aldehydes, all the compounds were screened for their antibacterial activities, cytotoxicity and in silico investigations. The compound, 2-(4-nitrobenzylidene)-1-(quinazolin-4-yl)hydrazine (1b), displayed significant antimicrobial activity against several susceptible and resistant bacteria without any cytotoxicity. Moreover, scanning electron microscopy (SEM) revealed the complete destruction of Staphylococcus aureus and Escherichia coli following exposure to this compound after 2 h exposure. The in silico studies confirmed the better binding energy of these compounds in comparison with the reference drugs in complex with topoisomerase IV and bacterial ribosomal receptor. Compound 1b can be considered a promising lead compound for the development of broad-spectrum antibacterial medications after further studies.

Extended sister-chromosome catenation leads to massive reorganization of the E. coli genome.

In bacteria, chromosome segregation occurs progressively from the origin to terminus within minutes of replication of each locus. Between replication and segregation, sister loci are held in an apparent cohesive state by topological links. The decatenation activity of topoisomerase IV (Topo IV) is required for segregation of replicated loci, yet little is known about the structuring of the chromosome maintained in a cohesive state. In this work, we investigated chromosome folding in cells with altered decatenation activities. Within minutes after Topo IV inactivation, massive chromosome reorganization occurs, associated with increased in contacts between nearby loci, likely trans-contacts between sister chromatids, and in long-range contacts between the terminus and distant loci. We deciphered the respective roles of Topo III, MatP and MukB when TopoIV activity becomes limiting. Topo III reduces short-range inter-sister contacts suggesting its activity near replication forks. MatP, the terminus macrodomain organizing system, and MukB, the Escherichia coli SMC, promote long-range contacts with the terminus. We propose that the large-scale conformational changes observed under these conditions reveal defective decatenation attempts involving the terminus area. Our results support a model of spatial and temporal partitioning of the tasks required for sister chromosome segregation.

The MukB-topoisomerase IV interaction mutually suppresses their catalytic activities.

The bacterial condensin MukB and the cellular chromosomal decatenase, topoisomerase IV interact and this interaction is required for proper condensation and topological ordering of the chromosome. Here, we show that Topo IV stimulates MukB DNA condensation by stabilizing loops in DNA: MukB alone can condense nicked plasmid DNA into a protein-DNA complex that has greater electrophoretic mobility than that of the DNA alone, but both MukB and Topo IV are required for a similar condensation of a linear DNA representing long stretches of the chromosome. Remarkably, we show that rather than MukB stimulating the decatenase activity of Topo IV, as has been argued previously, in stoichiometric complexes of the two enzymes each inhibits the activity of the other: the ParC subunit of Topo IV inhibits the MukF-stimulated ATPase activity of MukB and MukB inhibits both DNA crossover trapping and DNA cleavage by Topo IV. These observations suggest that when in complex on the DNA, Topo IV inhibits the motor function of MukB and the two proteins provide a stable scaffold for chromosomal DNA condensation.

Design and synthesis of ciprofloxacin-sulfonamide hybrids to manipulate ciprofloxacin pharmacological qualities: Potency and side effects.

Fluoroquinolones are a class of antibacterial agents used clinically to treat a wide array of bacterial infections. Although being potent, susceptibility to CNS side effects limits their use. It was observed that improvements in absorption, activity and side effects were achieved via modifications at the N atom of the C7 of the side chain. To meet the increasing demand for development of new antibacterial agents, nineteen novel ciprofloxacin-sulfonamide hybrid molecules were designed, synthesized and characterized by IR, 1H NMR and 13C NMR as potential antibacterial agents with dual DNA gyrase/topoisomerase IV inhibitory activity. Most of the synthesized compounds showed significant antibacterial activity that was revealed by testing their inhibitory activity against DNA gyrase, DNA topoisomerase IV as well as their minimum inhibitory concentration against Staphylococcus aureus. Six ciprofloxacin-sulfonamide hybrids (3f, 5d, 7a, 7d, 7e and 9b) showed potent inhibitory activity against DNA topoisomerase IV, compared to ciprofloxacin (IC50: 0.55 µM), with IC50 range: 0.23-0.44 µM. DNA gyrase was also efficiently inhibited by five ciprofloxacin-sulfonamide hybrids (3f, 5d, 5e, 7a and 7d) with IC50 range: 0.43-1.1 µM (IC50 of ciprofloxacin: 0.83 µM). Compounds 3a and 3b showed a marked improvement in the antibacterial activity over ciprofloxacin against both Gram-positive and Gram-negative pathogens, namely, Staphylococcus aureus Newman and Escherichia coli ATCC8739, with MIC = 0.324 and 0.422 µM, respectively, that is 4.2-fold and 3.2-fold lower than ciprofloxacin (MIC = 1.359 µM) against the Gram-positive Staphylococcus aureus, and MIC = 0.025 and 0.013 µM, respectively, that is 10.2-fold and 19.6-fold lower than ciprofloxacin (MIC = 0.255 µM) against the Gram-negative Escherichia coli ATCC8739. Also, the most active compounds showed lower CNS and convulsive side effects compared to ciprofloxacin with a concomitant decrease in GABA expression.

Optimization of TopoIV Potency, ADMET Properties, and hERG Inhibition of 5-Amino-1,3-dioxane-Linked Novel Bacterial Topoisomerase Inhibitors: Identification of a Lead with In Vivo Efficacy against MRSA.

Novel bacterial topoisomerase inhibitors (NBTIs) are among the most promising new antibiotics in preclinical/clinical development. We previously reported dioxane-linked NBTIs with potent antistaphylococcal activity and reduced hERG inhibition, a key safety liability. Herein, polarity-focused optimization enabled the delineation of clear structure-property relationships for both microsomal metabolic stability and hERG inhibition, resulting in the identification of lead compound 79. This molecule demonstrates potent antibacterial activity against diverse Gram-positive pathogens, inhibition of both DNA gyrase and topoisomerase IV, a low frequency of resistance, a favorable in vitro cardiovascular safety profile, and in vivo efficacy in a murine model of methicillin-resistant Staphylococcus aureus infection.

Design, synthesis, and antibacterial evaluation of new quinoline-1,3,4-oxadiazole and quinoline-1,2,4-triazole hybrids as potential inhibitors of DNA gyrase and topoisomerase IV.

DNA gyrase and topoisomerase IV (topo IV) inhibitors are among the most interesting antibacterial drug classes without antibacterial pipeline representative. Twenty-four new quinoline-1,3,4-oxadiazole and quinoline-1,2,4-triazole hybrids were developed and tested against DNA gyrase and topoisomerase IV from Escherichia coli and Staphylococcus aureus. The most potent compounds 4c, 4e, 4f, and 5e displayed an IC50 of 34, 26, 32, and 90 nM against E. coli DNA gyrase, respectively (novobiocin, IC50 = 170 nM). The activities of 4c, 4e, 4f, and 5e on DNA gyrase from S. aureus were weaker than those on E. coli gyrase. Compound 4e showed IC50 values (0.47 µM and 0.92 µM) against E. coli topo IV and S. aureus topo IV, respectively in comparison to novobiocin (IC50 = 11, 27 µM, respectively). Antibacterial activity against Gram-positive and Gram-negative bacterial strains has been studied. Some compounds have demonstrated superior antibacterial activity to ciprofloxacin against some of the bacterial strain studied. The most active compounds in this study showed no cytotoxic effect with cell viability>86%. Finally, a molecular docking analysis was performed to investigate the binding mode and interactions of the most active compounds to the active site of DNA gyrase and topoisomerase IV (topo IV) enzymes.

What makes a type IIA topoisomerase a gyrase or a Topo IV?

Type IIA topoisomerases catalyze a variety of different reactions: eukaryotic topoisomerase II relaxes DNA in an ATP-dependent reaction, whereas the bacterial representatives gyrase and topoisomerase IV (Topo IV) preferentially introduce negative supercoils into DNA (gyrase) or decatenate DNA (Topo IV). Gyrase and Topo IV perform separate, dedicated tasks during replication: gyrase removes positive supercoils in front, Topo IV removes pre-catenanes behind the replication fork. Despite their well-separated cellular functions, gyrase and Topo IV have an overlapping activity spectrum: gyrase is also able to catalyze DNA decatenation, although less efficiently than Topo IV. The balance between supercoiling and decatenation activities is different for gyrases from different organisms. Both enzymes consist of a conserved topoisomerase core and structurally divergent C-terminal domains (CTDs). Deletion of the entire CTD, mutation of a conserved motif and even by just a single point mutation within the CTD converts gyrase into a Topo IV-like enzyme, implicating the CTDs as the major determinant for function. Here, we summarize the structural and mechanistic features that make a type IIA topoisomerase a gyrase or a Topo IV, and discuss the implications for type IIA topoisomerase evolution.

Topological stress is responsible for the detrimental outcomes of head-on replication-transcription conflicts.

Conflicts between the replication and transcription machineries have profound effects on chromosome duplication, genome organization, and evolution across species. Head-on conflicts (lagging-strand genes) are significantly more detrimental than codirectional conflicts (leading-strand genes). The fundamental reason for this difference is unknown. Here, we report that topological stress significantly contributes to this difference. We find that head-on, but not codirectional, conflict resolution requires the relaxation of positive supercoils by the type II topoisomerases DNA gyrase and Topo IV, at least in the Gram-positive model bacterium Bacillus subtilis. Interestingly, our data suggest that after positive supercoil resolution, gyrase introduces excessive negative supercoils at head-on conflict regions, driving pervasive R-loop formation. Altogether, our results reveal a fundamental mechanistic difference between the two types of encounters, addressing a long-standing question in the field of replication-transcription conflicts.

Changes in the topology of DNA replication intermediates: Important discrepancies between in vitro and in vivo.

The topology of DNA duplexes changes during replication and also after deproteinization in vitro. Here we describe these changes and then discuss for the first time how the distribution of superhelical stress affects the DNA topology of replication intermediates, taking into account the progression of replication forks. The high processivity of Topo IV to relax the left-handed (+) supercoiling that transiently accumulates ahead of the forks is not essential, since DNA gyrase and swiveling of the forks cooperate with Topo IV to accomplish this task in vivo. We conclude that despite Topo IV has a lower processivity to unlink the right-handed (+) crossings of pre-catenanes and fully replicated catenanes, this is indeed its main role in vivo. This would explain why in the absence of Topo IV replication goes-on, but fully replicated sister duplexes remain heavily catenated.

Structural insights into the biological activity of a thioxopyrimidine derivative.

Bacterial resistance to the main widespread antibiotics, such as those based on quinolones, is a concern of the scientific community, leading to the search for new classes of molecules that can be used as an alternative. Here, we investigate the crystalline and chemical characteristics of a thioxopyrimide to understand its demonstrated biological activity and to identify which portion of the molecule can be used as a framework to develop new antibiotics. For this purpose, structural studies of ethyl 4-methyl-2-phenyl-6-thioxo-1,6-dihydro-5-pyrimidinecarboxylate were carried out aided by Hirshfeld surface analysis, as well as theoretical calculations on frontier molecular orbitals, molecular electrostatic potential, and conformational stability, in addition to docking studies targeting topoisomerase IV. The docking results show a reasonable accommodation of the molecule at the topoisomerase IV binding site and interact mainly by hydrogen bonds between the thioxopyrimidine portion with Glu198, Thr292, and Gly225, aided by hydrophobic interactions involving the rest of the molecule. These results suggest a relationship between the antibacterial activity shown mainly with the 4-thioxopyrimidine portion, leading to the investigation of new compounds that use this scaffold.

New dual ATP-competitive inhibitors of bacterial DNA gyrase and topoisomerase IV active against ESKAPE pathogens.

The rise in multidrug-resistant bacteria defines the need for identification of new antibacterial agents that are less prone to resistance acquisition. Compounds that simultaneously inhibit multiple bacterial targets are more likely to suppress the evolution of target-based resistance than monotargeting compounds. The structurally similar ATP binding sites of DNA gyrase and topoisomerase Ⅳ offer an opportunity to accomplish this goal. Here we present the design and structure-activity relationship analysis of balanced, low nanomolar inhibitors of bacterial DNA gyrase and topoisomerase IV that show potent antibacterial activities against the ESKAPE pathogens. For inhibitor 31c, a crystal structure in complex with Staphylococcus aureus DNA gyrase B was obtained that confirms the mode of action of these compounds. The best inhibitor, 31h, does not show any in vitro cytotoxicity and has excellent potency against Gram-positive (MICs: range, 0.0078-0.0625 µg/mL) and Gram-negative pathogens (MICs: range, 1-2 µg/mL). Furthermore, 31h inhibits GyrB mutants that can develop resistance to other drugs. Based on these data, we expect that structural derivatives of 31h will represent a step toward clinically efficacious multitargeting antimicrobials that are not impacted by existing antimicrobial resistance.

Topoisomerase IV tracks behind the replication fork and the SeqA complex during DNA replication in Escherichia coli.

Topoisomerase IV (TopoIV) is a vital bacterial enzyme which disentangles newly replicated DNA and enables segregation of daughter chromosomes. In bacteria, DNA replication and segregation are concurrent processes. This means that TopoIV must continually remove inter-DNA linkages during replication. There exists a short time lag of about 10-20 min between replication and segregation in which the daughter chromosomes are intertwined. Exactly where TopoIV binds during the cell cycle has been the subject of much debate. We show here that TopoIV localizes to the origin proximal side of the fork trailing protein SeqA and follows the movement pattern of the replication machinery in the cell.

Novel Tetrazole-Based Antimicrobial Agents Targeting Clinical Bacteria Strains: Exploring the Inhibition of Staphylococcus aureus DNA Topoisomerase IV and Gyrase.

Eleven novel imide-tetrazoles were synthesized. In the initial stage of research, in silico structure-based pharmacological prediction was conducted. All compounds were screened for antimicrobial activity using standard and clinical strains. Within the studied group, compounds 1-3 were recognized as leading structures with the most promising results in antimicrobial studies. Minimal inhibitory concentration values for compounds 1, 2, 3 were within the range of 0.8-3.2 µg/mL for standard and clinical Gram-positive and Gram-negative bacterial strains, showing in some cases higher activity than the reference Ciprofloxacin. Additionally, all three inhibited the growth of all clinical Staphylococci panels: Staphylococcus aureus (T5592; T5591) and Staphylococcus epidermidis (5253; 4243) with MIC values of 0.8 µg/mL. Selected compounds were examined in topoisomerase IV decatenation assay and DNA gyrase supercoiling assay, followed by suitable molecular docking studies to explore the possible binding modes. In summary, the presented transition from substrate imide-thioureas to imide-tetrazole derivatives resulted in significant increase of antimicrobial properties. The compounds 1-3 proposed here provide a promising basis for further exploration towards novel antimicrobial drug candidates.

Design, Synthesis and Biological Evaluation of Novel Heterocyclic Fluoroquinolone Citrate Conjugates as Potential Inhibitors of Topoisomerase IV: A Computational Molecular Modeling Study.

BACKGROUND & OBJECTIVE: A facile and efficient method for the synthesis of novel derivatives of FQ citrate conjugates with 1,2,4-triazoles and 1,3,4-oxadiazole scaffolds 8-11 using conventional, as well as microwave irradiation methods, was reported. Based on these original building blocks, the new derivatives of 3, 7-disubstituted fluoroquinolones bearing the oxadiazolyl-triazole groups were obtained. These invaluable derivatives are of great interest in medicinal and pharmaceutical studies because of their important biological properties. METHODS: All the reactions were examined under conventional as well as microwave mediated conditions. The structures of obtained compounds were confirmed by 1H NMR, 13C NMR, IR HRMS spectroscopy, and elemental analysis. The antibacterial and antifungal activities of these compounds were screened against Gram-positive, Gram-negative bacteria, and fungal stains by the agar well diffusion method. Cytotoxic assay of the title compounds was evaluated against cervical carcinoma cell line (HeLa) by using the MTT assay. The crystal structure of the Quinolone-DNA cleavage complex of type IV topoisomerase from S. pneumoniae (PDB ID: 3RAE) complex was obtained from the Protein Database (PDB, http:// www.rcsb.org). Molecular properties prediction-drug likeness was studied by Molinspiration and Molsoft software, while lipophilicity and solubility parameters were studied using the Osiris program. RESULTS: A novel approach for the synthesis of benzylthio-1,2,4-triazole and 1,3,4-oxadiazoles core with regioisomeric norfloxacin citrate conjugates was developed. Among the title compounds, 11b, 10a reveal pronounced activity against S. pneumoniae with minimum inhibitory concentrations of 0.89, 0.96 mg/mL and MBCs of 2.95, 2.80 mg/mL, respectively. Minimum Fungicidal Concentration (MFC) has been determined for each compound against two fungal strains. Compound 11b showed maximum anti-cancer activity against HeLa cell line with IC50 value 11.3 ± 0.41 comparable to standard drug DXN. For binding mode, active site residues and docking energies (ΔG =-7.9 Kcal/mol) for ligand 9b exhibited the highest hydrogen bonding (3.59274 A˚), Pi- Alkyl (5.14468 A˚) interactions with amino acid LEU479 of 3RAE protein. The compounds following the Lipinski 'Rule of five' were synthesized for antimicrobial and anti-cancer screening as oral bioavailable drugs/leads. Maximum drug likeness model score 1.52, 1.41 was found for compounds 10d, 11b. CONCLUSION: The present work, through simple synthetic approaches, led to the development of novel hybrids of fluoroquinolone containing citrate-triazole-oxadiazole pharmacophores that exhibited remarkable biological activities against different microorganisms and cell lines. The compounds showed suitable druglike properties and are expected to present good bioavailability profile. An efficient combination of molecular modeling and biological activity provided an insight into QSAR guidelines that could aid in further development and optimization of the norfloxacin derivatives.