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Scientifica
Volume 2016 (2016), Article ID 6894758, 6 pages
http://dx.doi.org/10.1155/2016/6894758
Research Article

Modulation of Drug Resistance in Staphylococcus aureus with Coumarin Derivatives

1Post-Graduate Program in Natural and Synthetic Bioactive Products, Federal University of Paraíba, 58051-900 João Pessoa, PB, Brazil
2Laboratory of Genetics of Microorganisms, Molecular Biology Department, Federal University of Paraíba, 58051-900 João Pessoa, PB, Brazil
3Laboratory of Synthesis and Drug Delivery, Biological Science Department, State University of Paraíba, 58071-160 João Pessoa, PB, Brazil

Received 30 December 2015; Accepted 3 April 2016

Academic Editor: Antonio Espinosa

Copyright © 2016 Rodrigo Santos Aquino de Araújo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Semisynthetic and commercial coumarins were investigated for their antibacterial and adjuvant properties with antibiotic agents against norfloxacin, erythromycin, and tetracycline resistant Staphylococcus aureus as based on efflux mechanisms. The coumarins and certain commercial antibiotics had their Minimum Inhibitory Concentrations determined by broth microdilution assay against resistant S. aureus strains which overexpress efflux pump proteins. For evaluation of the modulatory activity, the antibiotics MICs were determined in the presence of the coumarin derivatives at subinhibitory concentration. Although the coumarins did not display relevant antibacterial activity (MIC ≥ 128 µg/mL), they did modulate the antibiotics activities. Various coumarins, especially the alkylated derivatives in combination with antibiotics at subinhibitory concentrations, modulated antibiotic activity, reducing the MIC for tetracycline and norfloxacin by 2 to 8 times. Polar Surface Area (PSA) studies were performed and the fact that the presence of apolar groups is an important factor for the modulatory activity of coumarins was corroborated. Docking on the Penicillin-Binding Protein from MRSA identified that 18 is a potential ligand presenting low . The results indicate that coumarin derivatives modulated antibiotic resistance and may be used as potential antibiotic adjuvants, acting by bacterial efflux pump inhibition in S. aureus.

1. Introduction

The development of mechanisms of resistance by the microorganisms is a natural process that occurs in greater intensity with the indiscriminate and inappropriate use of antimicrobials [1]. This natural adaptation phenomenon has become the principle problem worldwide for the treatment of infectious diseases, creating a continuous need for the discovery of new chemical entities with antibiotic properties [2].

Bacterial infections are a major cause of morbidity and mortality in hospitals, as in the case of nosocomial infections caused by Staphylococcus aureus, which cause about 12,000 deaths per year in the USA [3]. It is mentioned in the literature that S. aureus is a versatile pathogen with numerous virulence factors, making this microorganism capable of acquiring antibiotic resistance determinants [4]. Resistance to methicillin (MRSA) first appeared in 1961 [5], with currently described S. aureus strains having acquired resistance to all β-lactams, macrolides, aminoglycosides, tetracycline, rifampicin, quinolones, and even vancomycin [6].

Among the various mechanisms of bacterial resistance, transmembrane efflux proteins have been commonly associated with resistance to multiple antibiotics and other chemotherapeutic agents [7, 8]. These proteins actively extrude toxic substrates, including antibiotics, from the cell. Antibiotic activity-modifying agents or modulators are compounds that potentiate the activity of antibiotics against resistant strains; some of these agents act as efflux pump inhibitors (EPI) [9]. Some efflux pumps have been shown to be of clinical significance in bacterial infections [10], among which are the TetK pump (promoting resistance to tetracycline), the MsrA macrolide efflux protein (promoting macrolide resistance), and the NorA efflux pump (with resistance to fluoroquinolones) [11].

Currently, one of the best strategies to control bacterial resistance and extend the life of existing antibiotics is to associate them with modulators of drug resistance, similar to what is done with many β-lactam antibiotics that are administered in combination with potassium clavulanate. Compounds that potentiate the activity of antimicrobial agents present numerous benefits including the possibility of reducing the antibiotic concentration in prophylactic or therapeutic treatments of infection and reversion of cell resistance to conventional therapy.

Coumarins comprise a class of natural phenolic compounds characterized by a single benzene fused to an α-pyrone ring. They stand out for having great biological potential, as demonstrated in several studies, compounds with antifungal [12] and antibacterial activities [13], and also modulators of antibiotic resistance [14].

Given the above, and knowing the antimicrobial potential and modulating action of coumarin derivatives on antibiotic resistance, our goal was to develop semisynthetic coumarin derivatives in order to evaluate their antibiotic and/or antibiotic adjuvant properties against effluxing S. aureus strains.

2. Materials and Methods

2.1. Coumarin Derivatives and Antibiotics

All commercial coumarins (1,2-Benzopyrone 1; 3-Hydroxycoumarin 2; 4-Hydroxycoumarin 3; 6-Hydroxycoumarin 4; 7-Hydroxycoumarin 5; 6,7-Dihydroxycoumarin 6; Coumarin-3-carboxylic acid 7; 3,3′-Methylene-bis-(4-hydroxycoumarin) 8; 6-Methoxy-7-hydroxycoumarin 9; and 7,8-Dihydroxy-6-methoxycoumarin 10), reagents, and solvents were purchased from Sigma-Aldrich (Seelze, Germany) and used without further purification. Norfloxacin, erythromycin, and tetracycline were obtained from Sigma Chemical Co., USA, and their stock solutions were then prepared [15]. Compounds 1122 were synthesized according to previously reported procedures [12]. The stock solutions of coumarin derivatives 122 were prepared in DMSO. The highest concentration remaining after broth dilution (4%) did not inhibit bacterial growth, and a positive control with only DMSO 4% was tested and showed no interference with bacterial growth (data not shown).

2.2. Bacterial Strains

Four S. aureus strains were used for the biological tests: one standard strain (ATCC 6538) and three antibiotics resistant strains: SA-1199B, which over expresses the norA gene encoding the NorA efflux protein [16]; RN4220 harboring plasmid pUL5054, which carries the gene encoding the MsrA macrolide efflux protein [17]; and IS-58, which possesses the TetK tetracycline efflux protein [18]. The strains, kindly provided by Professor Simon Gibbons (University of London), were maintained in blood agar base (Laboratórios Difco Ltda., Brazil) slants. Prior to use, the cells were grown overnight at 37°C in brain heart infusion broth (BHI-Laboratórios Difco Ltda., Brazil).

2.3. Drug Susceptibility Testing and Modulation Assay

The Minimum Inhibitory Concentrations (MICs) of the antibiotics and coumarin derivatives were determined in BHI by broth microdilution assay using a suspension of ca. 105 cfu/mL and a drug concentration range from 0.5 to 256 µg/mL (twofold serial dilutions). For better visualization of the bacterial growth (after 24 h) we used resazurin (0.01%). To evaluate coumarin as a modulator of drug resistance “modulation assay,” we used a method that has been widely applied to identify potential EPIs and is valid provided that one uses specifically known effluxing strains. The MICs of the antibiotics were determined in the presence of the subinhibitory concentrations of coumarin compounds. Both assays were performed in triplicate [19, 20].

2.4. Molecular Model

Three-dimensional structures were drawn using HyperChem 8.0 software [21]; the structures had energy-minimization calculated employing the method MM+ force field without any restriction [22]. Subsequently, a new geometry optimization process, based on the semiempirical method AM1 (Austin Model 1) was performed [23]. The optimized structures were subjected to conformation analysis using the random search method with 1,000 interactions, 100 cycles of optimization, and 10 lowest minimum energy conformers. The selected dihedrals were evaluated for rotation in accordance with the standard (default) conditions of the program; the number of simultaneous variations was 1 to 8. Acyclic chains were submitted to rotations from 60 to 180°; torsion rings were in the range of 30 to 120° [24, 25]. The lowest energy conformers were selected and imported into VEJA ZZ. The drawn coumarins were imported into the VEJA ZZ program [26] for calculation of the Polar Surface Area (PSA).

2.5. Docking

The structure of the Penicillin-Binding Protein (PBP) from Methicillin Resistant Staphylococcus aureus (MRSA) in complex with the ligand quinazolinone (PDB id: 4CJN) [27] was downloaded from the Protein Data Bank (http://www.rcsb.org/pdb/home/home.do). Coumarin derivatives 11, 13, and 1619 were submitted to molecular docking using the Molegro Virtual Docker, v. 6.0.1 (MVD) [28].

The protein and compound structures were prepared using the default parameter settings in the software package (score function: MolDock Score; ligand evaluation: Internal ES, Internal H-Bond, and sp2–sp2 Torsions, all checked; number of runs: 10 runs; algorithm: MolDock SE; maximum interactions: 1500; max. population size: 50; max. steps: 300; neighbor distance factor: 1.00; max. number of poses returned: 5). The docking procedure was performed using a GRID of 15 Ǻ in radius and 0.30 in resolution to cover the ligand-biding site of the Penicillin-Binding Protein (PBP) structure.

Templates with features expected to be relevant for ligand binding (quinazoline) were generated to perform docking. The MolDock Score [GRID] algorithm was used as the score function, and the MolDock search algorithm was used [28].

3. Results and Discussion

Twelve coumarin derivatives (1122) were synthesized from commercial coumarins 4-hydroxy- (3), 6-hydroxy- (4), and 7-hydroxycoumarin (5) by alkylation, acetylation, and nitration procedures according to procedures previously described by our group [12] (Scheme 1).

Scheme 1: Synthesis of alkyl-, acetyl-, and nitro-coumarin derivatives. Reagents and conditions: (a) acetic anhydride, pyridine, rt, ultrasound irradiation; (b) allyl bromide, geranyl bromide, or prenyl bromide, K2CO3, acetonitrile, and reflux; (c) HNO3/AcOH, 0–5°C for 30 min and then 90 min at rt.

All commercial (110) and synthetic (1122) coumarins were evaluated for their in vitro antibacterial activity against the four Staphylococcus aureus strains: the standard (ATCC 6538) and the three resistant: IS-58 (TetK), RN4220 (MsrA), and SA-1199B (NorA); however, all of the commercial coumarins (110) and the eight synthetic coumarins (11, 12, 15, and 1822) showed no antibacterial activity (MIC > 256 µg/mL) against any S. aureus strain evaluated. Compounds 13, 14, 16, and 17 showed weak activity with MIC values of 128 µg/mL (13 against ATCC 6538 and SA-1199B, 16 against SA-1199B, and 14 and 17 against all strains).

Shakeel-U-Rehman and colleagues [29] demonstrated antibacterial activity of coumarin compounds (with a free hydroxyl at position 8) against resistant and susceptible strains of S. aureus and also reported reduction in activity with acetylation of coumarin precursors. This came into disagreement with our acetylated compound (14). Disagreement with our results was also seen in comparison with the studies of Céspedes et al. [30], who reported good antibacterial activities for compounds with dihydroxyls at positions C-7 and C-8 and methoxylation at C-6 against susceptible strains of S. aureus, unlike our compounds 6 and 10, which revealed no antibacterial activity against strains of this microorganism.

In the assay for bacterial resistance (modulation assay) the MICs of the antibiotics (tetracycline against IS-58 strain, erythromycin against RN4220 strain, and norfloxacin against SA-1199B strain) were determined in the absence and in the presence of the coumarin derivatives (compounds 122) which were incorporated into the growth medium at a concentration corresponding to of the MIC (subinhibitory concentration) [19]. The results of the compounds that showed modulatory activity are presented in Table 1.

Table 1: MIC values (μg/mL) of antibiotics in the absence and presence of coumarin derivatives against S. aureus strains: SA-1199B (NorA), RN4220 (MsrA), and IS-58 (TetK).

As can be seen in Table 1, some coumarins enhanced the activity of tetracycline (2x) and of norfloxacin (2x to 8x), thus reducing the concentration required to inhibit the growth of drug resistant (effluxing) strains.

Among the commercial coumarins evaluated only 1,2-Benzopyrone (1) showed modulatory activity, reducing the MIC of norfloxacin in twice. However, the synthesized compounds having allyl (11, 19), geranyl (13, 18), and prenyl (16 and 17) radicals showed good to significant modulatory activities, in particular for resistance to norfloxacin (Table 1).

The common characteristic of these modulatory compounds is the presence of an alkyl radical, which has been described as important for the drug resistance modulating activity of S. aureus, inhibiting the efflux system [31]. Brunel et al. [32] have also associated the presence of the geranyl group as an important characteristic for modulation of multiple drug resistance in Gram-negative bacteria.

Changing the position of these C-7 radicals (11, 13, and 16) to the C-6 position (17, 18, and 19) favored the modulation of the tetracycline resistance by two times but reduced (two to four times) the modifying activity of norfloxacin. With respect to the modulatory activity of coumarin for the RN4220 (MRSA) strain, no reduction was observed for the erythromycin MIC. However, Reynolds et al. [33] have suggested that the MRSA protein cannot be an efflux pump protein.

Acetylated and nitrated coumarins were all inactive, which leads us to believe that the presence of electron withdrawing groups (decreasing the electron density of the benzopyran ring) or polar radicals negatively contribute to both antibiotic and modulatory activities for this class of compounds.

These conclusions based on a preliminary SAR study were also proven by analyzing the calculations of the Polar Surfaces Area (PSA) of the compounds, obtained by the VEJA ZZ program [26]. It was observed that the absence of free hydroxyls, carboxylic acids, and other polar groups (i.e., nitro derivatives) seems to be an important factor for the modulatory activity of coumarins.

Figure 1 shows the performed PSA, which allows observing the compounds that have polar surfaces (black) (2 and 22) and which are less active than the nonpolar derivatives (grey) (13 and 16).

Figure 1: Representation of the PSA (Polar Surface Area) of active (13 and 16) and inactive (2 and 22) compounds using VEJA ZZ program. White represents polar surface and grey-dark represents no-polar surfaces.

Additionally, docking study of the most active coumarins (11, 13, and 1619) against the Penicillin-Binding Protein (PBP) from Methicillin Resistant Staphylococcus aureus (MRSA) (PDB id: 4CJN) was performed. Table 2 shows the results of the MolDock Score of these potential inhibitors, and Figure 2 shows the interactions observed in the complex of the best ligand (compound 18) with the active site of the PBP enzyme.

Table 2: Binding energy () values.
Figure 2: Interactions observed in the compound 18-PBP complex.

One of bacterial resistance pathways occurs by low-interaction of the antibiotic by PPBs and hydrolysis by β-lactamases. These bacterial proteins are fundamental in the synthesis of the cell wall of bacteria, common target of β-lactam antibiotics. This fact makes PPBs a target searched against a resistance, particularly in Gram-positive bacteria like Staphylococcus aureus and also in a higher selectivity to these unique bacteria structures [3438].

Molecular docking can be formulated as an optimization problem, where the task is to find the ligand-binding mode with the lowest energy. Table 2 shows the energies (kcal/mol) obtained from the interaction of the coumarins 11, 13, and 1619 and quinazolinone (compound complex with the enzyme in the crystal structure) and the PPB. We observed that compounds 17 ( = −6224 kcal/mol) and 18 ( = –91322 kcal/mol) generated lower energy scores which represent better protein-ligand bindings and stable complexes.

Compound 18 has shown greater energy value than the quinazolinone ( = −71098 kcal/mol) ligand complexed in the PDB. Figure 2 reports in 2D (a) and in 3D (b) the interactions formed between 18 and PPB: we can see hydrogen bond with LYS316 of chain B and two steric bonds (ASN307 of chain A and LYS273 of chain B).

4. Conclusions

Acting as antibiotic adjuvants, synthetic coumarin derivatives, especially alkylated derivatives, have shown promising antibiotic activity. They act by modulating antibiotic resistance through a mechanism that most likely involves inhibition of bacterial efflux pumps, thus affecting S. aureus resistance to tetracycline and norfloxacin.

The preliminary SAR study was confirmed by PSA analysis and reinforces the idea that coumarin derivatives substituted with electron donors groups are more powerful. These findings may help future design and synthesis of new derivatives, with more potent modulatory activity.

The docking study performed against the Penicillin-Binding Protein (PBP) from Methicillin Resistant Staphylococcus aureus (MRSA) identified that compound 18 is the best ligand with lower energy scores being one potential inhibitor of this important enzyme.

Considering the natural and inevitable development of resistance mechanisms in microorganisms, especially S. aureus, and the constant need for new agents with antibiotic properties, our results contribute to the problematic of bacterial infections. The discovery of new chemical entities associable with conventional antibiotics (antibiotic adjuvants) may allow reversion of cell resistance to conventional therapy (increased useful antibiotic life) and thus reduce antibiotic concentrations in prophylactic or therapeutic treatments.

Competing Interests

The authors declare that they have no competing interests.

Acknowledgments

The work was supported by CNPq. The authors also thank both the CNPq and CAPES for scholarships of Rodrigo Santos Aquino de Araújo, Ryldene Marques Duarte da Cruz, Vivyanne dos Santos Falcão-Silva, and Luciana Scotti.

References

  1. G. L. French, “The continuing crisis in antibiotic resistance,” International Journal of Antimicrobial Agents, vol. 36, no. 3, pp. S3–S7, 2010. View at Publisher · View at Google Scholar · View at Scopus
  2. L. P. Jordheim, S. Ben Larbi, O. Fendrich et al., “Gemcitabine is active against clinical multiresistant Staphylococcus aureus strains and is synergistic with gentamicin,” International Journal of Antimicrobial Agents, vol. 39, no. 5, pp. 444–447, 2012. View at Publisher · View at Google Scholar · View at Scopus
  3. G. A. Noskin, R. J. Rubin, J. J. Schentag et al., “The burden of Staphylococcus aureus infections on hospitals in the United States: an analysis of the 2000 and 2001 Nationwide Inpatient Sample Database,” Archives of Internal Medicine, vol. 156, no. 15, pp. 1756–1761, 2005. View at Google Scholar
  4. H. F. Chambers and F. R. DeLeo, “Waves of resistance: Staphylococcus aureus in the antibiotic era,” Nature Reviews Microbiology, vol. 7, no. 9, pp. 629–641, 2009. View at Publisher · View at Google Scholar · View at Scopus
  5. Y.-H. Fang, P.-R. Hsueh, J.-J. Hu et al., “Community-acquired methicillin-resistant Staphylococcus aureus in children in northern Taiwan,” Journal of Microbiology, Immunology and Infection, vol. 37, no. 1, pp. 29–34, 2004. View at Google Scholar · View at Scopus
  6. K. Hiramatsu, Y. Katayama, H. Yuzawa, and T. Ito, “Molecular genetics of methicillin-resistant Staphylococcus aureus,” International Journal of Medical Microbiology, vol. 292, no. 2, pp. 67–74, 2002. View at Publisher · View at Google Scholar · View at Scopus
  7. B. Marquez, “Bacterial efflux systems and efflux pumps inhibitors,” Biochimie, vol. 87, no. 12, pp. 1137–1147, 2005. View at Publisher · View at Google Scholar · View at Scopus
  8. L. J. V. Piddock, “Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria,” Clinical Microbiology Reviews, vol. 19, no. 2, pp. 382–402, 2006. View at Publisher · View at Google Scholar · View at Scopus
  9. S. Gibbons, “Anti-staphylococcal plant natural products,” Natural Product Reports, vol. 21, no. 2, pp. 263–277, 2004. View at Publisher · View at Google Scholar · View at Scopus
  10. X.-Z. Li and H. Nikaido, “Efflux-mediated drug resistance in bacteria,” Drugs, vol. 64, no. 2, pp. 159–204, 2004. View at Publisher · View at Google Scholar
  11. F. Van Bambeke, J.-M. Pagès, and V. J. Lee, “Inhibitors of bacterial efflux pumps as adjuvants in antibiotic treatments and diagnostic tools for detection of resistance by efflux,” Frontiers in Anti-Infective Drug Discovery, vol. 1, pp. 138–175, 2006. View at Publisher · View at Google Scholar · View at Scopus
  12. R. S. A. de Araújo, F. Q. S. Guerra, E. D. O. Lima et al., “Synthesis, Structure-activity Relationships (SAR) and in silico studies of coumarin derivatives with antifungal activity,” International Journal of Molecular Sciences, vol. 14, no. 1, pp. 1293–1309, 2013. View at Publisher · View at Google Scholar · View at Scopus
  13. N. Hamdi, A. S. Al-Ayed, R. B. Said, and A. Fabienne, “Synthesis and characterization of new thiazolidinones containing coumarin moieties and their antibacterial and antioxidant activities,” Molecules, vol. 17, no. 8, pp. 9321–9334, 2012. View at Publisher · View at Google Scholar · View at Scopus
  14. B. S. F. Bazzaz, M. Iranshahi, M. Naderinasab, S. Hajian, Z. Sabeti, and E. Masumi, “Evaluation of the effects of galbanic acid from Ferula szowitsiana and conferol from F. badrakema, as modulators of multi-drug resistance in clinical isolates of Escherichia coli and Staphylococcus aureus,” Research in Pharmaceutical Sciences, vol. 5, no. 1, pp. 25–32, 2010. View at Google Scholar · View at Scopus
  15. CLSI—Clinical and Laboratory Standards Institute, “Performance standards for antimicrobial susceptibility testing; twenty-fifth informational supplement,” CLSI Document M100-S25, Clinical and Laboratory Standards Institute, Wayne, Pa, USA, 2015. View at Google Scholar
  16. G. W. Kaatz and S. M. Seo, “Inducible NorA-mediated multidrug resistance in Staphylococcus aureus,” Antimicrobial Agents and Chemotherapy, vol. 39, no. 12, pp. 2650–2655, 1995. View at Publisher · View at Google Scholar · View at Scopus
  17. J. I. Ross, A. M. Farrell, E. A. Eady, J. H. Cove, and W. J. Cunliffe, “Characterisation and molecular cloning of the novel macrolide-streptogramin B resistance determinant from Staphylococcus epidermidis,” Journal of Antimicrobial Chemotherapy, vol. 24, no. 6, pp. 851–862, 1989. View at Publisher · View at Google Scholar · View at Scopus
  18. S. Gibbons and E. E. Udo, “The effect of reserpine, a modulator of multidrug efflux pumps, on the in vitro activity of tetracycline against clinical isolates of methicillin resistant Staphylococcus aureus (MRSA) possessing the tet(K) determinant,” Phytotherapy Research, vol. 14, no. 2, pp. 139–140, 2000. View at Publisher · View at Google Scholar · View at Scopus
  19. M. Stavri, L. J. V. Piddock, and S. Gibbons, “Bacterial efflux pump inhibitors from natural sources,” Journal of Antimicrobial Chemotherapy, vol. 59, no. 6, pp. 1247–1260, 2007. View at Publisher · View at Google Scholar · View at Scopus
  20. V. S. Falcão-Silva, D. A. Silva, M. D. F. V. Souza, and J. P. Siqueira Jr., “Modulation of drug resistance in Staphylococcus aureus by a kaempferol glycoside from Herissantia tiubae (Malvaceae),” Phytotherapy Research, vol. 23, no. 10, pp. 1367–1370, 2009. View at Publisher · View at Google Scholar · View at Scopus
  21. Hyperchem Program Release 8.0 for Windows 1999–2005, Hybercube Inc, Gainesville, Fla, USA.
  22. N. L. Allinger, “Conformational analysis. 130. MM2. A hydrocarbon force field utilizing V1 and V2 torsional terms,” Journal of the American Chemical Society, vol. 99, no. 25, pp. 8127–8134, 1977. View at Publisher · View at Google Scholar · View at Scopus
  23. M. J. S. Dewar, G. Zoebisch, E. F. Healy, and J. J. P. Stewart, “The development and use of quantum-mechanical molecular-models. 76. AM1—a new general-purpose quantum-mechanical molecular model,” Journal of the American Chemical Society, vol. 107, pp. 3902–3909, 1985. View at Google Scholar
  24. N. C. Cohen, Guidebook on Molecular Modeling in Drug Design, Academic Press, San Diego, Calif, USA, 1996.
  25. A. R. Leach, Molecular Modeling: Principles and Applications, Prentice Hall, London, UK, 2001.
  26. A. Pedretti, L. Villa, and G. Vistoli, “VEGA-an open platform to develop chemo-bio-informatics applications, using plug-in architecture and script programming,” Journal of Computer-Aided Molecular Design, vol. 18, no. 3, pp. 167–173, 2004. View at Publisher · View at Google Scholar · View at Scopus
  27. R. Bouley, M. Kumarasiri, Z. Peng et al., “Discovery of antibiotic (E)-3-(3-carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-one,” Journal of the American Chemical Society, vol. 137, no. 5, pp. 1738–1741, 2015. View at Google Scholar
  28. R. Thomsen and M. H. Christensen, “MolDock: a new technique for high-accuracy molecular docking,” Journal of Medicinal Chemistry, vol. 49, no. 11, pp. 3315–3321, 2006. View at Publisher · View at Google Scholar · View at Scopus
  29. Shakeel-U-Rehman, R. Khan, K. A. Bhat, A. F. Raja, A. S. Shawl, and M. S. Alam, “Isolation, characterisation and antibacterial activity studies of coumarins from Rhododendron lepidotum Wall. ex G. Don, Ericaceae,” Brazilian Journal of Pharmacognosy, vol. 20, no. 6, pp. 886–890, 2010. View at Publisher · View at Google Scholar · View at Scopus
  30. C. L. Céspedes, J. G. Avila, A. Martínez, B. Serrato, J. C. Calderón-Mugica, and R. Salgado-Garciglia, “Antifungal and antibacterial activities of Mexican tarragon (Tagetes lucida),” Journal of Agricultural and Food Chemistry, vol. 54, no. 10, pp. 3521–3527, 2006. View at Publisher · View at Google Scholar · View at Scopus
  31. A. Kumar, I. A. Khan, S. Koul et al., “Novel structural analogues of piperidine as inhibitors of the NorA efflux pump of Staphylococcus aureus,” Journal of Antimicrobial Chemotherapy, vol. 61, pp. 1270–1276, 2008. View at Google Scholar
  32. J. M. Brunel, A. Lieutaud, V. Lome, J.-M. Pagès, and J.-M. Bolla, “Polyamino geranic derivatives as new chemosensitizers to combat antibiotic resistant Gram-negative bacteria,” Bioorganic & Medicinal Chemistry, vol. 21, no. 5, pp. 1174–1179, 2013. View at Publisher · View at Google Scholar
  33. E. Reynolds, J. I. Ross, and J. H. Cove, “Msr(A) and related macrolide/streptogramin resistance determinants: incomplete transporters?” International Journal of Antimicrobial Agents, vol. 22, no. 3, pp. 228–236, 2003. View at Publisher · View at Google Scholar · View at Scopus
  34. A. Zervosen, E. Sauvage, J.-M. Frère, P. Charlier, and A. Luxen, “Development of new drugs for an old target—the penicillin binding proteins,” Molecules, vol. 17, no. 11, pp. 12478–12505, 2012. View at Publisher · View at Google Scholar · View at Scopus
  35. M. B. Fernandes, J. E. Gonçalves, M. T. Scotti, A. A. de Oliveira, L. C. Tavares, and S. Storpirtis, “Caco-2 cells cytotoxicity of nifuroxazide derivatives with potential activity against Methicillin-resistant Staphylococcus aureus (MRSA),” Toxicology in Vitro, vol. 26, no. 3, pp. 535–540, 2012. View at Publisher · View at Google Scholar · View at Scopus
  36. A. L. Greninger, S. S. Chatterjee, L. C. Chan et al., “Whole-genome sequencing of methicillin-resistant Staphylococcus aureus resistant to fifth-generation cephalosporins reveals potential non-meca mechanisms of resistance,” Plos ONE, vol. 11, no. 2, Article ID e0149541, 2016. View at Publisher · View at Google Scholar
  37. S. B. Hong, M. H. Rhee, B. Yun, Y. H. Lim, H. G. Song, and K. S. Shin, “Synergistic anti-bacterial effects of Phellinus baumii ethyl acetate extracts and beta-lactam antimicrobial agents against methicillin-resistant Staphylococcus aureus,” Annals of Laboratory Medicine, vol. 36, no. 2, pp. 111–116, 2016. View at Publisher · View at Google Scholar
  38. P. Lavanya, S. Ramaiah, and A. Anbarasu, “A molecular docking and dynamics study to screen potent anti-staphylococcal compounds against ceftaroline resistant MRSA,” Journal of Cellular Biochemistry, vol. 117, no. 2, pp. 542–548, 2016. View at Publisher · View at Google Scholar