Research ArticleModulation of Drug Resistance in Staphylococcus aureus withCoumarin Derivatives

Rodrigo Santos Aquino de Arauacutejo1 Joseacute Maria Barbosa-Filho1 Marcus Tullius Scotti1

Luciana Scotti1 Ryldene Marques Duarte da Cruz2 Vivyanne dos Santos Falcatildeo-Silva12

Joseacute Pinto de Siqueira-Juacutenior12 and Francisco Jaime Bezerra Mendonccedila-Junior13

1Post-Graduate Program inNatural and Synthetic Bioactive Products Federal University of Paraıba 58051-900 Joao Pessoa PB Brazil2Laboratory of Genetics of Microorganisms Molecular Biology Department Federal University of Paraıba58051-900 Joao Pessoa PB Brazil3Laboratory of Synthesis and Drug Delivery Biological Science Department State University of Paraıba58071-160 Joao Pessoa PB Brazil

Correspondence should be addressed to Jose Pinto de Siqueira-Junior jpsiquolcombrand Francisco Jaime Bezerra Mendonca-Junior franciscojbmendoncayahoocombr

Received 30 December 2015 Accepted 3 April 2016

Academic Editor Antonio Espinosa

Copyright copy 2016 Rodrigo Santos Aquino de Araujo et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Semisynthetic and commercial coumarins were investigated for their antibacterial and adjuvant properties with antibiotic agentsagainst norfloxacin erythromycin and tetracycline resistant Staphylococcus aureus as based on efflux mechanisms The coumarinsand certain commercial antibiotics had theirMinimum Inhibitory Concentrations determined by brothmicrodilution assay againstresistant S aureus strains which overexpress efflux pump proteins For evaluation of the modulatory activity the antibiotics MICswere determined in the presence of the coumarin derivatives at subinhibitory concentration Although the coumarins did notdisplay relevant antibacterial activity (MICge 128120583gmL) they didmodulate the antibiotics activities Various coumarins especiallythe alkylated derivatives in combination with antibiotics at subinhibitory concentrations modulated antibiotic activity reducingthe MIC for tetracycline and norfloxacin by 2 to 8 times Polar Surface Area (PSA) studies were performed and the fact that thepresence of apolar groups is an important factor for the modulatory activity of coumarins was corroborated Docking on thePenicillin-Binding Protein from MRSA identified that 18 is a potential ligand presenting low 119864binding The results indicate thatcoumarin derivatives modulated antibiotic resistance and may be used as potential antibiotic adjuvants acting by bacterial effluxpump inhibition in S aureus

1 Introduction

The development of mechanisms of resistance by the micro-organisms is a natural process that occurs in greater intensitywith the indiscriminate and inappropriate use of antimicrobi-als [1] This natural adaptation phenomenon has become theprinciple problem worldwide for the treatment of infectiousdiseases creating a continuous need for the discovery of newchemical entities with antibiotic properties [2]

Bacterial infections are a major cause of morbidity andmortality in hospitals as in the case of nosocomial infectionscaused by Staphylococcus aureus which cause about 12000

deaths per year in the USA [3] It is mentioned in theliterature that S aureus is a versatile pathogen with numerousvirulence factors making this microorganism capable ofacquiring antibiotic resistance determinants [4] Resistanceto methicillin (MRSA) first appeared in 1961 [5] with cur-rently described S aureus strains having acquired resistanceto all 120573-lactams macrolides aminoglycosides tetracyclinerifampicin quinolones and even vancomycin [6]

Among the various mechanisms of bacterial resistancetransmembrane efflux proteins have been commonly associ-ated with resistance to multiple antibiotics and other chemo-therapeutic agents [7 8]These proteins actively extrude toxic

Hindawi Publishing CorporationScientificaVolume 2016 Article ID 6894758 6 pageshttpdxdoiorg10115520166894758

2 Scientifica

substrates including antibiotics from the cell Antibioticactivity-modifying agents or modulators are compoundsthat potentiate the activity of antibiotics against resistantstrains some of these agents act as efflux pump inhibitors(EPI) [9] Some efflux pumps have been shown to be ofclinical significance in bacterial infections [10] among whichare the TetK pump (promoting resistance to tetracycline)the MsrA macrolide efflux protein (promoting macrolideresistance) and the NorA efflux pump (with resistance tofluoroquinolones) [11]

Currently one of the best strategies to control bacterialresistance and extend the life of existing antibiotics is toassociate them with modulators of drug resistance similarto what is done with many 120573-lactam antibiotics that areadministered in combination with potassium clavulanateCompounds that potentiate the activity of antimicrobialagents present numerous benefits including the possibilityof reducing the antibiotic concentration in prophylactic ortherapeutic treatments of infection and reversion of cellresistance to conventional therapy

Coumarins comprise a class of natural phenolic com-pounds characterized by a single benzene fused to an 120572-pyrone ring They stand out for having great biologicalpotential as demonstrated in several studies compoundswith antifungal [12] and antibacterial activities [13] and alsomodulators of antibiotic resistance [14]

Given the above and knowing the antimicrobial potentialand modulating action of coumarin derivatives on antibioticresistance our goal was to develop semisynthetic coumarinderivatives in order to evaluate their antibiotic andor antibi-otic adjuvant properties against effluxing S aureus strains

2 Materials and Methods

21 Coumarin Derivatives and Antibiotics All commercialcoumarins (12-Benzopyrone 1 3-Hydroxycoumarin 2 4-Hy-droxycoumarin 3 6-Hydroxycoumarin 4 7-Hydroxycouma-rin 5 67-Dihydroxycoumarin 6 Coumarin-3-carboxylicacid 7 331015840-Methylene-bis-(4-hydroxycoumarin) 8 6-Me-thoxy-7-hydroxycoumarin 9 and 78-Dihydroxy-6-methox-ycoumarin 10) reagents and solvents were purchased fromSigma-Aldrich (Seelze Germany) and used without furtherpurification Norfloxacin erythromycin and tetracyclinewere obtained from Sigma Chemical Co USA and theirstock solutions were then prepared [15] Compounds 11ndash22 were synthesized according to previously reported pro-cedures [12] The stock solutions of coumarin derivatives1ndash22 were prepared in DMSO The highest concentrationremaining after broth dilution (4) did not inhibit bacterialgrowth and a positive control with onlyDMSO4was testedand showed no interference with bacterial growth (data notshown)

22 Bacterial Strains Four S aureus strains were used for thebiological tests one standard strain (ATCC 6538) and threeantibiotics resistant strains SA-1199B which over expressesthe 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(Laboratorios Difco Ltda Brazil) slants Prior to use the cellswere grown overnight at 37∘C in brain heart infusion broth(BHI-Laboratorios Difco Ltda Brazil)

23 Drug Susceptibility Testing and Modulation Assay TheMinimum Inhibitory Concentrations (MICs) of the antibi-otics and coumarin derivatives were determined in BHIby broth microdilution assay using a suspension of ca105 cfumL and a drug concentration range from 05 to256120583gmL (twofold serial dilutions) For better visualizationof the bacterial growth (after 24 h) we used resazurin (001)To evaluate coumarin as a modulator of drug resistanceldquomodulation assayrdquo we used a method that has been widelyapplied to identify potential EPIs and is valid provided thatone uses specifically known effluxing strains The MICsof the antibiotics were determined in the presence of thesubinhibitory concentrations of coumarin compounds Bothassays were performed in triplicate [19 20]

24 Molecular Model Three-dimensional structures weredrawn using HyperChem 80 software [21] the structureshad energy-minimization calculated employing the methodMM+ force field without any restriction [22] Subsequently anew geometry optimization process based on the semiempir-ical method AM1 (Austin Model 1) was performed [23] Theoptimized structures were subjected to conformation analysisusing the random search method with 1000 interactions 100cycles of optimization and 10 lowest minimum energy con-formers The selected dihedrals were evaluated for rotationin accordance with the standard (default) conditions of theprogram the number of simultaneous variations was 1 to 8Acyclic chains were submitted to rotations from 60 to 180∘torsion rings were in the range of 30 to 120∘ [24 25] Thelowest energy conformers were selected and imported intoVEJAZZThedrawn coumarinswere imported into theVEJAZZ program [26] for calculation of the Polar Surface Area(PSA)

25 Docking The structure of the Penicillin-Binding Pro-tein (PBP) from Methicillin Resistant Staphylococcus aureus(MRSA) in complex with the ligand quinazolinone (PDBid 4CJN) [27] was downloaded from the Protein DataBank (httpwwwrcsborgpdbhomehomedo) Coumarinderivatives 11 13 and 16ndash19 were submitted to moleculardocking using the Molegro Virtual Docker v 601 (MVD)[28]

The protein and compound structures were preparedusing the default parameter settings in the software package(score function MolDock Score ligand evaluation InternalES Internal H-Bond and sp2ndashsp2 Torsions all checkednumber of runs 10 runs algorithm MolDock SE maximuminteractions 1500 max population size 50 max steps300 neighbor distance factor 100 max number of posesreturned 5) The docking procedure was performed using

Scientifica 3

O O

HO

HO

12

345

6

78

O O

RO

O O

AcO

O O

a b

c

15 = 4-OAc14 = 6-OAc12 = 7-OAc

19 = 6-allyl18 = 6-geranyl17 = 6-prenyl16 = 7-prenyl13 = 7-geranyl11 = 7-allyl

5 = 7-OH4 = 6-OH3 = 4-OH

O2

N

22 = 4-OH 3-NO2

21 = 6-OH 57-NO2

20 = 7-OH 6-NO2

Scheme 1 Synthesis of alkyl- acetyl- and nitro-coumarin derivatives Reagents and conditions (a) acetic anhydride pyridine rt ultrasoundirradiation (b) allyl bromide geranyl bromide or prenyl bromide K

2CO3 acetonitrile and reflux (c) HNO

3AcOH 0ndash5∘C for 30min and

then 90min at rt

a GRID of 15gt in radius and 030 in resolution to coverthe ligand-biding site of the Penicillin-Binding Protein (PBP)structure

Templates with features expected to be relevant for ligandbinding (quinazoline) were generated to perform dockingThe MolDock Score [GRID] algorithm was used as the scorefunction and the MolDock search algorithm was used [28]

3 Results and Discussion

Twelve coumarin derivatives (11ndash22) were synthesized fromcommercial coumarins 4-hydroxy- (3) 6-hydroxy- (4)and 7-hydroxycoumarin (5) by alkylation acetylation andnitration procedures according to procedures previouslydescribed by our group [12] (Scheme 1)

All commercial (1ndash10) and synthetic (11ndash22) coumarinswere evaluated for their in vitro antibacterial activity againstthe four Staphylococcus aureus strains the standard (ATCC6538) and the three resistant IS-58 (TetK) RN4220 (MsrA)and SA-1199B (NorA) however all of the commercialcoumarins (1ndash10) and the eight synthetic coumarins (1112 15 and 18ndash22) showed no antibacterial activity (MICgt 256120583gmL) against any S aureus strain evaluated Com-pounds 13 14 16 and 17 showed weak activity with MICvalues of 128 120583gmL (13 against ATCC 6538 and SA-1199B 16against SA-1199B and 14 and 17 against all strains)

Shakeel-U-Rehman and colleagues [29] demonstratedantibacterial activity of coumarin compounds (with a freehydroxyl at position 8) against resistant and susceptiblestrains of S aureus and also reported reduction in activitywith acetylation of coumarin precursors This came intodisagreement with our acetylated compound (14) Disagree-ment with our results was also seen in comparison withthe studies of Cespedes et al [30] who reported goodantibacterial activities for compounds with dihydroxyls at

positions C-7 and C-8 and methoxylation at C-6 againstsusceptible strains of S aureus unlike our compounds 6 and10 which revealed no antibacterial activity against strains ofthis microorganism

In the assay for bacterial resistance (modulation assay)the MICs of the antibiotics (tetracycline against IS-58 strainerythromycin against RN4220 strain and norfloxacin againstSA-1199B strain) were determined in the absence and inthe presence of the coumarin derivatives (compounds 1ndash22) which were incorporated into the growth medium ata concentration corresponding to 14 of the MIC (subin-hibitory concentration) [19] The results of the compoundsthat showed modulatory activity are presented in Table 1

As can be seen in Table 1 some coumarins enhanced theactivity of tetracycline (2x) and of norfloxacin (2x to 8x) thusreducing the concentration required to inhibit the growth ofdrug resistant (effluxing) strains

Among the commercial coumarins evaluated only 12-Benzopyrone (1) showed modulatory activity reducing theMIC of norfloxacin in twice However the synthesizedcompounds having allyl (11 19) geranyl (13 18) and prenyl(16 and 17) radicals showed good to significant modulatoryactivities in particular for resistance to norfloxacin (Table 1)

The common characteristic of these modulatory com-pounds is the presence of an alkyl radical which has beendescribed as important for the drug resistance modulatingactivity of S aureus inhibiting the efflux system [31] Brunel etal [32] have also associated the presence of the geranyl groupas an important characteristic for modulation of multipledrug resistance in Gram-negative bacteria

Changing the position of theseC-7 radicals (11 13 and 16)to the C-6 position (17 18 and 19) favored the modulationof the tetracycline resistance by two times but reduced(two to four times) the modifying activity of norfloxacinWith respect to the modulatory activity of coumarin for

4 Scientifica

Table 1 MIC values (120583gmL) of antibiotics in the absence and presence of coumarin derivatives against S aureus strains SA-1199B (NorA)RN4220 (MsrA) and IS-58 (TetK)

Antibiotic + coumarin MIC (120583gmL)IS-58 (TetK)Tetracycline

RN4220 (MrsA)Erythromycin

SA-1199B (NorA)Norfloxacin

Antibiotics alone 64 gt256 128Antibiotics + 1 64 gt256 64 (2x)Antibiotics + 11 64 gt256 32 (4x)Antibiotics + 13 64 gt256 32 (4x)Antibiotics + 16 64 gt256 16 (8x)Antibiotics + 17 32 (2x) gt256 64 (2x)Antibiotics + 18 32 (2x) gt256 64 (2x)Antibiotics + 19 64 gt256 64 (2x)

Figure 1 Representation of the PSA (Polar Surface Area) of active(13 and 16) and inactive (2 and 22) compounds using VEJA ZZprogram White represents polar surface and grey-dark representsno-polar surfaces

the RN4220 (MRSA) strain no reduction was observed forthe erythromycin MIC However Reynolds et al [33] havesuggested that the MRSA protein cannot be an efflux pumpprotein

Acetylated and nitrated coumarins were all inactivewhich leads us to believe that the presence of electronwithdrawing groups (decreasing the electron density of thebenzopyran ring) or polar radicals negatively contribute toboth antibiotic and modulatory activities for this class ofcompounds

These conclusions based on a preliminary SAR studywere also proven by analyzing the calculations of the PolarSurfaces Area (PSA) of the compounds obtained by theVEJA ZZ program [26] It was observed that the absence offree hydroxyls carboxylic acids and other polar groups (ienitro derivatives) seems to be an important factor for themodulatory activity of coumarins

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

Table 2 Binding energy (119864binding) values

Compounds MolDock Score (kcalmol)Quinazolinone minus7109811 minus4545313 minus5835816 minus5802117 minus6222418 minus9132219 minus55676

Additionally docking study of the most active coumarins(11 13 and 16ndash19) against the Penicillin-Binding Protein(PBP) from Methicillin Resistant Staphylococcus aureus(MRSA) (PDB id 4CJN) was performed Table 2 shows theresults of theMolDock Score of these potential inhibitors andFigure 2 shows the interactions observed in the complex ofthe best ligand (compound 18) with the active site of the PBPenzyme

One of bacterial resistance pathways occurs by low-interaction of the antibiotic by PPBs and hydrolysis by 120573-lactamases These bacterial proteins are fundamental in thesynthesis of the cell wall of bacteria common target of 120573-lactam antibiotics This fact makes PPBs a target searchedagainst a resistance particularly in Gram-positive bacterialike Staphylococcus aureus and also in a higher selectivity tothese unique bacteria structures [34ndash38]

Molecular docking can be formulated as an optimizationproblem where the task is to find the ligand-binding modewith the lowest energy Table 2 shows the energies (kcalmol)obtained from the interaction of the coumarins 11 13 and 16ndash19 and quinazolinone (compound complex with the enzymein the crystal structure) and the PPB We observed thatcompounds 17 (119864binding = minus6224 kcalmol) and 18 (119864binding =ndash91322 kcalmol) generated lower energy scores which repre-sent better protein-ligand bindings and stable complexes

Compound 18 has shown greater energy value than thequinazolinone (119864binding =minus71098 kcalmol) ligand complexedin the PDB Figure 2 reports in 2D (a) and in 3D (b)the interactions formed between 18 and PPB we can see

Scientifica 5

(a) (b)

Figure 2 Interactions observed in the compound 18-PBP complex

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 deriva-tives especially alkylated derivatives have shown promis-ing antibiotic activity They act by modulating antibioticresistance through a mechanism that most likely involvesinhibition of bacterial efflux pumps thus affecting S aureusresistance to tetracycline and norfloxacin

The preliminary SAR study was confirmed by PSAanalysis and reinforces the idea that coumarin derivativessubstituted with electron donors groups are more powerfulThese findings may help future design and synthesis of newderivatives with more potent modulatory activity

The docking study performed against the Penicillin-Binding Protein (PBP) from Methicillin Resistant Staphylo-coccus aureus (MRSA) identified that compound 18 is the bestligand with lower energy scores being one potential inhibitorof this important enzyme

Considering the natural and inevitable developmentof resistance mechanisms in microorganisms especially Saureus and the constant need for new agents with antibioticproperties our results contribute to the problematic of bacte-rial infections The discovery of new chemical entities asso-ciable with conventional antibiotics (antibiotic adjuvants)may allow reversion of cell resistance to conventional therapy(increased useful antibiotic life) and thus reduce antibioticconcentrations 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 thankboth the CNPq and CAPES for scholarships of Rodrigo

Santos Aquino de Araujo Ryldene Marques Duarte da CruzVivyanne dos Santos Falcao-Silva and Luciana Scotti

References

[1] G L French ldquoThe continuing crisis in antibiotic resistancerdquoInternational Journal of Antimicrobial Agents vol 36 no 3 ppS3ndashS7 2010

[2] L P Jordheim S Ben Larbi O Fendrich et al ldquoGemcitabineis active against clinical multiresistant Staphylococcus aureusstrains and is synergistic with gentamicinrdquo International Journalof Antimicrobial Agents vol 39 no 5 pp 444ndash447 2012

[3] G A Noskin R J Rubin J J Schentag et al ldquoThe burdenof Staphylococcus aureus infections on hospitals in the UnitedStates an analysis of the 2000 and 2001 Nationwide InpatientSample Databaserdquo Archives of Internal Medicine vol 156 no 15pp 1756ndash1761 2005

[4] H F Chambers and F R DeLeo ldquoWaves of resistanceStaphylococcus aureus in the antibiotic erardquo Nature ReviewsMicrobiology vol 7 no 9 pp 629ndash641 2009

[5] Y-H Fang P-R Hsueh J-J Hu et al ldquoCommunity-acquiredmethicillin-resistant Staphylococcus aureus in children innorthern Taiwanrdquo Journal of Microbiology Immunology andInfection vol 37 no 1 pp 29ndash34 2004

[6] K Hiramatsu Y Katayama H Yuzawa and T Ito ldquoMoleculargenetics of methicillin-resistant Staphylococcus aureusrdquo Inter-national Journal of Medical Microbiology vol 292 no 2 pp 67ndash74 2002

[7] B Marquez ldquoBacterial efflux systems and efflux pumpsinhibitorsrdquo Biochimie vol 87 no 12 pp 1137ndash1147 2005

[8] L J V Piddock ldquoClinically relevant chromosomally encodedmultidrug resistance efflux pumps in bacteriardquo Clinical Micro-biology Reviews vol 19 no 2 pp 382ndash402 2006

[9] S Gibbons ldquoAnti-staphylococcal plant natural productsrdquoNatu-ral Product Reports vol 21 no 2 pp 263ndash277 2004

[10] X-Z Li and H Nikaido ldquoEfflux-mediated drug resistance inbacteriardquo Drugs vol 64 no 2 pp 159ndash204 2004

[11] F Van Bambeke J-M Pages and V J Lee ldquoInhibitors ofbacterial efflux pumps as adjuvants in antibiotic treatments and

6 Scientifica

diagnostic tools for detection of resistance by effluxrdquo Frontiersin Anti-Infective Drug Discovery vol 1 pp 138ndash175 2006

[12] R S A deAraujo FQ SGuerra EDO Lima et al ldquoSynthesisStructure-activity Relationships (SAR) and in silico studies ofcoumarin derivatives with antifungal activityrdquo InternationalJournal of Molecular Sciences vol 14 no 1 pp 1293ndash1309 2013

[13] N Hamdi A S Al-Ayed R B Said and A Fabienne ldquoSyn-thesis and characterization of new thiazolidinones containingcoumarin moieties and their antibacterial and antioxidantactivitiesrdquoMolecules vol 17 no 8 pp 9321ndash9334 2012

[14] B S F Bazzaz M Iranshahi M Naderinasab S Hajian ZSabeti and E Masumi ldquoEvaluation of the effects of galbanicacid from Ferula szowitsiana and conferol from F badrakemaas modulators of multi-drug resistance in clinical isolates ofEscherichia coli and Staphylococcus aureusrdquo Research in Phar-maceutical Sciences vol 5 no 1 pp 25ndash32 2010

[15] CLSImdashClinical and Laboratory Standards InstituteldquoPerformance standards for antimicrobial susceptibility testingtwenty-fifth informational supplementrdquo CLSI DocumentM100-S25 Clinical and Laboratory Standards Institute WaynePa USA 2015

[16] G W Kaatz and S M Seo ldquoInducible NorA-mediatedmultidrug resistance in Staphylococcus aureusrdquo AntimicrobialAgents and Chemotherapy vol 39 no 12 pp 2650ndash2655 1995

[17] J I Ross A M Farrell E A Eady J H Cove andW J Cunliffe ldquoCharacterisation and molecular cloning ofthe novel macrolide-streptogramin B resistance determinantfrom Staphylococcus epidermidisrdquo Journal of AntimicrobialChemotherapy vol 24 no 6 pp 851ndash862 1989

[18] S Gibbons and E E Udo ldquoThe effect of reserpine a modulatorofmultidrug efflux pumps on the in vitro activity of tetracyclineagainst clinical isolates of methicillin resistant Staphylococcusaureus (MRSA) possessing the tet(K) determinantrdquo Phytother-apy Research vol 14 no 2 pp 139ndash140 2000

[19] M Stavri L J V Piddock and S Gibbons ldquoBacterial effluxpump inhibitors from natural sourcesrdquo Journal of AntimicrobialChemotherapy vol 59 no 6 pp 1247ndash1260 2007

[20] V S Falcao-Silva D A SilvaM D F V Souza and J P SiqueiraJr ldquoModulation of drug resistance in Staphylococcus aureus bya kaempferol glycoside from Herissantia tiubae (Malvaceae)rdquoPhytotherapy Research vol 23 no 10 pp 1367ndash1370 2009

[21] Hyperchem Program Release 80 for Windows 1999ndash2005Hybercube Inc Gainesville Fla USA

[22] N L Allinger ldquoConformational analysis 130 MM2 A hydro-carbon force field utilizing V1 and V2 torsional termsrdquo Journalof the American Chemical Society vol 99 no 25 pp 8127ndash81341977

[23] M J S Dewar G Zoebisch E F Healy and J J PStewart ldquoThe development and use of quantum-mechanicalmolecular-models 76 AM1mdasha new general-purpose quantum-mechanical molecular modelrdquo Journal of the American Chemi-cal Society vol 107 pp 3902ndash3909 1985

[24] N C CohenGuidebook onMolecular Modeling in Drug DesignAcademic Press San Diego Calif USA 1996

[25] A R Leach Molecular Modeling Principles and ApplicationsPrentice Hall London UK 2001

[26] A Pedretti L Villa and G Vistoli ldquoVEGA-an open platformto develop chemo-bio-informatics applications using plug-inarchitecture and script programmingrdquo Journal of Computer-Aided Molecular Design vol 18 no 3 pp 167ndash173 2004

[27] R BouleyM Kumarasiri Z Peng et al ldquoDiscovery of antibiotic(E)-3-(3-carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-onerdquo Journal of the American Chemical Society vol 137 no 5pp 1738ndash1741 2015

[28] R Thomsen and M H Christensen ldquoMolDock a new tech-nique for high-accuracy molecular dockingrdquo Journal of Medici-nal Chemistry vol 49 no 11 pp 3315ndash3321 2006

[29] Shakeel-U-Rehman R Khan K A Bhat A F Raja A S Shawland M S Alam ldquoIsolation characterisation and antibacterialactivity studies of coumarins from Rhododendron lepidotumWall ex G Don Ericaceaerdquo Brazilian Journal of Pharmacog-nosy vol 20 no 6 pp 886ndash890 2010

[30] C L Cespedes J G Avila A Martınez B Serrato J CCalderon-Mugica and R Salgado-Garciglia ldquoAntifungal andantibacterial activities of Mexican tarragon (Tagetes lucida)rdquoJournal of Agricultural and Food Chemistry vol 54 no 10 pp3521ndash3527 2006

[31] A Kumar I A Khan S Koul et al ldquoNovel structural analoguesof piperidine as inhibitors of the NorA efflux pump of Staphylo-coccus aureusrdquo Journal of Antimicrobial Chemotherapy vol 61pp 1270ndash1276 2008

[32] J M Brunel A Lieutaud V Lome J-M Pages and J-M BollaldquoPolyamino geranic derivatives as new chemosensitizers tocombat antibiotic resistant Gram-negative bacteriardquo Bioorganicamp Medicinal Chemistry vol 21 no 5 pp 1174ndash1179 2013

[33] E Reynolds J I Ross and J H Cove ldquoMsr(A) and relatedmacrolidestreptogramin resistance determinants incompletetransportersrdquo International Journal of Antimicrobial Agentsvol 22 no 3 pp 228ndash236 2003

[34] A Zervosen E Sauvage J-M Frere P Charlier and A LuxenldquoDevelopment of new drugs for an old targetmdashthe penicillinbinding proteinsrdquo Molecules vol 17 no 11 pp 12478ndash125052012

[35] M B Fernandes J E Goncalves M T Scotti A A deOliveira L C Tavares and S Storpirtis ldquoCaco-2 cells cytotox-icity of nifuroxazide derivatives with potential activity againstMethicillin-resistant Staphylococcus aureus (MRSA)rdquo Toxicol-ogy in Vitro vol 26 no 3 pp 535ndash540 2012

[36] A L Greninger S S Chatterjee L C Chan et al ldquoWhole-genome sequencing of methicillin-resistant Staphylococcusaureus resistant to fifth-generation cephalosporins revealspotential non-meca mechanisms of resistancerdquo Plos ONE vol11 no 2 Article ID e0149541 2016

[37] S B Hong M H Rhee B Yun Y H Lim H G Song andK S Shin ldquoSynergistic anti-bacterial effects of Phellinus baumiiethyl acetate extracts and beta-lactam antimicrobial agentsagainst methicillin-resistant Staphylococcus aureusrdquo Annals ofLaboratory Medicine vol 36 no 2 pp 111ndash116 2016

[38] P Lavanya S Ramaiah and A Anbarasu ldquoAmolecular dockingand dynamics study to screen potent anti-staphylococcal com-pounds against ceftaroline resistant MRSArdquo Journal of CellularBiochemistry vol 117 no 2 pp 542ndash548 2016

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Microbiology

2 Scientifica

substrates including antibiotics from the cell Antibioticactivity-modifying agents or modulators are compoundsthat potentiate the activity of antibiotics against resistantstrains some of these agents act as efflux pump inhibitors(EPI) [9] Some efflux pumps have been shown to be ofclinical significance in bacterial infections [10] among whichare the TetK pump (promoting resistance to tetracycline)the MsrA macrolide efflux protein (promoting macrolideresistance) and the NorA efflux pump (with resistance tofluoroquinolones) [11]

Currently one of the best strategies to control bacterialresistance and extend the life of existing antibiotics is toassociate them with modulators of drug resistance similarto what is done with many 120573-lactam antibiotics that areadministered in combination with potassium clavulanateCompounds that potentiate the activity of antimicrobialagents present numerous benefits including the possibilityof reducing the antibiotic concentration in prophylactic ortherapeutic treatments of infection and reversion of cellresistance to conventional therapy

Coumarins comprise a class of natural phenolic com-pounds characterized by a single benzene fused to an 120572-pyrone ring They stand out for having great biologicalpotential as demonstrated in several studies compoundswith antifungal [12] and antibacterial activities [13] and alsomodulators of antibiotic resistance [14]

Given the above and knowing the antimicrobial potentialand modulating action of coumarin derivatives on antibioticresistance our goal was to develop semisynthetic coumarinderivatives in order to evaluate their antibiotic andor antibi-otic adjuvant properties against effluxing S aureus strains

2 Materials and Methods

21 Coumarin Derivatives and Antibiotics All commercialcoumarins (12-Benzopyrone 1 3-Hydroxycoumarin 2 4-Hy-droxycoumarin 3 6-Hydroxycoumarin 4 7-Hydroxycouma-rin 5 67-Dihydroxycoumarin 6 Coumarin-3-carboxylicacid 7 331015840-Methylene-bis-(4-hydroxycoumarin) 8 6-Me-thoxy-7-hydroxycoumarin 9 and 78-Dihydroxy-6-methox-ycoumarin 10) reagents and solvents were purchased fromSigma-Aldrich (Seelze Germany) and used without furtherpurification Norfloxacin erythromycin and tetracyclinewere obtained from Sigma Chemical Co USA and theirstock solutions were then prepared [15] Compounds 11ndash22 were synthesized according to previously reported pro-cedures [12] The stock solutions of coumarin derivatives1ndash22 were prepared in DMSO The highest concentrationremaining after broth dilution (4) did not inhibit bacterialgrowth and a positive control with onlyDMSO4was testedand showed no interference with bacterial growth (data notshown)

22 Bacterial Strains Four S aureus strains were used for thebiological tests one standard strain (ATCC 6538) and threeantibiotics resistant strains SA-1199B which over expressesthe 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(Laboratorios Difco Ltda Brazil) slants Prior to use the cellswere grown overnight at 37∘C in brain heart infusion broth(BHI-Laboratorios Difco Ltda Brazil)

23 Drug Susceptibility Testing and Modulation Assay TheMinimum Inhibitory Concentrations (MICs) of the antibi-otics and coumarin derivatives were determined in BHIby broth microdilution assay using a suspension of ca105 cfumL and a drug concentration range from 05 to256120583gmL (twofold serial dilutions) For better visualizationof the bacterial growth (after 24 h) we used resazurin (001)To evaluate coumarin as a modulator of drug resistanceldquomodulation assayrdquo we used a method that has been widelyapplied to identify potential EPIs and is valid provided thatone uses specifically known effluxing strains The MICsof the antibiotics were determined in the presence of thesubinhibitory concentrations of coumarin compounds Bothassays were performed in triplicate [19 20]

24 Molecular Model Three-dimensional structures weredrawn using HyperChem 80 software [21] the structureshad energy-minimization calculated employing the methodMM+ force field without any restriction [22] Subsequently anew geometry optimization process based on the semiempir-ical method AM1 (Austin Model 1) was performed [23] Theoptimized structures were subjected to conformation analysisusing the random search method with 1000 interactions 100cycles of optimization and 10 lowest minimum energy con-formers The selected dihedrals were evaluated for rotationin accordance with the standard (default) conditions of theprogram the number of simultaneous variations was 1 to 8Acyclic chains were submitted to rotations from 60 to 180∘torsion rings were in the range of 30 to 120∘ [24 25] Thelowest energy conformers were selected and imported intoVEJAZZThedrawn coumarinswere imported into theVEJAZZ program [26] for calculation of the Polar Surface Area(PSA)

25 Docking The structure of the Penicillin-Binding Pro-tein (PBP) from Methicillin Resistant Staphylococcus aureus(MRSA) in complex with the ligand quinazolinone (PDBid 4CJN) [27] was downloaded from the Protein DataBank (httpwwwrcsborgpdbhomehomedo) Coumarinderivatives 11 13 and 16ndash19 were submitted to moleculardocking using the Molegro Virtual Docker v 601 (MVD)[28]

The protein and compound structures were preparedusing the default parameter settings in the software package(score function MolDock Score ligand evaluation InternalES Internal H-Bond and sp2ndashsp2 Torsions all checkednumber of runs 10 runs algorithm MolDock SE maximuminteractions 1500 max population size 50 max steps300 neighbor distance factor 100 max number of posesreturned 5) The docking procedure was performed using

Scientifica 3

O O

HO

HO

12

345

6

78

O O

RO

O O

AcO

O O

a b

c

15 = 4-OAc14 = 6-OAc12 = 7-OAc

19 = 6-allyl18 = 6-geranyl17 = 6-prenyl16 = 7-prenyl13 = 7-geranyl11 = 7-allyl

5 = 7-OH4 = 6-OH3 = 4-OH

O2

N

22 = 4-OH 3-NO2

21 = 6-OH 57-NO2

20 = 7-OH 6-NO2

Scheme 1 Synthesis of alkyl- acetyl- and nitro-coumarin derivatives Reagents and conditions (a) acetic anhydride pyridine rt ultrasoundirradiation (b) allyl bromide geranyl bromide or prenyl bromide K

2CO3 acetonitrile and reflux (c) HNO

3AcOH 0ndash5∘C for 30min and

then 90min at rt

a GRID of 15gt in radius and 030 in resolution to coverthe ligand-biding site of the Penicillin-Binding Protein (PBP)structure

Templates with features expected to be relevant for ligandbinding (quinazoline) were generated to perform dockingThe MolDock Score [GRID] algorithm was used as the scorefunction and the MolDock search algorithm was used [28]

3 Results and Discussion

Twelve coumarin derivatives (11ndash22) were synthesized fromcommercial coumarins 4-hydroxy- (3) 6-hydroxy- (4)and 7-hydroxycoumarin (5) by alkylation acetylation andnitration procedures according to procedures previouslydescribed by our group [12] (Scheme 1)

All commercial (1ndash10) and synthetic (11ndash22) coumarinswere evaluated for their in vitro antibacterial activity againstthe four Staphylococcus aureus strains the standard (ATCC6538) and the three resistant IS-58 (TetK) RN4220 (MsrA)and SA-1199B (NorA) however all of the commercialcoumarins (1ndash10) and the eight synthetic coumarins (1112 15 and 18ndash22) showed no antibacterial activity (MICgt 256120583gmL) against any S aureus strain evaluated Com-pounds 13 14 16 and 17 showed weak activity with MICvalues of 128 120583gmL (13 against ATCC 6538 and SA-1199B 16against SA-1199B and 14 and 17 against all strains)

Shakeel-U-Rehman and colleagues [29] demonstratedantibacterial activity of coumarin compounds (with a freehydroxyl at position 8) against resistant and susceptiblestrains of S aureus and also reported reduction in activitywith acetylation of coumarin precursors This came intodisagreement with our acetylated compound (14) Disagree-ment with our results was also seen in comparison withthe studies of Cespedes et al [30] who reported goodantibacterial activities for compounds with dihydroxyls at

positions C-7 and C-8 and methoxylation at C-6 againstsusceptible strains of S aureus unlike our compounds 6 and10 which revealed no antibacterial activity against strains ofthis microorganism

In the assay for bacterial resistance (modulation assay)the MICs of the antibiotics (tetracycline against IS-58 strainerythromycin against RN4220 strain and norfloxacin againstSA-1199B strain) were determined in the absence and inthe presence of the coumarin derivatives (compounds 1ndash22) which were incorporated into the growth medium ata concentration corresponding to 14 of the MIC (subin-hibitory concentration) [19] The results of the compoundsthat showed modulatory activity are presented in Table 1

As can be seen in Table 1 some coumarins enhanced theactivity of tetracycline (2x) and of norfloxacin (2x to 8x) thusreducing the concentration required to inhibit the growth ofdrug resistant (effluxing) strains

Among the commercial coumarins evaluated only 12-Benzopyrone (1) showed modulatory activity reducing theMIC of norfloxacin in twice However the synthesizedcompounds having allyl (11 19) geranyl (13 18) and prenyl(16 and 17) radicals showed good to significant modulatoryactivities in particular for resistance to norfloxacin (Table 1)

The common characteristic of these modulatory com-pounds is the presence of an alkyl radical which has beendescribed as important for the drug resistance modulatingactivity of S aureus inhibiting the efflux system [31] Brunel etal [32] have also associated the presence of the geranyl groupas an important characteristic for modulation of multipledrug resistance in Gram-negative bacteria

Changing the position of theseC-7 radicals (11 13 and 16)to the C-6 position (17 18 and 19) favored the modulationof the tetracycline resistance by two times but reduced(two to four times) the modifying activity of norfloxacinWith respect to the modulatory activity of coumarin for

4 Scientifica

Table 1 MIC values (120583gmL) of antibiotics in the absence and presence of coumarin derivatives against S aureus strains SA-1199B (NorA)RN4220 (MsrA) and IS-58 (TetK)

Antibiotic + coumarin MIC (120583gmL)IS-58 (TetK)Tetracycline

RN4220 (MrsA)Erythromycin

SA-1199B (NorA)Norfloxacin

Antibiotics alone 64 gt256 128Antibiotics + 1 64 gt256 64 (2x)Antibiotics + 11 64 gt256 32 (4x)Antibiotics + 13 64 gt256 32 (4x)Antibiotics + 16 64 gt256 16 (8x)Antibiotics + 17 32 (2x) gt256 64 (2x)Antibiotics + 18 32 (2x) gt256 64 (2x)Antibiotics + 19 64 gt256 64 (2x)

Figure 1 Representation of the PSA (Polar Surface Area) of active(13 and 16) and inactive (2 and 22) compounds using VEJA ZZprogram White represents polar surface and grey-dark representsno-polar surfaces

the RN4220 (MRSA) strain no reduction was observed forthe erythromycin MIC However Reynolds et al [33] havesuggested that the MRSA protein cannot be an efflux pumpprotein

Acetylated and nitrated coumarins were all inactivewhich leads us to believe that the presence of electronwithdrawing groups (decreasing the electron density of thebenzopyran ring) or polar radicals negatively contribute toboth antibiotic and modulatory activities for this class ofcompounds

These conclusions based on a preliminary SAR studywere also proven by analyzing the calculations of the PolarSurfaces Area (PSA) of the compounds obtained by theVEJA ZZ program [26] It was observed that the absence offree hydroxyls carboxylic acids and other polar groups (ienitro derivatives) seems to be an important factor for themodulatory activity of coumarins

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

Table 2 Binding energy (119864binding) values

Compounds MolDock Score (kcalmol)Quinazolinone minus7109811 minus4545313 minus5835816 minus5802117 minus6222418 minus9132219 minus55676

Additionally docking study of the most active coumarins(11 13 and 16ndash19) against the Penicillin-Binding Protein(PBP) from Methicillin Resistant Staphylococcus aureus(MRSA) (PDB id 4CJN) was performed Table 2 shows theresults of theMolDock Score of these potential inhibitors andFigure 2 shows the interactions observed in the complex ofthe best ligand (compound 18) with the active site of the PBPenzyme

One of bacterial resistance pathways occurs by low-interaction of the antibiotic by PPBs and hydrolysis by 120573-lactamases These bacterial proteins are fundamental in thesynthesis of the cell wall of bacteria common target of 120573-lactam antibiotics This fact makes PPBs a target searchedagainst a resistance particularly in Gram-positive bacterialike Staphylococcus aureus and also in a higher selectivity tothese unique bacteria structures [34ndash38]

Molecular docking can be formulated as an optimizationproblem where the task is to find the ligand-binding modewith the lowest energy Table 2 shows the energies (kcalmol)obtained from the interaction of the coumarins 11 13 and 16ndash19 and quinazolinone (compound complex with the enzymein the crystal structure) and the PPB We observed thatcompounds 17 (119864binding = minus6224 kcalmol) and 18 (119864binding =ndash91322 kcalmol) generated lower energy scores which repre-sent better protein-ligand bindings and stable complexes

Compound 18 has shown greater energy value than thequinazolinone (119864binding =minus71098 kcalmol) ligand complexedin the PDB Figure 2 reports in 2D (a) and in 3D (b)the interactions formed between 18 and PPB we can see

Scientifica 5

(a) (b)

Figure 2 Interactions observed in the compound 18-PBP complex

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 deriva-tives especially alkylated derivatives have shown promis-ing antibiotic activity They act by modulating antibioticresistance through a mechanism that most likely involvesinhibition of bacterial efflux pumps thus affecting S aureusresistance to tetracycline and norfloxacin

The preliminary SAR study was confirmed by PSAanalysis and reinforces the idea that coumarin derivativessubstituted with electron donors groups are more powerfulThese findings may help future design and synthesis of newderivatives with more potent modulatory activity

The docking study performed against the Penicillin-Binding Protein (PBP) from Methicillin Resistant Staphylo-coccus aureus (MRSA) identified that compound 18 is the bestligand with lower energy scores being one potential inhibitorof this important enzyme

Considering the natural and inevitable developmentof resistance mechanisms in microorganisms especially Saureus and the constant need for new agents with antibioticproperties our results contribute to the problematic of bacte-rial infections The discovery of new chemical entities asso-ciable with conventional antibiotics (antibiotic adjuvants)may allow reversion of cell resistance to conventional therapy(increased useful antibiotic life) and thus reduce antibioticconcentrations 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 thankboth the CNPq and CAPES for scholarships of Rodrigo

Santos Aquino de Araujo Ryldene Marques Duarte da CruzVivyanne dos Santos Falcao-Silva and Luciana Scotti

References

[1] G L French ldquoThe continuing crisis in antibiotic resistancerdquoInternational Journal of Antimicrobial Agents vol 36 no 3 ppS3ndashS7 2010

[2] L P Jordheim S Ben Larbi O Fendrich et al ldquoGemcitabineis active against clinical multiresistant Staphylococcus aureusstrains and is synergistic with gentamicinrdquo International Journalof Antimicrobial Agents vol 39 no 5 pp 444ndash447 2012

[3] G A Noskin R J Rubin J J Schentag et al ldquoThe burdenof Staphylococcus aureus infections on hospitals in the UnitedStates an analysis of the 2000 and 2001 Nationwide InpatientSample Databaserdquo Archives of Internal Medicine vol 156 no 15pp 1756ndash1761 2005

[4] H F Chambers and F R DeLeo ldquoWaves of resistanceStaphylococcus aureus in the antibiotic erardquo Nature ReviewsMicrobiology vol 7 no 9 pp 629ndash641 2009

[5] Y-H Fang P-R Hsueh J-J Hu et al ldquoCommunity-acquiredmethicillin-resistant Staphylococcus aureus in children innorthern Taiwanrdquo Journal of Microbiology Immunology andInfection vol 37 no 1 pp 29ndash34 2004

[6] K Hiramatsu Y Katayama H Yuzawa and T Ito ldquoMoleculargenetics of methicillin-resistant Staphylococcus aureusrdquo Inter-national Journal of Medical Microbiology vol 292 no 2 pp 67ndash74 2002

[7] B Marquez ldquoBacterial efflux systems and efflux pumpsinhibitorsrdquo Biochimie vol 87 no 12 pp 1137ndash1147 2005

[8] L J V Piddock ldquoClinically relevant chromosomally encodedmultidrug resistance efflux pumps in bacteriardquo Clinical Micro-biology Reviews vol 19 no 2 pp 382ndash402 2006

[9] S Gibbons ldquoAnti-staphylococcal plant natural productsrdquoNatu-ral Product Reports vol 21 no 2 pp 263ndash277 2004

[10] X-Z Li and H Nikaido ldquoEfflux-mediated drug resistance inbacteriardquo Drugs vol 64 no 2 pp 159ndash204 2004

[11] F Van Bambeke J-M Pages and V J Lee ldquoInhibitors ofbacterial efflux pumps as adjuvants in antibiotic treatments and

6 Scientifica

diagnostic tools for detection of resistance by effluxrdquo Frontiersin Anti-Infective Drug Discovery vol 1 pp 138ndash175 2006

[12] R S A deAraujo FQ SGuerra EDO Lima et al ldquoSynthesisStructure-activity Relationships (SAR) and in silico studies ofcoumarin derivatives with antifungal activityrdquo InternationalJournal of Molecular Sciences vol 14 no 1 pp 1293ndash1309 2013

[13] N Hamdi A S Al-Ayed R B Said and A Fabienne ldquoSyn-thesis and characterization of new thiazolidinones containingcoumarin moieties and their antibacterial and antioxidantactivitiesrdquoMolecules vol 17 no 8 pp 9321ndash9334 2012

[14] B S F Bazzaz M Iranshahi M Naderinasab S Hajian ZSabeti and E Masumi ldquoEvaluation of the effects of galbanicacid from Ferula szowitsiana and conferol from F badrakemaas modulators of multi-drug resistance in clinical isolates ofEscherichia coli and Staphylococcus aureusrdquo Research in Phar-maceutical Sciences vol 5 no 1 pp 25ndash32 2010

[15] CLSImdashClinical and Laboratory Standards InstituteldquoPerformance standards for antimicrobial susceptibility testingtwenty-fifth informational supplementrdquo CLSI DocumentM100-S25 Clinical and Laboratory Standards Institute WaynePa USA 2015

[16] G W Kaatz and S M Seo ldquoInducible NorA-mediatedmultidrug resistance in Staphylococcus aureusrdquo AntimicrobialAgents and Chemotherapy vol 39 no 12 pp 2650ndash2655 1995

[17] J I Ross A M Farrell E A Eady J H Cove andW J Cunliffe ldquoCharacterisation and molecular cloning ofthe novel macrolide-streptogramin B resistance determinantfrom Staphylococcus epidermidisrdquo Journal of AntimicrobialChemotherapy vol 24 no 6 pp 851ndash862 1989

[18] S Gibbons and E E Udo ldquoThe effect of reserpine a modulatorofmultidrug efflux pumps on the in vitro activity of tetracyclineagainst clinical isolates of methicillin resistant Staphylococcusaureus (MRSA) possessing the tet(K) determinantrdquo Phytother-apy Research vol 14 no 2 pp 139ndash140 2000

[19] M Stavri L J V Piddock and S Gibbons ldquoBacterial effluxpump inhibitors from natural sourcesrdquo Journal of AntimicrobialChemotherapy vol 59 no 6 pp 1247ndash1260 2007

[20] V S Falcao-Silva D A SilvaM D F V Souza and J P SiqueiraJr ldquoModulation of drug resistance in Staphylococcus aureus bya kaempferol glycoside from Herissantia tiubae (Malvaceae)rdquoPhytotherapy Research vol 23 no 10 pp 1367ndash1370 2009

[21] Hyperchem Program Release 80 for Windows 1999ndash2005Hybercube Inc Gainesville Fla USA

[22] N L Allinger ldquoConformational analysis 130 MM2 A hydro-carbon force field utilizing V1 and V2 torsional termsrdquo Journalof the American Chemical Society vol 99 no 25 pp 8127ndash81341977

[23] M J S Dewar G Zoebisch E F Healy and J J PStewart ldquoThe development and use of quantum-mechanicalmolecular-models 76 AM1mdasha new general-purpose quantum-mechanical molecular modelrdquo Journal of the American Chemi-cal Society vol 107 pp 3902ndash3909 1985

[24] N C CohenGuidebook onMolecular Modeling in Drug DesignAcademic Press San Diego Calif USA 1996

[25] A R Leach Molecular Modeling Principles and ApplicationsPrentice Hall London UK 2001

[26] A Pedretti L Villa and G Vistoli ldquoVEGA-an open platformto develop chemo-bio-informatics applications using plug-inarchitecture and script programmingrdquo Journal of Computer-Aided Molecular Design vol 18 no 3 pp 167ndash173 2004

[27] R BouleyM Kumarasiri Z Peng et al ldquoDiscovery of antibiotic(E)-3-(3-carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-onerdquo Journal of the American Chemical Society vol 137 no 5pp 1738ndash1741 2015

[28] R Thomsen and M H Christensen ldquoMolDock a new tech-nique for high-accuracy molecular dockingrdquo Journal of Medici-nal Chemistry vol 49 no 11 pp 3315ndash3321 2006

[29] Shakeel-U-Rehman R Khan K A Bhat A F Raja A S Shawland M S Alam ldquoIsolation characterisation and antibacterialactivity studies of coumarins from Rhododendron lepidotumWall ex G Don Ericaceaerdquo Brazilian Journal of Pharmacog-nosy vol 20 no 6 pp 886ndash890 2010

[30] C L Cespedes J G Avila A Martınez B Serrato J CCalderon-Mugica and R Salgado-Garciglia ldquoAntifungal andantibacterial activities of Mexican tarragon (Tagetes lucida)rdquoJournal of Agricultural and Food Chemistry vol 54 no 10 pp3521ndash3527 2006

[31] A Kumar I A Khan S Koul et al ldquoNovel structural analoguesof piperidine as inhibitors of the NorA efflux pump of Staphylo-coccus aureusrdquo Journal of Antimicrobial Chemotherapy vol 61pp 1270ndash1276 2008

[32] J M Brunel A Lieutaud V Lome J-M Pages and J-M BollaldquoPolyamino geranic derivatives as new chemosensitizers tocombat antibiotic resistant Gram-negative bacteriardquo Bioorganicamp Medicinal Chemistry vol 21 no 5 pp 1174ndash1179 2013

[33] E Reynolds J I Ross and J H Cove ldquoMsr(A) and relatedmacrolidestreptogramin resistance determinants incompletetransportersrdquo International Journal of Antimicrobial Agentsvol 22 no 3 pp 228ndash236 2003

[34] A Zervosen E Sauvage J-M Frere P Charlier and A LuxenldquoDevelopment of new drugs for an old targetmdashthe penicillinbinding proteinsrdquo Molecules vol 17 no 11 pp 12478ndash125052012

[35] M B Fernandes J E Goncalves M T Scotti A A deOliveira L C Tavares and S Storpirtis ldquoCaco-2 cells cytotox-icity of nifuroxazide derivatives with potential activity againstMethicillin-resistant Staphylococcus aureus (MRSA)rdquo Toxicol-ogy in Vitro vol 26 no 3 pp 535ndash540 2012

[36] A L Greninger S S Chatterjee L C Chan et al ldquoWhole-genome sequencing of methicillin-resistant Staphylococcusaureus resistant to fifth-generation cephalosporins revealspotential non-meca mechanisms of resistancerdquo Plos ONE vol11 no 2 Article ID e0149541 2016

[37] S B Hong M H Rhee B Yun Y H Lim H G Song andK S Shin ldquoSynergistic anti-bacterial effects of Phellinus baumiiethyl acetate extracts and beta-lactam antimicrobial agentsagainst methicillin-resistant Staphylococcus aureusrdquo Annals ofLaboratory Medicine vol 36 no 2 pp 111ndash116 2016

[38] P Lavanya S Ramaiah and A Anbarasu ldquoAmolecular dockingand dynamics study to screen potent anti-staphylococcal com-pounds against ceftaroline resistant MRSArdquo Journal of CellularBiochemistry vol 117 no 2 pp 542ndash548 2016

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Scientifica 3

O O

HO

HO

12

345

6

78

O O

RO

O O

AcO

O O

a b

c

15 = 4-OAc14 = 6-OAc12 = 7-OAc

19 = 6-allyl18 = 6-geranyl17 = 6-prenyl16 = 7-prenyl13 = 7-geranyl11 = 7-allyl

5 = 7-OH4 = 6-OH3 = 4-OH

O2

N

22 = 4-OH 3-NO2

21 = 6-OH 57-NO2

20 = 7-OH 6-NO2

Scheme 1 Synthesis of alkyl- acetyl- and nitro-coumarin derivatives Reagents and conditions (a) acetic anhydride pyridine rt ultrasoundirradiation (b) allyl bromide geranyl bromide or prenyl bromide K

2CO3 acetonitrile and reflux (c) HNO

3AcOH 0ndash5∘C for 30min and

then 90min at rt

a GRID of 15gt in radius and 030 in resolution to coverthe ligand-biding site of the Penicillin-Binding Protein (PBP)structure

Templates with features expected to be relevant for ligandbinding (quinazoline) were generated to perform dockingThe MolDock Score [GRID] algorithm was used as the scorefunction and the MolDock search algorithm was used [28]

3 Results and Discussion

Twelve coumarin derivatives (11ndash22) were synthesized fromcommercial coumarins 4-hydroxy- (3) 6-hydroxy- (4)and 7-hydroxycoumarin (5) by alkylation acetylation andnitration procedures according to procedures previouslydescribed by our group [12] (Scheme 1)

All commercial (1ndash10) and synthetic (11ndash22) coumarinswere evaluated for their in vitro antibacterial activity againstthe four Staphylococcus aureus strains the standard (ATCC6538) and the three resistant IS-58 (TetK) RN4220 (MsrA)and SA-1199B (NorA) however all of the commercialcoumarins (1ndash10) and the eight synthetic coumarins (1112 15 and 18ndash22) showed no antibacterial activity (MICgt 256120583gmL) against any S aureus strain evaluated Com-pounds 13 14 16 and 17 showed weak activity with MICvalues of 128 120583gmL (13 against ATCC 6538 and SA-1199B 16against SA-1199B and 14 and 17 against all strains)

Shakeel-U-Rehman and colleagues [29] demonstratedantibacterial activity of coumarin compounds (with a freehydroxyl at position 8) against resistant and susceptiblestrains of S aureus and also reported reduction in activitywith acetylation of coumarin precursors This came intodisagreement with our acetylated compound (14) Disagree-ment with our results was also seen in comparison withthe studies of Cespedes et al [30] who reported goodantibacterial activities for compounds with dihydroxyls at

positions C-7 and C-8 and methoxylation at C-6 againstsusceptible strains of S aureus unlike our compounds 6 and10 which revealed no antibacterial activity against strains ofthis microorganism

In the assay for bacterial resistance (modulation assay)the MICs of the antibiotics (tetracycline against IS-58 strainerythromycin against RN4220 strain and norfloxacin againstSA-1199B strain) were determined in the absence and inthe presence of the coumarin derivatives (compounds 1ndash22) which were incorporated into the growth medium ata concentration corresponding to 14 of the MIC (subin-hibitory concentration) [19] The results of the compoundsthat showed modulatory activity are presented in Table 1

As can be seen in Table 1 some coumarins enhanced theactivity of tetracycline (2x) and of norfloxacin (2x to 8x) thusreducing the concentration required to inhibit the growth ofdrug resistant (effluxing) strains

Among the commercial coumarins evaluated only 12-Benzopyrone (1) showed modulatory activity reducing theMIC of norfloxacin in twice However the synthesizedcompounds having allyl (11 19) geranyl (13 18) and prenyl(16 and 17) radicals showed good to significant modulatoryactivities in particular for resistance to norfloxacin (Table 1)

The common characteristic of these modulatory com-pounds is the presence of an alkyl radical which has beendescribed as important for the drug resistance modulatingactivity of S aureus inhibiting the efflux system [31] Brunel etal [32] have also associated the presence of the geranyl groupas an important characteristic for modulation of multipledrug resistance in Gram-negative bacteria

Changing the position of theseC-7 radicals (11 13 and 16)to the C-6 position (17 18 and 19) favored the modulationof the tetracycline resistance by two times but reduced(two to four times) the modifying activity of norfloxacinWith respect to the modulatory activity of coumarin for

4 Scientifica

Table 1 MIC values (120583gmL) of antibiotics in the absence and presence of coumarin derivatives against S aureus strains SA-1199B (NorA)RN4220 (MsrA) and IS-58 (TetK)

Antibiotic + coumarin MIC (120583gmL)IS-58 (TetK)Tetracycline

RN4220 (MrsA)Erythromycin

SA-1199B (NorA)Norfloxacin

Antibiotics alone 64 gt256 128Antibiotics + 1 64 gt256 64 (2x)Antibiotics + 11 64 gt256 32 (4x)Antibiotics + 13 64 gt256 32 (4x)Antibiotics + 16 64 gt256 16 (8x)Antibiotics + 17 32 (2x) gt256 64 (2x)Antibiotics + 18 32 (2x) gt256 64 (2x)Antibiotics + 19 64 gt256 64 (2x)

Figure 1 Representation of the PSA (Polar Surface Area) of active(13 and 16) and inactive (2 and 22) compounds using VEJA ZZprogram White represents polar surface and grey-dark representsno-polar surfaces

the RN4220 (MRSA) strain no reduction was observed forthe erythromycin MIC However Reynolds et al [33] havesuggested that the MRSA protein cannot be an efflux pumpprotein

Acetylated and nitrated coumarins were all inactivewhich leads us to believe that the presence of electronwithdrawing groups (decreasing the electron density of thebenzopyran ring) or polar radicals negatively contribute toboth antibiotic and modulatory activities for this class ofcompounds

These conclusions based on a preliminary SAR studywere also proven by analyzing the calculations of the PolarSurfaces Area (PSA) of the compounds obtained by theVEJA ZZ program [26] It was observed that the absence offree hydroxyls carboxylic acids and other polar groups (ienitro derivatives) seems to be an important factor for themodulatory activity of coumarins

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

Table 2 Binding energy (119864binding) values

Compounds MolDock Score (kcalmol)Quinazolinone minus7109811 minus4545313 minus5835816 minus5802117 minus6222418 minus9132219 minus55676

Additionally docking study of the most active coumarins(11 13 and 16ndash19) against the Penicillin-Binding Protein(PBP) from Methicillin Resistant Staphylococcus aureus(MRSA) (PDB id 4CJN) was performed Table 2 shows theresults of theMolDock Score of these potential inhibitors andFigure 2 shows the interactions observed in the complex ofthe best ligand (compound 18) with the active site of the PBPenzyme

One of bacterial resistance pathways occurs by low-interaction of the antibiotic by PPBs and hydrolysis by 120573-lactamases These bacterial proteins are fundamental in thesynthesis of the cell wall of bacteria common target of 120573-lactam antibiotics This fact makes PPBs a target searchedagainst a resistance particularly in Gram-positive bacterialike Staphylococcus aureus and also in a higher selectivity tothese unique bacteria structures [34ndash38]

Molecular docking can be formulated as an optimizationproblem where the task is to find the ligand-binding modewith the lowest energy Table 2 shows the energies (kcalmol)obtained from the interaction of the coumarins 11 13 and 16ndash19 and quinazolinone (compound complex with the enzymein the crystal structure) and the PPB We observed thatcompounds 17 (119864binding = minus6224 kcalmol) and 18 (119864binding =ndash91322 kcalmol) generated lower energy scores which repre-sent better protein-ligand bindings and stable complexes

Compound 18 has shown greater energy value than thequinazolinone (119864binding =minus71098 kcalmol) ligand complexedin the PDB Figure 2 reports in 2D (a) and in 3D (b)the interactions formed between 18 and PPB we can see

Scientifica 5

(a) (b)

Figure 2 Interactions observed in the compound 18-PBP complex

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 deriva-tives especially alkylated derivatives have shown promis-ing antibiotic activity They act by modulating antibioticresistance through a mechanism that most likely involvesinhibition of bacterial efflux pumps thus affecting S aureusresistance to tetracycline and norfloxacin

The preliminary SAR study was confirmed by PSAanalysis and reinforces the idea that coumarin derivativessubstituted with electron donors groups are more powerfulThese findings may help future design and synthesis of newderivatives with more potent modulatory activity

The docking study performed against the Penicillin-Binding Protein (PBP) from Methicillin Resistant Staphylo-coccus aureus (MRSA) identified that compound 18 is the bestligand with lower energy scores being one potential inhibitorof this important enzyme

Considering the natural and inevitable developmentof resistance mechanisms in microorganisms especially Saureus and the constant need for new agents with antibioticproperties our results contribute to the problematic of bacte-rial infections The discovery of new chemical entities asso-ciable with conventional antibiotics (antibiotic adjuvants)may allow reversion of cell resistance to conventional therapy(increased useful antibiotic life) and thus reduce antibioticconcentrations 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 thankboth the CNPq and CAPES for scholarships of Rodrigo

Santos Aquino de Araujo Ryldene Marques Duarte da CruzVivyanne dos Santos Falcao-Silva and Luciana Scotti

References

[1] G L French ldquoThe continuing crisis in antibiotic resistancerdquoInternational Journal of Antimicrobial Agents vol 36 no 3 ppS3ndashS7 2010

[2] L P Jordheim S Ben Larbi O Fendrich et al ldquoGemcitabineis active against clinical multiresistant Staphylococcus aureusstrains and is synergistic with gentamicinrdquo International Journalof Antimicrobial Agents vol 39 no 5 pp 444ndash447 2012

[3] G A Noskin R J Rubin J J Schentag et al ldquoThe burdenof Staphylococcus aureus infections on hospitals in the UnitedStates an analysis of the 2000 and 2001 Nationwide InpatientSample Databaserdquo Archives of Internal Medicine vol 156 no 15pp 1756ndash1761 2005

[4] H F Chambers and F R DeLeo ldquoWaves of resistanceStaphylococcus aureus in the antibiotic erardquo Nature ReviewsMicrobiology vol 7 no 9 pp 629ndash641 2009

[5] Y-H Fang P-R Hsueh J-J Hu et al ldquoCommunity-acquiredmethicillin-resistant Staphylococcus aureus in children innorthern Taiwanrdquo Journal of Microbiology Immunology andInfection vol 37 no 1 pp 29ndash34 2004

[6] K Hiramatsu Y Katayama H Yuzawa and T Ito ldquoMoleculargenetics of methicillin-resistant Staphylococcus aureusrdquo Inter-national Journal of Medical Microbiology vol 292 no 2 pp 67ndash74 2002

[7] B Marquez ldquoBacterial efflux systems and efflux pumpsinhibitorsrdquo Biochimie vol 87 no 12 pp 1137ndash1147 2005

[8] L J V Piddock ldquoClinically relevant chromosomally encodedmultidrug resistance efflux pumps in bacteriardquo Clinical Micro-biology Reviews vol 19 no 2 pp 382ndash402 2006

[9] S Gibbons ldquoAnti-staphylococcal plant natural productsrdquoNatu-ral Product Reports vol 21 no 2 pp 263ndash277 2004

[10] X-Z Li and H Nikaido ldquoEfflux-mediated drug resistance inbacteriardquo Drugs vol 64 no 2 pp 159ndash204 2004

[11] F Van Bambeke J-M Pages and V J Lee ldquoInhibitors ofbacterial efflux pumps as adjuvants in antibiotic treatments and

6 Scientifica

diagnostic tools for detection of resistance by effluxrdquo Frontiersin Anti-Infective Drug Discovery vol 1 pp 138ndash175 2006

[12] R S A deAraujo FQ SGuerra EDO Lima et al ldquoSynthesisStructure-activity Relationships (SAR) and in silico studies ofcoumarin derivatives with antifungal activityrdquo InternationalJournal of Molecular Sciences vol 14 no 1 pp 1293ndash1309 2013

[13] N Hamdi A S Al-Ayed R B Said and A Fabienne ldquoSyn-thesis and characterization of new thiazolidinones containingcoumarin moieties and their antibacterial and antioxidantactivitiesrdquoMolecules vol 17 no 8 pp 9321ndash9334 2012

[14] B S F Bazzaz M Iranshahi M Naderinasab S Hajian ZSabeti and E Masumi ldquoEvaluation of the effects of galbanicacid from Ferula szowitsiana and conferol from F badrakemaas modulators of multi-drug resistance in clinical isolates ofEscherichia coli and Staphylococcus aureusrdquo Research in Phar-maceutical Sciences vol 5 no 1 pp 25ndash32 2010

[15] CLSImdashClinical and Laboratory Standards InstituteldquoPerformance standards for antimicrobial susceptibility testingtwenty-fifth informational supplementrdquo CLSI DocumentM100-S25 Clinical and Laboratory Standards Institute WaynePa USA 2015

[16] G W Kaatz and S M Seo ldquoInducible NorA-mediatedmultidrug resistance in Staphylococcus aureusrdquo AntimicrobialAgents and Chemotherapy vol 39 no 12 pp 2650ndash2655 1995

[17] J I Ross A M Farrell E A Eady J H Cove andW J Cunliffe ldquoCharacterisation and molecular cloning ofthe novel macrolide-streptogramin B resistance determinantfrom Staphylococcus epidermidisrdquo Journal of AntimicrobialChemotherapy vol 24 no 6 pp 851ndash862 1989

[18] S Gibbons and E E Udo ldquoThe effect of reserpine a modulatorofmultidrug efflux pumps on the in vitro activity of tetracyclineagainst clinical isolates of methicillin resistant Staphylococcusaureus (MRSA) possessing the tet(K) determinantrdquo Phytother-apy Research vol 14 no 2 pp 139ndash140 2000

[19] M Stavri L J V Piddock and S Gibbons ldquoBacterial effluxpump inhibitors from natural sourcesrdquo Journal of AntimicrobialChemotherapy vol 59 no 6 pp 1247ndash1260 2007

[20] V S Falcao-Silva D A SilvaM D F V Souza and J P SiqueiraJr ldquoModulation of drug resistance in Staphylococcus aureus bya kaempferol glycoside from Herissantia tiubae (Malvaceae)rdquoPhytotherapy Research vol 23 no 10 pp 1367ndash1370 2009

[21] Hyperchem Program Release 80 for Windows 1999ndash2005Hybercube Inc Gainesville Fla USA

[22] N L Allinger ldquoConformational analysis 130 MM2 A hydro-carbon force field utilizing V1 and V2 torsional termsrdquo Journalof the American Chemical Society vol 99 no 25 pp 8127ndash81341977

[23] M J S Dewar G Zoebisch E F Healy and J J PStewart ldquoThe development and use of quantum-mechanicalmolecular-models 76 AM1mdasha new general-purpose quantum-mechanical molecular modelrdquo Journal of the American Chemi-cal Society vol 107 pp 3902ndash3909 1985

[24] N C CohenGuidebook onMolecular Modeling in Drug DesignAcademic Press San Diego Calif USA 1996

[25] A R Leach Molecular Modeling Principles and ApplicationsPrentice Hall London UK 2001

[26] A Pedretti L Villa and G Vistoli ldquoVEGA-an open platformto develop chemo-bio-informatics applications using plug-inarchitecture and script programmingrdquo Journal of Computer-Aided Molecular Design vol 18 no 3 pp 167ndash173 2004

[27] R BouleyM Kumarasiri Z Peng et al ldquoDiscovery of antibiotic(E)-3-(3-carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-onerdquo Journal of the American Chemical Society vol 137 no 5pp 1738ndash1741 2015

[28] R Thomsen and M H Christensen ldquoMolDock a new tech-nique for high-accuracy molecular dockingrdquo Journal of Medici-nal Chemistry vol 49 no 11 pp 3315ndash3321 2006

[29] Shakeel-U-Rehman R Khan K A Bhat A F Raja A S Shawland M S Alam ldquoIsolation characterisation and antibacterialactivity studies of coumarins from Rhododendron lepidotumWall ex G Don Ericaceaerdquo Brazilian Journal of Pharmacog-nosy vol 20 no 6 pp 886ndash890 2010

[30] C L Cespedes J G Avila A Martınez B Serrato J CCalderon-Mugica and R Salgado-Garciglia ldquoAntifungal andantibacterial activities of Mexican tarragon (Tagetes lucida)rdquoJournal of Agricultural and Food Chemistry vol 54 no 10 pp3521ndash3527 2006

[31] A Kumar I A Khan S Koul et al ldquoNovel structural analoguesof piperidine as inhibitors of the NorA efflux pump of Staphylo-coccus aureusrdquo Journal of Antimicrobial Chemotherapy vol 61pp 1270ndash1276 2008

[32] J M Brunel A Lieutaud V Lome J-M Pages and J-M BollaldquoPolyamino geranic derivatives as new chemosensitizers tocombat antibiotic resistant Gram-negative bacteriardquo Bioorganicamp Medicinal Chemistry vol 21 no 5 pp 1174ndash1179 2013

[33] E Reynolds J I Ross and J H Cove ldquoMsr(A) and relatedmacrolidestreptogramin resistance determinants incompletetransportersrdquo International Journal of Antimicrobial Agentsvol 22 no 3 pp 228ndash236 2003

[34] A Zervosen E Sauvage J-M Frere P Charlier and A LuxenldquoDevelopment of new drugs for an old targetmdashthe penicillinbinding proteinsrdquo Molecules vol 17 no 11 pp 12478ndash125052012

[35] M B Fernandes J E Goncalves M T Scotti A A deOliveira L C Tavares and S Storpirtis ldquoCaco-2 cells cytotox-icity of nifuroxazide derivatives with potential activity againstMethicillin-resistant Staphylococcus aureus (MRSA)rdquo Toxicol-ogy in Vitro vol 26 no 3 pp 535ndash540 2012

[36] A L Greninger S S Chatterjee L C Chan et al ldquoWhole-genome sequencing of methicillin-resistant Staphylococcusaureus resistant to fifth-generation cephalosporins revealspotential non-meca mechanisms of resistancerdquo Plos ONE vol11 no 2 Article ID e0149541 2016

[37] S B Hong M H Rhee B Yun Y H Lim H G Song andK S Shin ldquoSynergistic anti-bacterial effects of Phellinus baumiiethyl acetate extracts and beta-lactam antimicrobial agentsagainst methicillin-resistant Staphylococcus aureusrdquo Annals ofLaboratory Medicine vol 36 no 2 pp 111ndash116 2016

[38] P Lavanya S Ramaiah and A Anbarasu ldquoAmolecular dockingand dynamics study to screen potent anti-staphylococcal com-pounds against ceftaroline resistant MRSArdquo Journal of CellularBiochemistry vol 117 no 2 pp 542ndash548 2016

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 Scientifica

Table 1 MIC values (120583gmL) of antibiotics in the absence and presence of coumarin derivatives against S aureus strains SA-1199B (NorA)RN4220 (MsrA) and IS-58 (TetK)

Antibiotic + coumarin MIC (120583gmL)IS-58 (TetK)Tetracycline

RN4220 (MrsA)Erythromycin

SA-1199B (NorA)Norfloxacin

Antibiotics alone 64 gt256 128Antibiotics + 1 64 gt256 64 (2x)Antibiotics + 11 64 gt256 32 (4x)Antibiotics + 13 64 gt256 32 (4x)Antibiotics + 16 64 gt256 16 (8x)Antibiotics + 17 32 (2x) gt256 64 (2x)Antibiotics + 18 32 (2x) gt256 64 (2x)Antibiotics + 19 64 gt256 64 (2x)

Figure 1 Representation of the PSA (Polar Surface Area) of active(13 and 16) and inactive (2 and 22) compounds using VEJA ZZprogram White represents polar surface and grey-dark representsno-polar surfaces

the RN4220 (MRSA) strain no reduction was observed forthe erythromycin MIC However Reynolds et al [33] havesuggested that the MRSA protein cannot be an efflux pumpprotein

Acetylated and nitrated coumarins were all inactivewhich leads us to believe that the presence of electronwithdrawing groups (decreasing the electron density of thebenzopyran ring) or polar radicals negatively contribute toboth antibiotic and modulatory activities for this class ofcompounds

These conclusions based on a preliminary SAR studywere also proven by analyzing the calculations of the PolarSurfaces Area (PSA) of the compounds obtained by theVEJA ZZ program [26] It was observed that the absence offree hydroxyls carboxylic acids and other polar groups (ienitro derivatives) seems to be an important factor for themodulatory activity of coumarins

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

Table 2 Binding energy (119864binding) values

Compounds MolDock Score (kcalmol)Quinazolinone minus7109811 minus4545313 minus5835816 minus5802117 minus6222418 minus9132219 minus55676

Additionally docking study of the most active coumarins(11 13 and 16ndash19) against the Penicillin-Binding Protein(PBP) from Methicillin Resistant Staphylococcus aureus(MRSA) (PDB id 4CJN) was performed Table 2 shows theresults of theMolDock Score of these potential inhibitors andFigure 2 shows the interactions observed in the complex ofthe best ligand (compound 18) with the active site of the PBPenzyme

One of bacterial resistance pathways occurs by low-interaction of the antibiotic by PPBs and hydrolysis by 120573-lactamases These bacterial proteins are fundamental in thesynthesis of the cell wall of bacteria common target of 120573-lactam antibiotics This fact makes PPBs a target searchedagainst a resistance particularly in Gram-positive bacterialike Staphylococcus aureus and also in a higher selectivity tothese unique bacteria structures [34ndash38]

Molecular docking can be formulated as an optimizationproblem where the task is to find the ligand-binding modewith the lowest energy Table 2 shows the energies (kcalmol)obtained from the interaction of the coumarins 11 13 and 16ndash19 and quinazolinone (compound complex with the enzymein the crystal structure) and the PPB We observed thatcompounds 17 (119864binding = minus6224 kcalmol) and 18 (119864binding =ndash91322 kcalmol) generated lower energy scores which repre-sent better protein-ligand bindings and stable complexes

Compound 18 has shown greater energy value than thequinazolinone (119864binding =minus71098 kcalmol) ligand complexedin the PDB Figure 2 reports in 2D (a) and in 3D (b)the interactions formed between 18 and PPB we can see

Scientifica 5

(a) (b)

Figure 2 Interactions observed in the compound 18-PBP complex

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 deriva-tives especially alkylated derivatives have shown promis-ing antibiotic activity They act by modulating antibioticresistance through a mechanism that most likely involvesinhibition of bacterial efflux pumps thus affecting S aureusresistance to tetracycline and norfloxacin

The preliminary SAR study was confirmed by PSAanalysis and reinforces the idea that coumarin derivativessubstituted with electron donors groups are more powerfulThese findings may help future design and synthesis of newderivatives with more potent modulatory activity

The docking study performed against the Penicillin-Binding Protein (PBP) from Methicillin Resistant Staphylo-coccus aureus (MRSA) identified that compound 18 is the bestligand with lower energy scores being one potential inhibitorof this important enzyme

Considering the natural and inevitable developmentof resistance mechanisms in microorganisms especially Saureus and the constant need for new agents with antibioticproperties our results contribute to the problematic of bacte-rial infections The discovery of new chemical entities asso-ciable with conventional antibiotics (antibiotic adjuvants)may allow reversion of cell resistance to conventional therapy(increased useful antibiotic life) and thus reduce antibioticconcentrations 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 thankboth the CNPq and CAPES for scholarships of Rodrigo

Santos Aquino de Araujo Ryldene Marques Duarte da CruzVivyanne dos Santos Falcao-Silva and Luciana Scotti

References

[1] G L French ldquoThe continuing crisis in antibiotic resistancerdquoInternational Journal of Antimicrobial Agents vol 36 no 3 ppS3ndashS7 2010

[2] L P Jordheim S Ben Larbi O Fendrich et al ldquoGemcitabineis active against clinical multiresistant Staphylococcus aureusstrains and is synergistic with gentamicinrdquo International Journalof Antimicrobial Agents vol 39 no 5 pp 444ndash447 2012

[3] G A Noskin R J Rubin J J Schentag et al ldquoThe burdenof Staphylococcus aureus infections on hospitals in the UnitedStates an analysis of the 2000 and 2001 Nationwide InpatientSample Databaserdquo Archives of Internal Medicine vol 156 no 15pp 1756ndash1761 2005

[4] H F Chambers and F R DeLeo ldquoWaves of resistanceStaphylococcus aureus in the antibiotic erardquo Nature ReviewsMicrobiology vol 7 no 9 pp 629ndash641 2009

[5] Y-H Fang P-R Hsueh J-J Hu et al ldquoCommunity-acquiredmethicillin-resistant Staphylococcus aureus in children innorthern Taiwanrdquo Journal of Microbiology Immunology andInfection vol 37 no 1 pp 29ndash34 2004

[6] K Hiramatsu Y Katayama H Yuzawa and T Ito ldquoMoleculargenetics of methicillin-resistant Staphylococcus aureusrdquo Inter-national Journal of Medical Microbiology vol 292 no 2 pp 67ndash74 2002

[7] B Marquez ldquoBacterial efflux systems and efflux pumpsinhibitorsrdquo Biochimie vol 87 no 12 pp 1137ndash1147 2005

[8] L J V Piddock ldquoClinically relevant chromosomally encodedmultidrug resistance efflux pumps in bacteriardquo Clinical Micro-biology Reviews vol 19 no 2 pp 382ndash402 2006

[9] S Gibbons ldquoAnti-staphylococcal plant natural productsrdquoNatu-ral Product Reports vol 21 no 2 pp 263ndash277 2004

[10] X-Z Li and H Nikaido ldquoEfflux-mediated drug resistance inbacteriardquo Drugs vol 64 no 2 pp 159ndash204 2004

[11] F Van Bambeke J-M Pages and V J Lee ldquoInhibitors ofbacterial efflux pumps as adjuvants in antibiotic treatments and

6 Scientifica

diagnostic tools for detection of resistance by effluxrdquo Frontiersin Anti-Infective Drug Discovery vol 1 pp 138ndash175 2006

[12] R S A deAraujo FQ SGuerra EDO Lima et al ldquoSynthesisStructure-activity Relationships (SAR) and in silico studies ofcoumarin derivatives with antifungal activityrdquo InternationalJournal of Molecular Sciences vol 14 no 1 pp 1293ndash1309 2013

[13] N Hamdi A S Al-Ayed R B Said and A Fabienne ldquoSyn-thesis and characterization of new thiazolidinones containingcoumarin moieties and their antibacterial and antioxidantactivitiesrdquoMolecules vol 17 no 8 pp 9321ndash9334 2012

[14] B S F Bazzaz M Iranshahi M Naderinasab S Hajian ZSabeti and E Masumi ldquoEvaluation of the effects of galbanicacid from Ferula szowitsiana and conferol from F badrakemaas modulators of multi-drug resistance in clinical isolates ofEscherichia coli and Staphylococcus aureusrdquo Research in Phar-maceutical Sciences vol 5 no 1 pp 25ndash32 2010

[15] CLSImdashClinical and Laboratory Standards InstituteldquoPerformance standards for antimicrobial susceptibility testingtwenty-fifth informational supplementrdquo CLSI DocumentM100-S25 Clinical and Laboratory Standards Institute WaynePa USA 2015

[16] G W Kaatz and S M Seo ldquoInducible NorA-mediatedmultidrug resistance in Staphylococcus aureusrdquo AntimicrobialAgents and Chemotherapy vol 39 no 12 pp 2650ndash2655 1995

[17] J I Ross A M Farrell E A Eady J H Cove andW J Cunliffe ldquoCharacterisation and molecular cloning ofthe novel macrolide-streptogramin B resistance determinantfrom Staphylococcus epidermidisrdquo Journal of AntimicrobialChemotherapy vol 24 no 6 pp 851ndash862 1989

[18] S Gibbons and E E Udo ldquoThe effect of reserpine a modulatorofmultidrug efflux pumps on the in vitro activity of tetracyclineagainst clinical isolates of methicillin resistant Staphylococcusaureus (MRSA) possessing the tet(K) determinantrdquo Phytother-apy Research vol 14 no 2 pp 139ndash140 2000

[19] M Stavri L J V Piddock and S Gibbons ldquoBacterial effluxpump inhibitors from natural sourcesrdquo Journal of AntimicrobialChemotherapy vol 59 no 6 pp 1247ndash1260 2007

[20] V S Falcao-Silva D A SilvaM D F V Souza and J P SiqueiraJr ldquoModulation of drug resistance in Staphylococcus aureus bya kaempferol glycoside from Herissantia tiubae (Malvaceae)rdquoPhytotherapy Research vol 23 no 10 pp 1367ndash1370 2009

[21] Hyperchem Program Release 80 for Windows 1999ndash2005Hybercube Inc Gainesville Fla USA

[22] N L Allinger ldquoConformational analysis 130 MM2 A hydro-carbon force field utilizing V1 and V2 torsional termsrdquo Journalof the American Chemical Society vol 99 no 25 pp 8127ndash81341977

[23] M J S Dewar G Zoebisch E F Healy and J J PStewart ldquoThe development and use of quantum-mechanicalmolecular-models 76 AM1mdasha new general-purpose quantum-mechanical molecular modelrdquo Journal of the American Chemi-cal Society vol 107 pp 3902ndash3909 1985

[24] N C CohenGuidebook onMolecular Modeling in Drug DesignAcademic Press San Diego Calif USA 1996

[25] A R Leach Molecular Modeling Principles and ApplicationsPrentice Hall London UK 2001

[26] A Pedretti L Villa and G Vistoli ldquoVEGA-an open platformto develop chemo-bio-informatics applications using plug-inarchitecture and script programmingrdquo Journal of Computer-Aided Molecular Design vol 18 no 3 pp 167ndash173 2004

[27] R BouleyM Kumarasiri Z Peng et al ldquoDiscovery of antibiotic(E)-3-(3-carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-onerdquo Journal of the American Chemical Society vol 137 no 5pp 1738ndash1741 2015

[28] R Thomsen and M H Christensen ldquoMolDock a new tech-nique for high-accuracy molecular dockingrdquo Journal of Medici-nal Chemistry vol 49 no 11 pp 3315ndash3321 2006

[29] Shakeel-U-Rehman R Khan K A Bhat A F Raja A S Shawland M S Alam ldquoIsolation characterisation and antibacterialactivity studies of coumarins from Rhododendron lepidotumWall ex G Don Ericaceaerdquo Brazilian Journal of Pharmacog-nosy vol 20 no 6 pp 886ndash890 2010

[30] C L Cespedes J G Avila A Martınez B Serrato J CCalderon-Mugica and R Salgado-Garciglia ldquoAntifungal andantibacterial activities of Mexican tarragon (Tagetes lucida)rdquoJournal of Agricultural and Food Chemistry vol 54 no 10 pp3521ndash3527 2006

[31] A Kumar I A Khan S Koul et al ldquoNovel structural analoguesof piperidine as inhibitors of the NorA efflux pump of Staphylo-coccus aureusrdquo Journal of Antimicrobial Chemotherapy vol 61pp 1270ndash1276 2008

[32] J M Brunel A Lieutaud V Lome J-M Pages and J-M BollaldquoPolyamino geranic derivatives as new chemosensitizers tocombat antibiotic resistant Gram-negative bacteriardquo Bioorganicamp Medicinal Chemistry vol 21 no 5 pp 1174ndash1179 2013

[33] E Reynolds J I Ross and J H Cove ldquoMsr(A) and relatedmacrolidestreptogramin resistance determinants incompletetransportersrdquo International Journal of Antimicrobial Agentsvol 22 no 3 pp 228ndash236 2003

[34] A Zervosen E Sauvage J-M Frere P Charlier and A LuxenldquoDevelopment of new drugs for an old targetmdashthe penicillinbinding proteinsrdquo Molecules vol 17 no 11 pp 12478ndash125052012

[35] M B Fernandes J E Goncalves M T Scotti A A deOliveira L C Tavares and S Storpirtis ldquoCaco-2 cells cytotox-icity of nifuroxazide derivatives with potential activity againstMethicillin-resistant Staphylococcus aureus (MRSA)rdquo Toxicol-ogy in Vitro vol 26 no 3 pp 535ndash540 2012

[36] A L Greninger S S Chatterjee L C Chan et al ldquoWhole-genome sequencing of methicillin-resistant Staphylococcusaureus resistant to fifth-generation cephalosporins revealspotential non-meca mechanisms of resistancerdquo Plos ONE vol11 no 2 Article ID e0149541 2016

[37] S B Hong M H Rhee B Yun Y H Lim H G Song andK S Shin ldquoSynergistic anti-bacterial effects of Phellinus baumiiethyl acetate extracts and beta-lactam antimicrobial agentsagainst methicillin-resistant Staphylococcus aureusrdquo Annals ofLaboratory Medicine vol 36 no 2 pp 111ndash116 2016

[38] P Lavanya S Ramaiah and A Anbarasu ldquoAmolecular dockingand dynamics study to screen potent anti-staphylococcal com-pounds against ceftaroline resistant MRSArdquo Journal of CellularBiochemistry vol 117 no 2 pp 542ndash548 2016

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Scientifica 5

(a) (b)

Figure 2 Interactions observed in the compound 18-PBP complex

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 deriva-tives especially alkylated derivatives have shown promis-ing antibiotic activity They act by modulating antibioticresistance through a mechanism that most likely involvesinhibition of bacterial efflux pumps thus affecting S aureusresistance to tetracycline and norfloxacin

The preliminary SAR study was confirmed by PSAanalysis and reinforces the idea that coumarin derivativessubstituted with electron donors groups are more powerfulThese findings may help future design and synthesis of newderivatives with more potent modulatory activity

The docking study performed against the Penicillin-Binding Protein (PBP) from Methicillin Resistant Staphylo-coccus aureus (MRSA) identified that compound 18 is the bestligand with lower energy scores being one potential inhibitorof this important enzyme

Considering the natural and inevitable developmentof resistance mechanisms in microorganisms especially Saureus and the constant need for new agents with antibioticproperties our results contribute to the problematic of bacte-rial infections The discovery of new chemical entities asso-ciable with conventional antibiotics (antibiotic adjuvants)may allow reversion of cell resistance to conventional therapy(increased useful antibiotic life) and thus reduce antibioticconcentrations 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 thankboth the CNPq and CAPES for scholarships of Rodrigo

Santos Aquino de Araujo Ryldene Marques Duarte da CruzVivyanne dos Santos Falcao-Silva and Luciana Scotti

References

[1] G L French ldquoThe continuing crisis in antibiotic resistancerdquoInternational Journal of Antimicrobial Agents vol 36 no 3 ppS3ndashS7 2010

[2] L P Jordheim S Ben Larbi O Fendrich et al ldquoGemcitabineis active against clinical multiresistant Staphylococcus aureusstrains and is synergistic with gentamicinrdquo International Journalof Antimicrobial Agents vol 39 no 5 pp 444ndash447 2012

[3] G A Noskin R J Rubin J J Schentag et al ldquoThe burdenof Staphylococcus aureus infections on hospitals in the UnitedStates an analysis of the 2000 and 2001 Nationwide InpatientSample Databaserdquo Archives of Internal Medicine vol 156 no 15pp 1756ndash1761 2005

[4] H F Chambers and F R DeLeo ldquoWaves of resistanceStaphylococcus aureus in the antibiotic erardquo Nature ReviewsMicrobiology vol 7 no 9 pp 629ndash641 2009

[5] Y-H Fang P-R Hsueh J-J Hu et al ldquoCommunity-acquiredmethicillin-resistant Staphylococcus aureus in children innorthern Taiwanrdquo Journal of Microbiology Immunology andInfection vol 37 no 1 pp 29ndash34 2004

[6] K Hiramatsu Y Katayama H Yuzawa and T Ito ldquoMoleculargenetics of methicillin-resistant Staphylococcus aureusrdquo Inter-national Journal of Medical Microbiology vol 292 no 2 pp 67ndash74 2002

[7] B Marquez ldquoBacterial efflux systems and efflux pumpsinhibitorsrdquo Biochimie vol 87 no 12 pp 1137ndash1147 2005

[8] L J V Piddock ldquoClinically relevant chromosomally encodedmultidrug resistance efflux pumps in bacteriardquo Clinical Micro-biology Reviews vol 19 no 2 pp 382ndash402 2006

[9] S Gibbons ldquoAnti-staphylococcal plant natural productsrdquoNatu-ral Product Reports vol 21 no 2 pp 263ndash277 2004

[10] X-Z Li and H Nikaido ldquoEfflux-mediated drug resistance inbacteriardquo Drugs vol 64 no 2 pp 159ndash204 2004

[11] F Van Bambeke J-M Pages and V J Lee ldquoInhibitors ofbacterial efflux pumps as adjuvants in antibiotic treatments and

6 Scientifica

diagnostic tools for detection of resistance by effluxrdquo Frontiersin Anti-Infective Drug Discovery vol 1 pp 138ndash175 2006

[12] R S A deAraujo FQ SGuerra EDO Lima et al ldquoSynthesisStructure-activity Relationships (SAR) and in silico studies ofcoumarin derivatives with antifungal activityrdquo InternationalJournal of Molecular Sciences vol 14 no 1 pp 1293ndash1309 2013

[13] N Hamdi A S Al-Ayed R B Said and A Fabienne ldquoSyn-thesis and characterization of new thiazolidinones containingcoumarin moieties and their antibacterial and antioxidantactivitiesrdquoMolecules vol 17 no 8 pp 9321ndash9334 2012

[14] B S F Bazzaz M Iranshahi M Naderinasab S Hajian ZSabeti and E Masumi ldquoEvaluation of the effects of galbanicacid from Ferula szowitsiana and conferol from F badrakemaas modulators of multi-drug resistance in clinical isolates ofEscherichia coli and Staphylococcus aureusrdquo Research in Phar-maceutical Sciences vol 5 no 1 pp 25ndash32 2010

[15] CLSImdashClinical and Laboratory Standards InstituteldquoPerformance standards for antimicrobial susceptibility testingtwenty-fifth informational supplementrdquo CLSI DocumentM100-S25 Clinical and Laboratory Standards Institute WaynePa USA 2015

[16] G W Kaatz and S M Seo ldquoInducible NorA-mediatedmultidrug resistance in Staphylococcus aureusrdquo AntimicrobialAgents and Chemotherapy vol 39 no 12 pp 2650ndash2655 1995

[17] J I Ross A M Farrell E A Eady J H Cove andW J Cunliffe ldquoCharacterisation and molecular cloning ofthe novel macrolide-streptogramin B resistance determinantfrom Staphylococcus epidermidisrdquo Journal of AntimicrobialChemotherapy vol 24 no 6 pp 851ndash862 1989

[18] S Gibbons and E E Udo ldquoThe effect of reserpine a modulatorofmultidrug efflux pumps on the in vitro activity of tetracyclineagainst clinical isolates of methicillin resistant Staphylococcusaureus (MRSA) possessing the tet(K) determinantrdquo Phytother-apy Research vol 14 no 2 pp 139ndash140 2000

[19] M Stavri L J V Piddock and S Gibbons ldquoBacterial effluxpump inhibitors from natural sourcesrdquo Journal of AntimicrobialChemotherapy vol 59 no 6 pp 1247ndash1260 2007

[20] V S Falcao-Silva D A SilvaM D F V Souza and J P SiqueiraJr ldquoModulation of drug resistance in Staphylococcus aureus bya kaempferol glycoside from Herissantia tiubae (Malvaceae)rdquoPhytotherapy Research vol 23 no 10 pp 1367ndash1370 2009

[21] Hyperchem Program Release 80 for Windows 1999ndash2005Hybercube Inc Gainesville Fla USA

[22] N L Allinger ldquoConformational analysis 130 MM2 A hydro-carbon force field utilizing V1 and V2 torsional termsrdquo Journalof the American Chemical Society vol 99 no 25 pp 8127ndash81341977

[23] M J S Dewar G Zoebisch E F Healy and J J PStewart ldquoThe development and use of quantum-mechanicalmolecular-models 76 AM1mdasha new general-purpose quantum-mechanical molecular modelrdquo Journal of the American Chemi-cal Society vol 107 pp 3902ndash3909 1985

[24] N C CohenGuidebook onMolecular Modeling in Drug DesignAcademic Press San Diego Calif USA 1996

[25] A R Leach Molecular Modeling Principles and ApplicationsPrentice Hall London UK 2001

[26] A Pedretti L Villa and G Vistoli ldquoVEGA-an open platformto develop chemo-bio-informatics applications using plug-inarchitecture and script programmingrdquo Journal of Computer-Aided Molecular Design vol 18 no 3 pp 167ndash173 2004

[27] R BouleyM Kumarasiri Z Peng et al ldquoDiscovery of antibiotic(E)-3-(3-carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-onerdquo Journal of the American Chemical Society vol 137 no 5pp 1738ndash1741 2015

[28] R Thomsen and M H Christensen ldquoMolDock a new tech-nique for high-accuracy molecular dockingrdquo Journal of Medici-nal Chemistry vol 49 no 11 pp 3315ndash3321 2006

[29] Shakeel-U-Rehman R Khan K A Bhat A F Raja A S Shawland M S Alam ldquoIsolation characterisation and antibacterialactivity studies of coumarins from Rhododendron lepidotumWall ex G Don Ericaceaerdquo Brazilian Journal of Pharmacog-nosy vol 20 no 6 pp 886ndash890 2010

[30] C L Cespedes J G Avila A Martınez B Serrato J CCalderon-Mugica and R Salgado-Garciglia ldquoAntifungal andantibacterial activities of Mexican tarragon (Tagetes lucida)rdquoJournal of Agricultural and Food Chemistry vol 54 no 10 pp3521ndash3527 2006

[31] A Kumar I A Khan S Koul et al ldquoNovel structural analoguesof piperidine as inhibitors of the NorA efflux pump of Staphylo-coccus aureusrdquo Journal of Antimicrobial Chemotherapy vol 61pp 1270ndash1276 2008

[32] J M Brunel A Lieutaud V Lome J-M Pages and J-M BollaldquoPolyamino geranic derivatives as new chemosensitizers tocombat antibiotic resistant Gram-negative bacteriardquo Bioorganicamp Medicinal Chemistry vol 21 no 5 pp 1174ndash1179 2013

[33] E Reynolds J I Ross and J H Cove ldquoMsr(A) and relatedmacrolidestreptogramin resistance determinants incompletetransportersrdquo International Journal of Antimicrobial Agentsvol 22 no 3 pp 228ndash236 2003

[34] A Zervosen E Sauvage J-M Frere P Charlier and A LuxenldquoDevelopment of new drugs for an old targetmdashthe penicillinbinding proteinsrdquo Molecules vol 17 no 11 pp 12478ndash125052012

[35] M B Fernandes J E Goncalves M T Scotti A A deOliveira L C Tavares and S Storpirtis ldquoCaco-2 cells cytotox-icity of nifuroxazide derivatives with potential activity againstMethicillin-resistant Staphylococcus aureus (MRSA)rdquo Toxicol-ogy in Vitro vol 26 no 3 pp 535ndash540 2012

[36] A L Greninger S S Chatterjee L C Chan et al ldquoWhole-genome sequencing of methicillin-resistant Staphylococcusaureus resistant to fifth-generation cephalosporins revealspotential non-meca mechanisms of resistancerdquo Plos ONE vol11 no 2 Article ID e0149541 2016

[37] S B Hong M H Rhee B Yun Y H Lim H G Song andK S Shin ldquoSynergistic anti-bacterial effects of Phellinus baumiiethyl acetate extracts and beta-lactam antimicrobial agentsagainst methicillin-resistant Staphylococcus aureusrdquo Annals ofLaboratory Medicine vol 36 no 2 pp 111ndash116 2016

[38] P Lavanya S Ramaiah and A Anbarasu ldquoAmolecular dockingand dynamics study to screen potent anti-staphylococcal com-pounds against ceftaroline resistant MRSArdquo Journal of CellularBiochemistry vol 117 no 2 pp 542ndash548 2016

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 Scientifica

diagnostic tools for detection of resistance by effluxrdquo Frontiersin Anti-Infective Drug Discovery vol 1 pp 138ndash175 2006

[12] R S A deAraujo FQ SGuerra EDO Lima et al ldquoSynthesisStructure-activity Relationships (SAR) and in silico studies ofcoumarin derivatives with antifungal activityrdquo InternationalJournal of Molecular Sciences vol 14 no 1 pp 1293ndash1309 2013

[13] N Hamdi A S Al-Ayed R B Said and A Fabienne ldquoSyn-thesis and characterization of new thiazolidinones containingcoumarin moieties and their antibacterial and antioxidantactivitiesrdquoMolecules vol 17 no 8 pp 9321ndash9334 2012

[14] B S F Bazzaz M Iranshahi M Naderinasab S Hajian ZSabeti and E Masumi ldquoEvaluation of the effects of galbanicacid from Ferula szowitsiana and conferol from F badrakemaas modulators of multi-drug resistance in clinical isolates ofEscherichia coli and Staphylococcus aureusrdquo Research in Phar-maceutical Sciences vol 5 no 1 pp 25ndash32 2010

[15] CLSImdashClinical and Laboratory Standards InstituteldquoPerformance standards for antimicrobial susceptibility testingtwenty-fifth informational supplementrdquo CLSI DocumentM100-S25 Clinical and Laboratory Standards Institute WaynePa USA 2015

[16] G W Kaatz and S M Seo ldquoInducible NorA-mediatedmultidrug resistance in Staphylococcus aureusrdquo AntimicrobialAgents and Chemotherapy vol 39 no 12 pp 2650ndash2655 1995

[17] J I Ross A M Farrell E A Eady J H Cove andW J Cunliffe ldquoCharacterisation and molecular cloning ofthe novel macrolide-streptogramin B resistance determinantfrom Staphylococcus epidermidisrdquo Journal of AntimicrobialChemotherapy vol 24 no 6 pp 851ndash862 1989

[18] S Gibbons and E E Udo ldquoThe effect of reserpine a modulatorofmultidrug efflux pumps on the in vitro activity of tetracyclineagainst clinical isolates of methicillin resistant Staphylococcusaureus (MRSA) possessing the tet(K) determinantrdquo Phytother-apy Research vol 14 no 2 pp 139ndash140 2000

[19] M Stavri L J V Piddock and S Gibbons ldquoBacterial effluxpump inhibitors from natural sourcesrdquo Journal of AntimicrobialChemotherapy vol 59 no 6 pp 1247ndash1260 2007

[20] V S Falcao-Silva D A SilvaM D F V Souza and J P SiqueiraJr ldquoModulation of drug resistance in Staphylococcus aureus bya kaempferol glycoside from Herissantia tiubae (Malvaceae)rdquoPhytotherapy Research vol 23 no 10 pp 1367ndash1370 2009

[21] Hyperchem Program Release 80 for Windows 1999ndash2005Hybercube Inc Gainesville Fla USA

[22] N L Allinger ldquoConformational analysis 130 MM2 A hydro-carbon force field utilizing V1 and V2 torsional termsrdquo Journalof the American Chemical Society vol 99 no 25 pp 8127ndash81341977

[23] M J S Dewar G Zoebisch E F Healy and J J PStewart ldquoThe development and use of quantum-mechanicalmolecular-models 76 AM1mdasha new general-purpose quantum-mechanical molecular modelrdquo Journal of the American Chemi-cal Society vol 107 pp 3902ndash3909 1985

[24] N C CohenGuidebook onMolecular Modeling in Drug DesignAcademic Press San Diego Calif USA 1996

[25] A R Leach Molecular Modeling Principles and ApplicationsPrentice Hall London UK 2001

[26] A Pedretti L Villa and G Vistoli ldquoVEGA-an open platformto develop chemo-bio-informatics applications using plug-inarchitecture and script programmingrdquo Journal of Computer-Aided Molecular Design vol 18 no 3 pp 167ndash173 2004

[27] R BouleyM Kumarasiri Z Peng et al ldquoDiscovery of antibiotic(E)-3-(3-carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-onerdquo Journal of the American Chemical Society vol 137 no 5pp 1738ndash1741 2015

[28] R Thomsen and M H Christensen ldquoMolDock a new tech-nique for high-accuracy molecular dockingrdquo Journal of Medici-nal Chemistry vol 49 no 11 pp 3315ndash3321 2006

[29] Shakeel-U-Rehman R Khan K A Bhat A F Raja A S Shawland M S Alam ldquoIsolation characterisation and antibacterialactivity studies of coumarins from Rhododendron lepidotumWall ex G Don Ericaceaerdquo Brazilian Journal of Pharmacog-nosy vol 20 no 6 pp 886ndash890 2010

[30] C L Cespedes J G Avila A Martınez B Serrato J CCalderon-Mugica and R Salgado-Garciglia ldquoAntifungal andantibacterial activities of Mexican tarragon (Tagetes lucida)rdquoJournal of Agricultural and Food Chemistry vol 54 no 10 pp3521ndash3527 2006

[31] A Kumar I A Khan S Koul et al ldquoNovel structural analoguesof piperidine as inhibitors of the NorA efflux pump of Staphylo-coccus aureusrdquo Journal of Antimicrobial Chemotherapy vol 61pp 1270ndash1276 2008

[32] J M Brunel A Lieutaud V Lome J-M Pages and J-M BollaldquoPolyamino geranic derivatives as new chemosensitizers tocombat antibiotic resistant Gram-negative bacteriardquo Bioorganicamp Medicinal Chemistry vol 21 no 5 pp 1174ndash1179 2013

[33] E Reynolds J I Ross and J H Cove ldquoMsr(A) and relatedmacrolidestreptogramin resistance determinants incompletetransportersrdquo International Journal of Antimicrobial Agentsvol 22 no 3 pp 228ndash236 2003

[34] A Zervosen E Sauvage J-M Frere P Charlier and A LuxenldquoDevelopment of new drugs for an old targetmdashthe penicillinbinding proteinsrdquo Molecules vol 17 no 11 pp 12478ndash125052012

[35] M B Fernandes J E Goncalves M T Scotti A A deOliveira L C Tavares and S Storpirtis ldquoCaco-2 cells cytotox-icity of nifuroxazide derivatives with potential activity againstMethicillin-resistant Staphylococcus aureus (MRSA)rdquo Toxicol-ogy in Vitro vol 26 no 3 pp 535ndash540 2012

[36] A L Greninger S S Chatterjee L C Chan et al ldquoWhole-genome sequencing of methicillin-resistant Staphylococcusaureus resistant to fifth-generation cephalosporins revealspotential non-meca mechanisms of resistancerdquo Plos ONE vol11 no 2 Article ID e0149541 2016

[37] S B Hong M H Rhee B Yun Y H Lim H G Song andK S Shin ldquoSynergistic anti-bacterial effects of Phellinus baumiiethyl acetate extracts and beta-lactam antimicrobial agentsagainst methicillin-resistant Staphylococcus aureusrdquo Annals ofLaboratory Medicine vol 36 no 2 pp 111ndash116 2016

[38] P Lavanya S Ramaiah and A Anbarasu ldquoAmolecular dockingand dynamics study to screen potent anti-staphylococcal com-pounds against ceftaroline resistant MRSArdquo Journal of CellularBiochemistry vol 117 no 2 pp 542ndash548 2016

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology