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ABSTRACT NorA, an extensively studied efflux pump in Staphylococcus aureus, has been connected to fluoroquinolone, antiseptic, and disinfection resistance. Several studies have also emphasized how efflux pumps, including NorA, function as the first line of defense of S. aureus against antibiotics. In this study, we have screened some chemically synthesized indole derivatives for their activity as efflux pump inhibitors (EPIs). The derivative SMJ-5 was found to be a potent NorA efflux pump inhibitor among the screened indole derivatives, owing to increased ethidium bromide and norfloxacin accumulation in norA over-expressing S. aureus. The combination of SMJ-5 and ciprofloxacin demonstrated the eradication of S. aureus biofilm and prolonged the post-antibiotic effect more than ciprofloxacin alone. SMJ-5 was able to inhibit staphyloxanthin virulence. In in vitro time-kill trials and in vivo efficacy investigations, the combination enhanced the bactericidal activity of ciprofloxacin against S. aureus. Additionally, reverse transcription PCR results revealed that SMJ-5 also inhibits the NorA efflux pump indirectly at the transcriptional level. IMPORTANCE The NorA efflux pump is the most effective resistance mechanism in S. aureus. The clinical importance of NorA efflux pumps is demonstrated by the expression of pump genes in S. aureus strains in response to fluoroquinolones and biocides. Along with the repercussions of decreased fluoroquinolone sensitivity, increasing expression of efflux pump genes by their substrate necessitates the importance of efflux pump inhibitors. Reserpine and verapamil are clinically used to treat ailments and have proven NorA inhibitors, but, unfortunately, the concentration needed for these drugs to inhibit the pump is not safe in clinical settings. In the current study, we have screened some indole derivatives, and among them, SMJ-5 was reported to potentiate norfloxacin and ciprofloxacin at their sub-inhibitory concentration by inhibiting the norA gene transcriptionally. Here we highlight the promising points of this study, which could serve as a model to design a therapeutic EPI candidate against norA over-expressing S. aureus.

Objective: Efflux pumps are transmembrane proteins associated with bacterial resistance mechanisms. Bacteria use these proteins to actively transport antibiotics to the extracellular medium, preventing the pharmacological action of these drugs. This study aimed to evaluate in vitro the antibacterial activity of 1,8-naphthyridines sulfonamides, as well as their ability to inhibit efflux systems of Staphylococcus aureus strains expressing different levels of the NorA efflux pump. Methods: The broth microdilution test was performed to assess antibacterial activity. Efflux pump inhibition was evaluated in silico by molecular docking and in vitro by fluorometric tests, and the minimum inhibitory concentration (MIC) was determined. The MIC was determined in the association between 1,8-naphthyridine and norfloxacin or ethidium bromide. Results: The 1,8-naphthyridines did not show direct antibacterial activity. However, they effectively reduced the MIC of multidrug-resistant bacteria by associating with norfloxacin and ethidium bromide, in addition to increasing the fluorescence emission. In silico analysis addressing the binding between NorA and 1,8-naphthyridines suggests that hydrogen bonds and hydrophilic interactions represent the interactions with the most favourable binding energy, corroborating the experimental data. Conclusion: Our data suggest that 1,8-naphthyridines sulfonamides inhibit bacterial resistance through molecular mechanisms associated with inhibition of the NorA efflux pump in S. aureus strains.

ABSTRACT Staphylococcus aureus is a versatile human commensal bacteria and pathogen that causes various community and hospital-acquired infections. The S. aureus efflux pump NorA which belongs to the major facilitator superfamily, confers resistance to a range of substrates. Many efflux pump inhibitors (EPIs) have been discovered, but none is clinically approved due to their undesirable toxicities. In this study, we have screened clinically approved drugs for possible NorA EPI-like activity. We identified six drugs that showed the best efflux pump inhibition in vitro, with a fractional inhibitory concentration index of ≤0.5, indicating synergism with hydrophilic fluoroquinolones. The mechanistic validation of efflux inhibitory potential was demonstrated in ethidium bromide-based accumulation and efflux inhibition assays. We further confirmed the functionality of EPIs by norfloxacin accumulation assay depicting more realistic proof of the conjecture. None of the EPIs disturbed membrane function or depleted the ATP synthesis levels in bacteria. Both raloxifene and pyrvinium displayed an increase in bactericidal activity of ciprofloxacin in time-kill kinetics, prolonged its post-antibiotic effect, and reduced the frequency of spontaneous resistant mutant development. The combination of EPIs with ciprofloxacin caused significant eradication of preformed biofilms. Moreover, in the murine thigh infection model, a single dose of pyrvinium combined with ciprofloxacin reduced the bacterial burden significantly compared to untreated control and ciprofloxacin alone, indicating the efficacy of the combination. Conclusively, this study represents approved drugs that can be repurposed and combined with antibiotics as NorA EPIs, having anti-biofilm properties to treat severe S. aureus infections at clinically relevant concentrations. IMPORTANCE Staphylococcus aureus is a frequent pathogen bacterium and the predominant cause of worsened nosocomial infections. Efflux pumps contribute to drug efflux and are reportedly associated with biofilm formation, thereby promoting difficult-to-treat biofilm-associated S. aureus infections. One strategy to combat these bacteria is to reduce active efflux and increase pathogen sensitivity to existing antibiotics. Repurposing approved drugs may solve the classical toxicity issues with previous efflux pump inhibitors and help reach sufficient plasma concentrations. We describe the in silico-based screening of FDA-approved drugs that identified six different molecules able to inhibit NorA pump (Major Facilitator Superfamily). Our study highlights that these compounds bind to and block the activity of the NorA pump and increase the sensitivity of S. aureus and methicillin-resistant S. aureus to fluoroquinolones. These drugs combined with fluoroquinolones significantly reduced the preformed biofilms and displayed significant efficacy in the murine thigh infection model when compared to untreated control and ciprofloxacin alone.

Cuminum cyminum L. (cumin) is valued for its aromatic and medicinal properties. There are several reports of antibacterial activity of C. cyminum essential oil (CcEO). Accordingly, the present study was conducted to investigate the mechanism(s) of action of the CcEO against multidrug-resistant (MDR) Staphylococcus aureus. Therefore, 10 S. aureus MDR isolates, obtained from different sources, were selected based on the antibiotic susceptibility patterns and the Clinical and Laboratory Standards Institute definition and subjected to the examinations. Our results exhibited promising bacteriostatic and bactericidal properties of the CcEO. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration values ranged from 5 to 10 and 10 to 20 μL ⋅ mL–1, respectively. Scanning electron microscope was used to assess the bacterial cell structure and morphology after the induction with 1/2 MIC concentration of the CcEO. The observed morphological changes appeared to be deformation of the cell membrane and destruction of the cells. In the case of quorum sensing inhibitory potential, treatment of S. aureus isolates with the sub-MIC concentrations (1/2 MIC) of the CcEO significantly reduced the hld expression (3.13-fold downregulation), which considerably controls S. aureus quorum-sensing accessory regulator system. Another virulence factor influenced by the CcEO was the polysaccharide intercellular adhesion production system, as an important component of cell–cell adhesion and biofilm formation. Consequently, the expression level of the intercellular adhesion (ica) locus in the S. aureus cells was examined following treatment with CcEO. The results showed significant decrease (−3.3-fold) in ica expression, indicating that the CcEO could potentially interfere with the process of biofilm formation. Using the ethidium bromide efflux inhibition assay, the S. aureus NorA efflux pump was phenotypically but not genotypically (in quantitative polymerase chain reaction assay) affected by the CcEO treatment. Using gas chromatography–mass spectrometry analysis, cuminic aldehyde (38.26%), α,β-dihydroxyethylbenzene (29.16%), 2-caren-10-al (11.20%), and γ-terpinene (6.49%) were the most detected compounds. The antibacterial and antivirulence action of the CcEO at sub-MIC concentrations means that no microbial resistance will be promoted and developed after the treatment with this agent. These findings revealed that the CcEO is a promising antibacterial agent to control infections caused by the MDR S. aureus strains.

Cuminum cyminum L. (cumin) is valued for its aromatic and medicinal properties. There are several reports of antibacterial activity of C. cyminum essential oil (CcEO). Accordingly, the present study was conducted to investigate the mechanism(s) of action of the CcEO against multidrug-resistant (MDR) Staphylococcus aureus. Therefore, 10 S. aureus MDR isolates, obtained from different sources, were selected based on the antibiotic susceptibility patterns and the Clinical and Laboratory Standards Institute definition and subjected to the examinations. Our results exhibited promising bacteriostatic and bactericidal properties of the CcEO. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration values ranged from 5 to 10 and 10 to 20 μL ⋅ mL–1, respectively. Scanning electron microscope was used to assess the bacterial cell structure and morphology after the induction with 1/2 MIC concentration of the CcEO. The observed morphological changes appeared to be deformation of the cell membrane and destruction of the cells. In the case of quorum sensing inhibitory potential, treatment of S. aureus isolates with the sub-MIC concentrations (1/2 MIC) of the CcEO significantly reduced the hld expression (3.13-fold downregulation), which considerably controls S. aureus quorum-sensing accessory regulator system. Another virulence factor influenced by the CcEO was the polysaccharide intercellular adhesion production system, as an important component of cell–cell adhesion and biofilm formation. Consequently, the expression level of the intercellular adhesion (ica) locus in the S. aureus cells was examined following treatment with CcEO. The results showed significant decrease (−3.3-fold) in ica expression, indicating that the CcEO could potentially interfere with the process of biofilm formation. Using the ethidium bromide efflux inhibition assay, the S. aureus NorA efflux pump was phenotypically but not genotypically (in quantitative polymerase chain reaction assay) affected by the CcEO treatment. Using gas chromatography–mass spectrometry analysis, cuminic aldehyde (38.26%), α,β-dihydroxyethylbenzene (29.16%), 2-caren-10-al (11.20%), and γ-terpinene (6.49%) were the most detected compounds. The antibacterial and antivirulence action of the CcEO at sub-MIC concentrations means that no microbial resistance will be promoted and developed after the treatment with this agent. These findings revealed that the CcEO is a promising antibacterial agent to control infections caused by the MDR S. aureus strains. [ABSTRACT FROM AUTHOR]

Baki Vijaya Bhaskar,1 Tirumalasetty Muni Chandra Babu,1 Netala Vasudeva Reddy,2 Wudayagiri Rajendra1 1Division of Molecular Biology, Department of Zoology, 2Department of Biotechnology, Sri Venkateswara University, Tirupati, Andhra Pradesh, India Abstract: Emerging drug resistance in clinical isolates of Staphylococcus aureus might be implicated to the overexpression of NorA efflux pump which is capable of extruding numerous structurally diverse compounds. However, NorA efflux pump is considered as a potential drug target for the development of efflux pump inhibitors. In the present study, NorA model was constructed based on the crystal structure of glycerol-3-phosphate transporter (PDBID: 1PW4). Molecular dynamics (MD) simulation was performed using NAMD2.7 for NorA which is embedded in the hydrated lipid bilayer. Structural design of NorA unveils amino (N)- and carboxyl (C)-terminal domains which are connected by long cytoplasmic loop. N and C domains are composed of six transmembrane α-helices (TM) which exhibits pseudo-twofold symmetry and possess voluminous substrate binding cavity between TM helices. Molecular docking of reserpine, totarol, ferruginol, salvin, thioxanthene, phenothiazine, omeprazole, verapamil, nalidixic acid, ciprofloxacin, levofloxacin, and acridine to NorA found that all the molecules were bound at the large hydrophobic cleft and indicated significant interactions with the key residues. In addition, structure-based virtual screening was employed which indicates that 14 potent novel lead molecules such as CID58685302, CID58685367, CID5799283, CID5578487, CID60028372, ZINC12196383, ZINC72140751, ZINC72137843, ZINC39227983, ZINC43742707, ZINC12196375, ZINC66166948, ZINC39228014, and ZINC14616160 have highest binding affinity for NorA. These lead molecules displayed considerable pharmacological properties as evidenced by Lipinski rule of five and prophecy of toxicity risk assessment. Thus, the present study will be helpful in designing and synthesis of a novel class of NorA efflux pump inhibitors that restore the susceptibilities of drug compounds. Keywords: Staphylococcus aureus, NorA efflux pump, molecular dynamics, virtual screening, docking

The alarming and rapid spread of antimicrobial resistance among bacteria represents a high risk for global health. Targeting factors involved in resistance to restore the activity of failing antibiotics is a promising strategy to overcome this urgent medical need. Efflux pump inhibitors are able to increase antibiotic concentrations in bacteria, thus they can be considered true antimicrobial resistance breakers. In this work, continuing our studies on inhibitors of the Staphylococcus aureus NorA pump, we designed, synthesized and biologically evaluated novel 2-phenylquinoline derivatives starting from our hits 1 and 2 . Two of the synthesized compounds ( 6 and 7 ) bearing a C-6 benzyloxy group showed the best NorA inhibition activity, thereby providing an excellent starting point to direct future chemical optimizations. [ABSTRACT FROM AUTHOR]

Toxins and venoms produced by living organisms have exhibited a variety of biological activities against microorganisms. In this study, we tested seven snake venoms from the family Viperidae for antibacterial activity and the activities of reversal of antibiotic resistance and inhibition of biofilm formation against 22 clinical isolates of Staphylococcus aureus. Bothrops moojeni venom exhibited anti staphylococcal activity with the lowest mean value of minimum inhibitory concentration (MIC). Moreover, reversal of antibiotic resistance was observed for combinations of B. moojeni venom (½ x MIC) and norfloxacin or ampicillin (both ½ x MIC) for 86.4% and 50% of the isolates, respectively. B. moojeni venom alone at ½ MIC inhibited 90% of biofilm formation, whereas in combination with ciprofloxacin, both at ½ MIC, a reduction on the NorA efflux pump activity was observed. The detection of in vitro mutants colonies of S. aureus resistant to B. moojeni venom was low and they did not survive. A phospholipase A2 was purified from the venom of B. moojeni and displayed anti-staphylococcal activity when tested alone or in combination with ciprofloxacin. The results presented here will contribute to the search for new antimicrobial agents against resistant S. aureus.

Multidrug efflux systems play a prominent role in medicine, as they are important contributors to bacterial antibiotic resistance. NorA is an efflux pump transporter from the major facilitator superfamily that expels numerous drug compounds across the inner membrane of Staphylococcus aureus (S. aureus). The design of novel inhibitors to combat drug efflux could offer new opportunities to avoid the problem of antibiotic resistance. In this study, we performed molecular modeling studies in an effort to discover novel NorA efflux pump inhibitors. A group of over 673 compounds from the PubChem database with a high (>80%) level of similarity to the chemical structure of capsaicin was used to study the binding affinity of small molecule compounds for the NorA efflux pump. Ten potential lead compounds displayed a good druggability profile, with one in particular (CID 44330438) providing new insight into the molecular mechanism of the inhibition of major facilitator superfamily (MFS) efflux pump transporters. It is our hope that the overall strategy described in this study, and the structural information of the potential novel inhibitors thus identified, will stimulate others to pursue the development of better drugs to tackle multidrug resistance in S. aureus.

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A total of eighteen piperic acid (PA) and 4-ethylpiperic acid (EPA) amides ( C1 – C18 ) with α-, β- and γ-amino acids were synthesized, characterized and evaluated for their efflux pump inhibitory activity against ciprofloxacin resistant Staphylococcus aureus . The amides were screened against NorA overexpressing S. aureus SA-1199B and wild type S. aureus SA-1199 using ethidium bromide as NorA efflux pump substrate. EPI C6 was found to be most potent and reduced the MIC of ciprofloxacin by 16 fold followed by C18 which showed 4 fold reduction of MIC. Ethidium bromide efflux inhibition and accumulation assay proved these compounds as NorA inhibitors. [ABSTRACT FROM AUTHOR]

Background and Objective: Antibiotic resistant phenotypes of bacteria have been shown to be related to efflux pumps. Research for finding compounds with an ability to inhibit these pumps seems worthwhile due to their ability to increase bacterial sensitivity to antibiotics or return sensitivity to resistant strains. The aim of this study was to evaluate inhibitory effect of Thymus daenensis essential oil (EO) on NorA efflux pump of staphylococcus aureus. Materials and Methods: Thymus daenensis essential oil was extracted from dried thyme leaves by hydrodistillation method and then the effect of the essential oil on bacterial efflux pump was evaluated using ethidium bromide-agar cartwheel method with respect to bacterial efflux capacity of ethidium bromide. Minimum inhibitory concentration (MIC) of Thymus daenensis essential oil was measured using microdilution test followed by the evaluation of the effect of sub-MIC concentration of essential oil on fluoroquinolone antibiotics. The efflux level of ethidium bromide, ciprofloxacin and norfloxacin (substances transmitted by NorA) was considered as the NorA efflux pump activity. Results: The cartwheel method showed that Thymus daenensis essential oil could reduce the bacterial efflux capacity of ethidium bromide. The results of checkerboard test indicated that the interactions between sub-MIC concentration of the essential oil and ethidium bromide, ciprofloxacin and norfloxacin were totally synergistic. Conclusion: Synergistic effect of Thymus daenensis essential oil with the other components demonstrated the potential of Thymus daenensis to enhance the antimicrobial activity of fluoroquinolone antibiotics against staphylococcus aureus. As these antibiotics are effluxed by NorA pump, this increased sensitivity to antibiotics is possibly due to NorA efflux pump inhibitors. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus causes a wide variety of infections worldwide. Methicillin-resistant S. aureus is one of most common causes of nosocomial and community acquired infections. The fluoroquinolones are an important class of antibiotics that used to treat infections caused by S. aureus. Today, a significant increase in the rate of ciprofloxacin resistance in methicillin-resistant S. aureus strains is concerning. The norA efflux pump is considered as contributors to antibiotic resistance. Here, we aimed to evaluate the expression of norA efflux pump in the presence of hexahydroquinoline derivative in methicillin and ciprofloxacin resistant S. aureus. We were determined minimum inhibitory concentration of ciprofloxacin and hexahydroquinoline derivative and their combination by broth microdilution method against ciprofloxacin resistant S. aureus. The expression of the norA efflux pump gene was evaluated by quantitative Real-time PCR. This study showed that minimum inhibitory concentrations of ciprofloxacin in the presence of hexahydroquinoline derivative in comparison to ciprofloxacin were decreased. Quantitative Real-time PCR identified the increased expression of norA efflux pump gene in methicillin and ciprofloxacin resistant S. aureus strain. The increased expression of norA efflux pump gene may have resulted in the effort of S. aureus to survive. The results showed that the hexahydroquinoline derivative enhanced the antibacterial effect of ciprofloxacin against methicillin and ciprofloxacin resistant S. aureus. Therefore, the derivatives may be used as inhibitors of antibiotic resistance for combination therapy.

The aim of this study is to explore the potential enhancing effect of ginsenoside 20(S)-Rh2 (Rh2) towards ciprofloxacin (CIP) against Staphylococcus aureus ( S. aureus ) infection in vitro and in vivo , and analyze the possible mechanisms through NorA inhibition from a target cellular pharmacokinetic view. In combination with non-toxic dosage of Rh2, the susceptibilities of S. aureus strains to CIP were significantly augmented, and the antibacterial kinetics of CIP in the S. aureus strains were markedly promoted. This enhancing effect of Rh2 towards CIP was also observed in S. aureus infected peritonitis mice, with elevated survival rate and reduced bacteria counts in blood. However, Rh2 did not influence the plasma concentrations of CIP. Further analysis indicated that Rh2 significantly promoted the accumulations of CIP in S. aureus , and inhibited the NorA mediated efflux of pyronin Y. The expressions of NorA gene on S. aureus were positively correlated with the enhancing effect of Rh2 with CIP. This is the first report of the enhancing effect of Rh2 with CIP for S. aureus infection in vitro and in vivo , of which it is probably that Rh2 inhibited NorA-mediated efflux and promoted the accumulation of CIP in the bacteria. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus causes a wide variety of infections worldwide. Methicillin-resistant S. aureus is one of most common causes of nosocomial and community acquired infections. The fluoroquinolones are an important class of antibiotics that used to treat infections caused by S. aureus. Today, a significant increase in the rate of ciprofloxacin resistance in methicillin-resistant S. aureus strains is concerning. The norA efflux pump is considered as contributors to antibiotic resistance. Here, we aimed to evaluate the expression of norA efflux pump in the presence of hexahydroquinoline derivative in methicillin and ciprofloxacin resistant S. aureus. We were determined minimum inhibitory concentration of ciprofloxacin and hexahydroquinoline derivative and their combination by broth microdilution method against ciprofloxacin resistant S. aureus. The expression of the norA efflux pump gene was evaluated by quantitative Real-time PCR. This study showed that minimum inhibitory concentrations of ciprofloxacin in the presence of hexahydroquinoline derivative in comparison to ciprofloxacin were decreased. Quantitative Real-time PCR identified the increased expression of norA efflux pump gene in methicillin and ciprofloxacin resistant S. aureus strain. The increased expression of norA efflux pump gene may have resulted in the effort of S. aureus to survive. The results showed that the hexahydroquinoline derivative enhanced the antibacterial effect of ciprofloxacin against methicillin and ciprofloxacin resistant S. aureus. Therefore, the derivatives may be used as inhibitors of antibiotic resistance for combination therapy. [ABSTRACT FROM AUTHOR]

[Display omitted] • Dihydroquinazoline analogues as NorA efflux pump inhibitor, synergistically reduced norfloxacin and Etbr MIC of S. aureus 1199b by 16 modulation folds. • Effective log reduction (>103 log) in bacterial kill kinetics assay in presence of these analogues at 4 h confirmed their bactericidal nature. • Dihydroquinazoline analogues, significantly able to down regulate the norA gene expression in the presence of sub-inhibitory concentration of norfloxacin. • The most important finding of this study was the ability of analogues to significantly reduce the intracellular S. aureus 1199b (>103 log) in human THP-1 monocytes in presence of norfloxacin. Staphylococcus aureus is an emerging human pathogen that has become difficult to treat due to its high resistance against wide range of drugs. Emergence of drug resistant isolates has further convoluted the treatment process. Among different resistance mechanisms, efflux pump proteins play a central role and has made itself a direct approach for therapeutic exploration. To demarcate the role of dihydroquinazoline analogues as NorA efflux pump inhibitor in S. aureus 1199B (NorA over producing) strain total seventeen analogues were synthesized and tested for their modulatory effects on norfloxacin and Etbr resistance. Further accumulation assays, bacterial time kill kinetics, cytotoxicity assay were also carried out. The intracellular killing ability of analogues, as EPI was determined using THP-1 monocytes. The binding interaction of analogues with NorA was also predicted. Dihydroquinazoline analogues notably reduced the MIC of norfloxacin and Etbr in S. aureus 1199B. In addition to their very low toxicity, they showed high Etbr and norfloxacin accumulation respectively. Further effective over time log reduction in bacterial kill kinetics in presence of these analogues confirmed their role as NorA efflux pump inhibitor. FESEM analysis clearly depicted their effect on the cell surface morphology owing to its lyses. The most significant finding of this study was the ability of analogues to significantly reduce the intracellular S. aureus 1199B in human THP-1 monocytes in presence of norfloxacin. Our study has shown for the first time the possibility of developing the dihydroquinazoline analogues as NorA efflux pump inhibitors for S. aureus and control its infection. [ABSTRACT FROM AUTHOR]

A series of novel 1,3,4-oxadiazole conjugates of Capsaicin were efficiently synthesized by one point modification of capsaicin. Compound 17i has demonstrated potential NorA efflux pump inhibitory potentials at minimum effective concentration of 12.5 μg/mL with four fold improved ciprofloxacin activity against S. aureus strain (SA1199B). [Display omitted] • A series of novel 1,3,4-oxadiazole conjugates of Capsaicin were prepared. • The compounds were evaluated for ciprofloxacin activity potentiating effect. • 17i has potentiated the activity of ciprofloxacin by four folds at MEC of 12.5 µg/mL. • 17i has decreased the EtBr efflux confirming NorA inhibition as the mode of action. • 17i has reduced the mutation prevention concentration of ciprofloxacin. • Compound 17i may serve as lead for exploration of new capsaicin based EPI. NorA efflux pump pertaining to the major facilitator superfamily (MFS) is known to play a key role in antibiotic and biocide resistance in Staphylococcus aureus (S. aureus). It accounts for the extrusion of antibiotics like fluoroquinolones (e.g. ciprofloxacin). Several compounds including synthetic and natural products have been identified as potential NorA efflux pump inhibitors (EPIs) and found to restore the antibacterial activity of antibiotics. However, none of the reported EPIs have reached to clinical approval probably due to their high toxicity profiles. Considering the NorA efflux pump inhibitory potential of capsaicin, a series of capsaicin-based 1,3,4 oxadiazole conjugates were prepared and evaluated for ciprofloxacin activity potentiating effect. Among the new capsaicinoids tested, 17i displayed a minimum effective concentration (MEC) of 12.5 µg/mL against NorA overexpressing S. aureus strain (SA1199B), whereas capsaicin showed MEC of 50 µg/mL. The kill kinetics curve for the combination showed that ciprofloxacin at a sub–inhibitory concentration (0.25 × MIC) was equipotent in effect, to its MIC. 17i has significantly decreased the ethidium bromide efflux confirming NorA inhibition as the mode of action. Mutation prevention concentration of the ciprofloxacin was reduced in combination with 17i. In silico studies revealed the binding efficiency and binding affinity of 17i with NorA. This compound may serve as a template for the further drug discovery. [ABSTRACT FROM AUTHOR]

The NorA efflux pump is one of the most studied efflux systems in Staphylococcus aureus and confers multidrug resistance to a variety of dyes and antimicrobial compounds. Hence, inhibition of the NorA efflux pump might be a viable option for restoring susceptibility to antibiotics like fluoroquinolones. Fluorescent real-time efflux assays are important tools to identify putative efflux pump inhibitors. Nevertheless, the number of available compounds for usage in Staphylococcus aureus is limited. Previously, a 3-dipropyloxacarbocyanine iodide (DiOC 3) efflux assay was published that circumvented problems associated with the usage of ethidium bromide, namely slow efflux and suggested mutagenicity. However, the DiOC 3 assay protocol was cuvette – based and therefore needs to be adapted to the 96-well plate format. Hence, we optimized this assay for usage with 96-well plates. The new assay allows for rapid high-throughput efflux pump inhibitor screening. • Fluorescent efflux assays are important tools to identify efflux pump inhibitors. • DiOC 3 acts as a fluorescent substrate in the S. aureus NorA efflux pump. • The DiOC 3 circumvent problems associated with the usage of ethidum bromide. • DiOC 3 cuvette assay doesn't allow rapid screening for efflux pumps inhibitors (EPI). • The novel 96-well plate DiOC 3 method is suitable for high-throughput EPI screening. [ABSTRACT FROM AUTHOR]

Fluorobenzylidene-imidazolidines were analysed as efflux pump inhibitors against S. aureus SA-1199B, which overproduces the NorA efflux pump. These compounds decreased the Minimum Inhibitory Concentration of norfloxacin and ciprofloxacin up to 6.4- and 3.2-fold, respectively. The experimental and molecular-docking analysis accounts for the mechanism of the inhibition of the efflux pump. • Modulation experiments with EtBr and molecular docking analysis support the inhibition mechanism. • This enhancement activity is mediated by the inhibition of the NorA efflux pump. • The synthetic imidazolines enhanced the activity of norfloxacin and ciprofloxacin against SA-1199B. Searching for new alternatives to antibiotic treatments is crucial to surmount the multidrug-resistant bacteria. In this work, the antimicrobial activity of synthetic imidazolidines was evaluated as well as their modulating effect on the resistance to fluoroquinolones in a S. aureus strain (SA-1199B), which overexpresses the norA gene that encodes the NorA efflux pump. Results showed weak antimicrobial activity (512 μg mL−1) for two fluorobenzylidene derivatives against this bacterial strain, while the other benzylidene derivatives were inactive. Despite this fact, both fluorinated compounds were able to enhance the activity of norfloxacin and ciprofloxacin against SA-1199B up to 6.4– and 3.2-fold, respectively. In addition, both derivatives potentiated the action of ethidium bromide against this strain, suggesting that the modulating effect probably involves the inhibition of the NorA efflux pump, which is in concordance with the fluorimetic assays and molecular docking analyses performed in this work. [ABSTRACT FROM AUTHOR]

• 50 Novel acrylohydrazides were synthesized for NorA efflux pump inhibitory activity. • All these 50 acrylohydrazides were tested for modulation study in vitro. • This study provided compound 19 as the most active one. • Compound 19 was further evaluated for time kill kinetics, PAE, MPC. • NorA binding affinity study in silico provided 19 and 52 as the best compounds. • This is the first report of acrylohydrazides as novel NorA efflux pump inhibitors. The NorA efflux pump decreases the intracellular concentration of fluoroquinolones (ciprofloxacin, norfloxacin) by effluxing them from Staphylococcus aureus cells. The synthesis of novel acrylohydrazide derivatives was achieved using well-known reactions and were characterized by various spectroscopy techniques. The synthesized 50 compounds were evaluated for the NorA efflux pump inhibition activity against S. aureus SA-1199B (norA ++) and K1758 (norA -) strains. The study provided two most active compounds viz. 19 and 52. Compound 19 was found to be most active in potentiating effect of norfloxacin and also it showed enhanced uptake, efflux inhibition in ethidium bromide assay. Further compound 19 also enhanced post antibiotic effect and reduced mutation prevention concentration of norfloxacin. The homology modeling study was performed to elucidate three-dimensional structure of NorA. Docking studies of potent molecules were done to find the binding affinity and interaction with active site residues. Further, all the tested compounds exhibited good ADME and drug-likeness properties in- silico. Based on the in-silico studies and detailed in vitro studies, acrylohydrazides derivatives may be considered as potential NorA EPI candidates. [ABSTRACT FROM AUTHOR]

Real-time fluorescent efflux assays are commonly used for measuring the efflux of bacterial pumps. Here we describe an optimized protocol for the NorA efflux pump in S. aureus using DiOC 3 instead of ethidium bromide. Glucose and sodium formate were tested as energy carriers. This novel method is fast and reproducible. [ABSTRACT FROM AUTHOR]

The use and misuse of antibiotics has resulted in critical conditions for drug-resistant bacteria emergency, accelerating the development of antimicrobial resistance (AMR). In this context, the co-administration of an antibiotic with a compound able to restore sufficient antibacterial activity may be a successful strategy. In particular, the identification of efflux pump inhibitors (EPIs) holds promise for new antibiotic resistance breakers (ARBs). Indeed, bacterial efflux pumps have a key role in AMR development; for instance, NorA efflux pump contributes to Staphylococcus aureus (S. aureus) resistance against fluoroquinolone antibiotics (e.g., ciprofloxacin) by promoting their active extrusion from the cells. Even though NorA efflux pump is known to be a potential target for EPIs development, the absence of structural information about this protein and the little knowledge available on its mechanism of action have strongly hampered rational drug discovery efforts in this area. In the present work, we investigated at the molecular level the substrate recognition pathway of NorA through a Supervised Molecular Dynamics (SuMD) approach, using a NorA homology model. Specific amino acids were identified as playing a key role in the efflux pump-mediated extrusion of its substrate, paving the way for a deeper understanding of both the mechanisms of action and the inhibition of such efflux pumps. [ABSTRACT FROM AUTHOR]

Multidrug efflux systems play a prominent role in medicine, as they are important contributors to bacterial antibiotic resistance. NorA is an efflux pump transporter from the major facilitator superfamily that expels numerous drug compounds across the inner membrane of Staphylococcus aureus (S. aureus). The design of novel inhibitors to combat drug efflux could offer new opportunities to avoid the problem of antibiotic resistance. In this study, we performed molecular modeling studies in an effort to discover novel NorA efflux pump inhibitors. A group of over 673 compounds from the PubChem database with a high (>80%) level of similarity to the chemical structure of capsaicin was used to study the binding affinity of small molecule compounds for the NorA efflux pump. Ten potential lead compounds displayed a good druggability profile, with one in particular (CID 44330438) providing new insight into the molecular mechanism of the inhibition of major facilitator superfamily (MFS) efflux pump transporters. It is our hope that the overall strategy described in this study, and the structural information of the potential novel inhibitors thus identified, will stimulate others to pursue the development of better drugs to tackle multidrug resistance in S. aureus. [ABSTRACT FROM AUTHOR]

Background: Methicillin-resistant and vancomycin-resistant Staphylococcus aureus are pathogens causing severe infectious diseases that pose real public health threats problems worldwide. In S. aureus, the most efficient multidrug-resistant system is the NorA efflux pump. For this reason, it is critical to identify efflux pump inhibitors., Objective: In this paper, we present an update of the new natural and synthetic compounds that act as modulators of antibiotic resistance through the inhibition of the S. aureus NorA efflux pump., Results: Several classes of compounds capable of restoring the antibiotic activity have been identified against resistant-S. aureus strains, acting as NorA efflux pump inhibitors. The most promising classes of compounds were quinolines, indoles, pyridines, phenols, and sulfur-containing heterocycles. However, the substantial degree structural diversity of these compounds makes it difficult to establish good structure- activity correlations that allow the design of compounds with more promising activities and properties., Conclusion: Despite substantial efforts put forth in the search for new antibiotic adjuvants that act as efflux pump inhibitors, and despite several promising results, there are currently no efflux pump inhibitors authorized for human or veterinary use, or in clinical trials. Unfortunately, it appears that infection control strategies have remained the same since the discovery of penicillin, and that most efforts remain focused on discovering new classes of antibiotics, rather than trying to prolong the life of available antibiotics, and simultaneously fighting mechanisms of bacterial resistance., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)