Cinnamic Acid Analogs as Intervention Catalysts for Overcoming Antifungal Tolerance

14 Pages·2017·1.52 MB·English

molecules
Communication
Cinnamic Acid Analogs as Intervention Catalysts for
Overcoming Antifungal Tolerance
JongH.Kim*,KathleenL.ChanandLuisaW.Cheng
FoodborneToxinDetectionandPreventionResearchUnit,WesternRegionalResearchCenter,USDA-ARS,
800BuchananSt.,Albany,CA94710,USA;[email protected](K.L.C.);[email protected](L.W.C.)
* Correspondence:[email protected];Tel.:[phone]
Received:28September2017;Accepted:19October2017;Published:21October2017
Abstract:Disruptionoffungalcellwallshouldbeaneffectiveinterventionstrategy. However,thecell
wall-disruptingechinocandindrugs,suchascaspofungin(CAS),cannotexterminatefilamentousfungal
pathogensduringtreatment.Forpotencyimprovementofcellwall-disruptingagents(CAS,octylgallate
(OG)), antifungal efficacy of thirty-three cinnamic acid derivatives was investigated against
Saccharomyces cerevisiae slt2∆, bck1∆, mutants of the mitogen-activated protein kinase (MAPK),
and MAPK kinase kinase, respectively, in cell wall integrity system, and glr1∆, mutant of
CAS-responsiveglutathionereductase. Cellwallmutantswerehighlysusceptibletofourcinnamic
acids(4-chloro-α-methyl-,4-methoxy-,4-methyl-,3-methylcinnamicacids),where4-chloro-α-methyl-
and4-methylcinnamicacidspossessedthehighestactivity. Structure-activityrelationshiprevealed
that 4-methylcinnamic acid, the deoxygenated structure of 4-methoxycinnamic acid, overcame
tolerance of glr1∆ to 4-methoxycinnamic acid, indicating the significance of para substitution
of methyl moiety for effective fungal control. The potential of compounds as chemosensitizers
(interventioncatalysts)tocellwalldisruptants(viz.,4-chloro-α-methyl-or4-methylcinnamicacids+
CASorOG)wasassessedaccordingtoClinicalLaboratoryStandardsInstituteM38-A.Synergistic
chemosensitization greatly lowers minimum inhibitory concentrations of the co-administered
drug/agents. 4-Chloro-α-methylcinnamicacidfurtherovercamefludioxoniltoleranceofAspergillus
fumigatus antioxidant MAPK mutants (sakA∆, mpkC∆). Collectively, 4-chloro-α-methyl- and
4-methylcinnamic acids possess chemosensitizing capability to augment antifungal efficacy of
conventional drug/agents, thus could be developed as target-based (i.e., cell wall disruption)
interventioncatalysts.
Keywords: antifungal; antioxidant system; caspofungin; cell wall integrity; chemosensitization;
cinnamicacids;interventioncatalysts;smallmolecules;synergism
1. Introduction
Mycotic diseases, such as human invasive aspergillosis caused by Aspergillus (e.g., Aspergillus
fumigatus,Aspergillusflavus),ormycotoxincontaminationbyfilamentousfungalpathogens(e.g.,Aspergillus
parasiticus—producinghepato-carcinogenicaflatoxins)areproblematicsinceeffectiveantifungalagents,
especiallythoseforcontrolofdrug/fungicide-resistantpathogens, areoftenverylimited. Studies
have shown that the fungal cell wall plays an important role during host infection, and thus has
long been a target of many antifungal agents [1]. Currently, the lipopeptide drug echinocandins
(e.g., caspofungin (CAS; Figure 1), micafungin, anidulafungin) are clinically applied to target the
cellwallintegritysystemoffungalpathogens[2]. However, duetoincreasingconcernsaboutthe
safetyofcertainantifungaldrugsthathavebeeninwideuseandtheimpactofrepeatedexposureto
thesecompoundsonhealthandfungalresistance,industriesconstantlyseeknewantifungalsordrug
potentiators(viz.,interventioncatalysts)withimprovedhealthandsafetyprofiles.
Molecules2017,22,1783;doi:10.3390/molecules22101783 www.mdpi.com/journal/molecules
Molecules 2017, 22, 1783 3 of 13
Molecules2017,22,1783 2of14
(a) (b) (c) (d)
(e) (f)

Figure 1. Structures of antifungal agents used in this study. (a) Cinnamic acid (Basic structure),
(b)F4-iCguhrloe r1o.- αSt-rmuectthuyrelcsi nonf aamntiicfuancigda,l( ac)g4e-nMtse uthsoedxy icni nthniasm sticudacyi.d (,a()d C)i4n-Mnaemthiyc laccinidn a(Bmaiscica csitdr,u(cet)uCrea)s,p (bof)u 4n-gin
Chloro-α-methylcinnamic acid, (c) 4-Methoxycinnamic acid, (d) 4-Methylcinnamic acid, (e)
(CAS),(f)Octylgallate(OG).
Caspofungin (CAS), (f) Octyl gallate (OG).
ThecellwallintegritypathwayiswellcharacterizedinthemodelfungusSaccharomycescerevisiae,
2. Results and Discussion
wheretheupstreammitogen-activatedproteinkinase(MAPK)signalingcascades(requiredforcellwall
ma2in.1t.e Indaenntciefi)caatrieonc oofn tthreo lMleodstb Pyotthenet eCniznynmameicp Aroctidesin vikai Ynaeasset CSc[r3ee].niGnegn: 4o-mCheloarnod-αf-umnetchtiyol-n, a4l-Mcoemthpoxayr-i,s ons
fur4th-Merertheyvle- aalnedd 3t-hMatetmhyalnciynngaemniecs Aincidths ecellwallintegritysysteminfungi,suchasAspergillusnidulans,
Aspergillusoryzae, A.fumigatus, andS.cerevisiae, arewellconserved[4,5]. Forexample, A.nidulans
To investigate the cell wall-interfering potential of cinnamic acids in fungi, the antifungal
mpkA∆(cellwallintegrityMAPKmutant)exhibitedhypersensitivitytothecellwallinterferingagent
efficacy of 33 analogs of cinnamic acid (0.5 mM cutoff; Table 1) was investigated by examining cell
calcwoaflllu ionrtewgrhiittye m(CuWtan)t[s5 ]o.f SSi.m cielraervihsiyaep e(srlst2eΔns aitnivde brcek1sΔpo, nensecotdoinCgW MwAaPsKa alsnodo MbsAePrvKe dkininasSe. kceinreavsies iae
slt2(∆M,AthPeKoKrKth),o rloesgpoeucstivceelllyw) ianl linin vtietgrori tyyeaMstA dPilKutmionu tbainotasosfatyhse. Tmhoed Se.l cyereeavsitsi[a5e] b.ck1Δ and slt2Δ have
beNeno tseewrvoinrtgh aysi ssctrheaetnitnhge tootohlesr fsoirg indaelnintigfycinagsc naedwe, cneallm welayll “dainsrtuiopxtiindga natg”enMtsA, PcoKnspidatehriwnga yt,hiessae lso
invmoluvteadntisn wfuenreg ahligshulsyc esputsicbeiplittiyblteo tcoe lclewll awlladlli spreurptutarbnitnsg[ 6r–e8ag].eInntsg seuncehr aals, iCntAaSc t[a1n9]t.i oIxni dthaen tyMeaAst PK
patphawthaoygiesnn Cec. easlbsiacraynsf,o CrAthSe terfefaetcmtievnetn aelsssoo tfricgeglelrws ainllcdreisarsuedp teaxnptrse,swsihoinle oMf GALPRK1,m wuhtiacthio ennscocaduess ethder ug
reseisntzaynmcee [g6l–u8t]a.thFioonree xraedmupctlaes,eS .recsepreovnissiibaelea fnotri omxiadinatnatinMinAg PcKellmuluart agnltust,aeth.gio.,nheo hg1om(MeoAstPasKis) o[2r0p].b s2
(MAThPeKrekfoirnea, sgel;uMtatAhiPoKneK r)e,dwuecrteasteo mleuratannttt (oglcre1lΔl)w waalls- dalissor uinpctliundgeadg ienn tthsis[ 6s–tu8d].yF. urthermore,mutants
oftheupstreamsensors,suchastransmembraneosmosensor(SHO1)orhistidinekinaseosmosensor
Table 1. Growth scores of yeast strains at 0.5 mM (cutoff) of cinnamic acid analogs during yeast
(SLN1),inthesamesignalingsystemarealsopartiallytoleranttoCW.Therefore,inadditiontothe
dilution bioassay (WT, Wild type; 0, No growth; 6, Full growth; See Experimental section).
cellwallintegritypathway,fungalsusceptibilitytocellwallinterferingagentsisalsodependentupon
theexistence/availabilityCionfnainmtiac cAtcicdos mponentsoftheantiWoTx idantMsltA2ΔP Ksigbcnka1Δli ngpgaltrh1Δw ays[6–8].
Group 1 (Highest activity):
A similar drug tolerance was also determined in the hum0 an yeast0 pathogen0 Candida0 albicans [9].
4-Chloro-α-methylcinnamic acid
CrosstalksbetweenM4A-MPeKthyrlcoinuntaems,ici a.eci.d, the“antioxidant”a1 nd“cellw0 allinteg0r ity”pat0h ways,have
recentlybeenreported4-[M10et,h1o1x]y.cinnamic acid 1 0 0 6
3-Methylcinnamic acid 1 0 0 6
Antifungal chemosensitization is an intervention strategy for effective control of fungal
Group 2 (Moderate activity):
6 2 2 4
pathogens,wherec2o-C-ahdlomro-iαn-misettrhaytlicoinnnaomfica accihde mosensitizer(viz.,naturalorsyntheticcompoundsthat
functionasinterventionα-Mcaettahylylcsintsn)aminicc areciads estheefficacyofth6e convent4io naldrug6 co-admi6n istered[12].
3,4-Dimethoxycinnamic acid 6 4 6 6
Achemosensitizeronitsowndoesnothavetopossesspotentantifungalactivity.However,sub-fungicidal
4-Ethoxy-3-methoxycinnamic acid 6 4 6 6
concentrationofachemoBesneznyslciitniznaemrisci agcnidi ficantlyincapacitates6 fungalde5f ensesyst5e mstoa6c onventional
drug,thusmaking3t,h4-eDiphaydthrooxgy-etnranhs-icginhnlaymsiuc ascciedp tibletothedru6g co-appl5ie d[12]. E5x amples6o fantifungal
3-Chloro-4-methoxycinnamic acid 6 5 6 6
chemosensitization include: (1) piperazinyl quinolone, which sensitizes the human pathogen C.
β-Methylcinnamic acid 6 5 6 6
albicanstotheazoledrugfluconazole(FLC),resultinginovercomingFLCresistanceofC.albicans[13],

(2)cyclobutene-dione(squarile)derivatives,whichsensitizetheC.albicansmajorfacilitatorsuperfamily
transporter(Mdr1p;responsibleforFLCresistance)toFLC[14],and(3)benzhydroxamicacid(inhibitor
Molecules2017,22,1783 3of14
ofmitochondrialalternativeoxidases),whichenhancesthesensitivityofRhizopusoryzaetotriazoles
(posaconazole,itraconazole)[15].
Thecinnamicacidderivativesaregenerallyrecognizedassafe(GRAS)reagent[16],andthus,
arecurrentlyusedasfoodadditives. Ofnote,cinnamicacidderivatives,suchascinnamicacidamides,
havealsobeendevelopedasantifungalagentsbytargetingcellwallbiosynthesis[17,18].
Inthisstudy,theeffectivenessofthirty-threecinnamicacidderivativesasinterventioncatalysts
wasinvestigated. Resultsshowed4-chloro-α-methyl-and4-methylcinnamicacids(Figure1)possessed
the highest chemosensitizing capability to augment the efficacy of cell wall-targeting antifungals
(CAS,octylgallate(OG))ortoovercomefungaltolerancetothefungicidefludioxonil. Itisconcluded
thatcinnamicacidderivativescouldbedevelopedaspromisinginterventioncatalystsforeffective
controloffungalpathogens.
2. ResultsandDiscussion
2.1. IdentificationoftheMostPotentCinnamicAcidsviaYeastScreening: 4-Chloro-α-methyl-,4-Methoxy-,
4-Methyl-and3-MethylcinnamicAcids
Toinvestigatethecellwall-interferingpotentialofcinnamicacidsinfungi,theantifungalefficacy
of33analogsofcinnamicacid(0.5mMcutoff;Table1)wasinvestigatedbyexaminingcellwallintegrity
mutants of S. cerevisiae (slt2∆ and bck1∆, encoding MAPK and MAPK kinase kinase (MAPKKK),
respectively)ininvitroyeastdilutionbioassays. TheS.cerevisiaebck1∆andslt2∆havebeenserving
asscreeningtoolsforidentifyingnewcellwalldisruptingagents,consideringthesemutantswere
highlysusceptibletocellwallperturbingreagentssuchasCAS[19]. IntheyeastpathogenC.albicans,
CAStreatmentalsotriggersincreasedexpressionofGLR1,whichencodestheenzymeglutathione
reductaseresponsibleformaintainingcellularglutathionehomeostasis[20]. Therefore,glutathione
reductasemutant(glr1∆)wasalsoincludedinthisstudy.
Initially,4-chloro-α-methyl-,4-methoxy-,4-methyl-and3-methylcinnamicacidswerefoundto
possessthehighestantifungalactivityagainstS.cerevisiaecellwallintegritymutants(viz.,nogrowth
of bck1∆ and slt2∆ at 0.5 mM) (Table 1). Test compounds were classified into three groups based
onthelevelofantifungalactivityagainstbck1∆andslt2∆mutants(at0.5mM)asfollows: Group1
(Fourcinnamicacids),4-chloro-α-methyl-,4-methoxy-,4-methyl-,3-methylcinnamicacids(Complete
growth inhibition of bck1∆ and slt2∆ (Growth score = 0)). The group 1 compounds also inhibited
the growth of WT (Growth score = 0 to 1); Group 2 (Eight cinnamic acids), 2-chloro-α-methyl-,
α-methyl-,3,4-dimethoxy-,4-ethoxy-3-methoxy-,benzyl-,3,4-dihydroxy-trans-,3-chloro-4-methoxy-,
β-methylcinnamic acids (Moderate growth inhibition of bck1∆ and slt2∆ (Growth score = 2 to 5));
Group3,theremainingtwenty-twocinnamicacids(includingcinnamicacidasthebasicstructure)
(Nogrowthinhibitionofbck1∆andslt2∆(Growthscore=6))(Table1).
Notably,theglr1∆mutantwashyper-tolerant(Growthscore=6)to4-methoxycinnamicacidwhen
compared to other strains (WT, bck1∆, slt2∆; Growth score = 0 to 1). However, this hyper-tolerance
was abolished by 4-methylcinnamic acid (Growth score = 0), the deoxygenated analog of
4-methoxycinnamic acid (at para methoxyl moiety; Table 1; Figure 2). In contrast, treatment with
theotherdeoxygenatedanalog3-methylcinnamicacid(Group1)couldnotabolishhyper-tolerance
of glr1∆ mutant (Growth score = 6; Table 1; Figure data not shown), indicating structure-activity
relationshipoftestcompounds,whereparasubstitutionofmethylmoietyiscrucialforovercomingthe
glr1∆hyper-toleranceto4-methoxycinnamicacid.
Molecules 2017, 22, 1783 4 of 13
Molecules2017,22,1783 4of14
Group 3 (No activity):
6 6 6 6
Cinnamic acid (Basic structure)
Table1.GrowthscMoreethsyol-ftryaenas-sctinsntraaminics aactid0 .5mM(cutoff)ofcin6n amicacid6a nalogsdu6 ringyeast6 dilution
bioassay(WT,Wild2ty-Mpeet;h0y,lcNinonagmroicw actihd; 6,Fullgrowth;SeeEx6 perimenta6l section).6 6
2-Methoxycinnamic acid 6 6 6 6
3-MeCthionxnyacimnniacmAicc aidcisd W6 T slt26 ∆ bck16∆ glr1∆6
3,4,5-Trimethoxycinnamic acid 6 6 6 6
4-HyGdrrooxuyp-3-1m(eHthiogxhyecsintnaacmtiivc iatcyi)d: 6 6 6 6
0 0 0 0
34-H-Cyhdrlooxryo--4α-m-metehtohxyyclcininnanmamic aicciadc id 6 6 6 6
24-H-Mydertohxyylccininnnamamic iacciadc id 61 06 06 0 6
43--HMyedtrhooxyxcyincninanmaicm aiccida cid 61 06 06 6 6
4-Hydroxycinnamic acid 6 6 6 6
3-Methylcinnamicacid 1 0 0 6
Cinnamyl acetate 6 6 6 6
Group2(Moderateactivity):
4-Acetoxy-3-methoxycinnamic acid 66 26 26 4 6
2-Chloro-α-methylcinnamicacid
4-Hexadecyloxy-3-methoxycinnamic acid 6 6 6 6
α-Methylcinnamicacid 6 4 6 6
2-Methoxy-α-methylcinnamic acid 6 6 6 6
M3,e4t-hDyli mα-metehtohyxlycicninnanmaimc aiccida cid 66 46 66 6 6
Eth4y-lE 4t-hhoyxdyro-x3y--m3-emthetohxoxyycciinnnnaammiicc aacicdi d 66 46 66 6 6
4-BenzylBoxeyn-3z-ymlceitnhonxaymcinicnaamciidc acid 66 56 56 6 6
3,35,-4D-iDmiehthyodxryo-4x-yh-ytdrraonxsy-cciinnnnaammici accaidc id 66 56 56 6 6
3-4C-Ahmloirnoo--42--mmeetthhyolcxinyncaimnnica amciidc acid 66 56 66 6 6
3,4-Diβh-yMdreotxhyyhylcdirnoncianmnaimciacc aicdid 66 56 66 6 6
3-Hydroxy-αG-mroerucapp3to(-Nβ-moeatchtyilvciintyn)a:mic acid 6 6 6 6
6 6 6 6
Cinnamicacid(Basicstructure)
Initially, 4-chloMreot-hαy-lm-teratnhsy-lc-i,n 4n-ammeitchaocxiyd-, 4-methyl- and6 3-methy6lcinnamic6 acids we6re found to
possess the highest an2-tMifuenthgyallc aincntiavmityic aagcaidinst S. cerevisiae cel6l wall inte6grity mu6tants (viz6., no growth
of bck1Δ and slt2Δ a2t -0M.5e mthoMx)y c(Tinanbalme 1ic).a Tciedst compounds we6re classifi6ed into th6ree grou6ps based on
3-Methoxycinnamicacid 6 6 6 6
the level of antifungal activity against bck1Δ and slt2Δ mutants (at 0.5 mM) as follows: Group 1 (Four
3,4,5-Trimethoxycinnamicacid 6 6 6 6
cinnamic acids), 4-chloro-α-methyl-, 4-methoxy-, 4-methyl-, 3-methylcinnamic acids (Complete
4-Hydroxy-3-methoxycinnamicacid 6 6 6 6
growth inhibition of bck1Δ and slt2Δ (Growth score = 0)). The group 1 compounds also inhibited the
3-Hydroxy-4-methoxycinnamicacid 6 6 6 6
growth of WT (Grow2t-hH yscdorroex y=c 0in tnoa 1m);i Gcarcoiudp 2 (Eight cinnam6ic acids),6 2-chloro-6α-methyl6-, α-methyl-
, 3,4-dimethoxy-, 43--eHthyodxroyx-3y-cminentahmoxicya-,c idbenzyl-, 3,4-dihy6droxy-tra6ns-, 3-ch6loro-4-m6ethoxy-, β-
methylcinnamic acid4s- H(Mydordoexryactien gnraomwictha cinidhibition of bck1Δ a6nd slt2Δ 6(Growth s6core = 2 t6o 5)); Group
Cinnamylacetate 6 6 6 6
3, the remaining twenty-two cinnamic acids (including cinnamic acid as the basic structure) (No
4-Acetoxy-3-methoxycinnamicacid 6 6 6 6
growth inhibition of bck1Δ and slt2Δ (Growth score = 6)) (Table 1).
4-Hexadecyloxy-3-methoxycinnamicacid 6 6 6 6
Notably, the glr1Δ mutant was hyper-tolerant (Growth score = 6) to 4-methoxycinnamic acid
2-Methoxy-α-methylcinnamicacid 6 6 6 6
when compared Mtoe tohtyhleαr -smtreatihnysl c(iWnnTa,m biccka1cΔid, slt2Δ; Growth6 score = 60 to 1). H6owever,6 this hyper-
tolerance waEst hayblo4li-shhyeddr obxyy 4-3--mmeetthhyolxcyincinnanmamic iaccaicdi d(Growth s6core = 0),6 the deox6ygenated6 analog of 4-
methoxycinna4m-Bice nazcyidlo x(ayt- 3p-amrae tmhoextyhcoixnynla mmiocieatcyid; Table 1; Fi6gure 2). I6n contras6t, treatme6nt with the
other deoxyg3e,5n-aDteimd eatnhaolxoyg-4 3-h-mydertohxyylcciinnnnaammiicc aaccidid (Group 61) could n6ot abolis6h hyper-6tolerance of
4-Amino-2-methylcinnamicacid 6 6 6 6
glr1Δ mutant (Growth score = 6; Table 1; Figure data not shown), indicating structure-activity
3,4-Dihydroxyhydrocinnamicacid 6 6 6 6
relationship of test compounds, where para substitution of methyl moiety is crucial for overcoming
3-Hydroxy-α-mercapto-β-methylcinnamicacid 6 6 6 6
the glr1Δ hyper-tolerance to 4-methoxycinnamic acid.

FigFuirgeur2e. 2.Y Yeaeasstt ddiilluuttiioonn bbiiooaassssaayy shsohwowinign dgifdfeifrfeenrteianlt isaulscseupstciebpilittiyb iolift yS. ocefreSv.isciearee svlits2iΔae, bsclkt21Δ∆,, abncdk1 ∆,
glr1Δ mutants to cinnamic acid analogs (0.5 mM).
andglr1∆mutantstocinnamicacidanalogs(0.5mM).

2.2. FungalToleranceto4-MethoxycinnamicAcidWasUniquetoGlutathioneReductaseMutant
GLR1 encodes the enzyme glutathione reductase (cytosolic and mitochondrial), which converts
oxidizedglutathione(GSSG)toreducedglutathione(GSH)[21](SupplementaryFigureS1). Cytosolic
Molecules2017,22,1783 5of14
glutathione reductase also determines the redox state of glutathione in the mitochondrial
inter-membrane space [22]. GLR1 is known to genetically interact with thirteen genes involved
in: (1)Cellredoxhomeostasis,(2)Cellularresponsetooxidativestress,(3)Proteinglutathionylation,
and(4)Glutathionemetabolicprocess(Table2;SupplementaryFigureS2)[22].Therefore,anotheryeast
bioassaywasperformedtoinvestigatewhetherGLR1interactinggenemutantswerealsohyper-tolerant
to4-methoxycinnamicacid.
Table2.ResponsesofS.cerevisiaeWTandgenedeletionmutantsto4-methoxy-or4-methylcinnamic
acid(0.5mM).
S.cerevisiae FunctionsofDeletedGenes 4-Methoxy- 4-Methyl-
WT Parental(Matahis3∆1leu2∆0met15∆0ura3∆0) 1 1
glr1∆ GLR1&GLR1interactinggenes: 4(Hyper-tolerant) 0
Glutathionereductase
trr1∆ Cytoplasmicthioredoxinreductase 0 0
trr2∆ Mitochondrialthioredoxinreductase 1 0
tsa1∆ Thioredoxinperoxidase 0 0
trx1∆ Cytoplasmicthioredoxinisoenzyme 0 0
trx2∆ Cytoplasmicthioredoxinisoenzyme 0 0
gsh1∆ γ-glutamylcysteinesynthetase 0 0
gsh2∆ Glutathionesynthetase 0 0
grx1∆ Glutathione-dependentdisulfideoxidoreductase 1 0
grx2∆ Cytoplasmicglutaredoxin 0 0
ahp1∆ Thiol-specificperoxiredoxin 1 0
prx1∆ Mitochondrialperoxiredoxin 0 0
tsa2∆ Cytoplasmicthioredoxinperoxidase 0 0
dot5∆ Nuclearthiolperoxidase 0 0
ycf1∆ VacuolarglutathioneS-conjugatetransporter 1 0
ctt1∆ CASresponsiveantioxidantgenes: 0 0
CytosoliccatalaseT
cta1∆ CatalaseA 0 0
sod1∆ CytosolicCu,Znsuperoxidedismutase 0 0
sod2∆ MitochondrialMnsuperoxidedismutase 0 0
Inapriorstudy,thecellwalldisruptingdrugCASalsoactivatedfungalantioxidantsystem,viz.,
enhancedexpressionofMn-superoxidedismutase(SOD2)andnuclearrelocationofMAPK(Hog1p)
thatleadstoincrementalincreaseofcatalase(CAT1)expression/activity[20]. Thus,fouradditional
mutantsofantioxidantgenes—twocatalases(catalasesA,T)andtwosuperoxidedimutases(cytosolic,
mitochondrial)mutants—werealsoincludedinthisassay(Table2).
AsshowninTable2,hyper-tolerantresponseto4-methoxycinnamicacidwasuniquetoglr1∆
mutant. All other mutants (including WT) were hyper-sensitive to the same treatment (Growth
score=0to1). Therefore,resultsindicatedthatfunctionsof“GLR1-interacting”or“CAS-responsive”
antioxidantgeneswerenotcorrelatedwithglr1∆hyper-toleranceto4-methoxycinnamicacid.
2.3. SupplementationofReducedGlutathione(GSH)DidNotAbolishHyper-Toleranceto
4-MethoxycinnamicAcid
TodeterminewhethersupplementationofGSH(Reducedglutathione;themetabolicproductof
glutathionereductase)canabolishthehyper-toleranceofglr1∆mutantto4-methoxycinnamicacid,
yeastbioassaywasperformedbyexogenouslyprovidingGSH(1mM)intoyeastbioassaymedium
(SeeFigure3). Forcomparison,theeffectofGSSG(Oxidizedglutathione;1mM)wasalsoinvestigated.
AsshowninFigure3,glr1∆hyper-toleranceto4-methoxycinnamicacidwasnotabolishedbyGSH
(orGSSG),thusindicatinglimitationofcellularGSHcausedbyGLR1mutationwasnotthedeterminant
ofglr1∆hyper-toleranceto4-methoxycinnamicacid.
Molecules2017,22,1783 6of14
Molecules 2017, 22, 1783 6 of 13

Figure 3. Glutathione supplementation test. The tolerance of S. cerevisiae glutathione reductase mutant
Figure3.Glutathionesupplementationtest.ThetoleranceofS.cerevisiaeglutathionereductasemutant
(glr1Δ) to 4-methoxycinnamic acid was not abolished by supplementation of reduced (GSH) or
(glr1∆)to4-methoxycinnamicacidwasnotabolishedbysupplementationofreduced(GSH)oroxidized
oxidized (GSSG) glutathione, indicating glutathione limitation was not the determinant of glr1Δ
(GSSG)glutathione,indicatingglutathionelimitationwasnotthedeterminantofglr1∆hyper-tolerance
hyper-tolerance to 4-methoxycinnamic acid.
to4-methoxycinnamicacid.
Currently, it is not well understood how the methoxyl- or methyl- moiety in cinnamic acid at
thCe uprarrean stulyb,sittitiustnioont wis eclolrurneldaeterdst owoidthh hoywpethr-etomleerathnocex yolr- ohrypmeert-hseynls-imtivoiiteyt,y reinspceicntnivaemlyi,c oafc igdlra1tΔt he
paramsuutbanstti. t4u-tMioenthisoxcyocrirnenlaatmedicw aictihd hisy pa enra-ttoulrearla pnhceenoorlihc yapceidr-, swenhsicithiv iist yp,rreevsipoeucstliyv eshlyo,wofng tlor 1p∆osmseustsa nt.
4-Mtheethraopxeyuctiincn paomteinctaiacli dsuischa ansa tpurreavlepnhtieonno olifc caocliodn, wcahrciicnhoigsepnreesvisi,o iunsclryeassheo iwn nintsouplions sseecsrsetthioenr,a petecu. tic
pot[e2n3t,i2a4l]s. uWchhaesreparse, veexncteiopnt ofofrc ofleown craerpcoinrtosg eonne smise,tianbcoreliassme inofi n4s-umlienthsyelccrientnioanm,iect ca.c[i2d3 ,2b4y] .cWerhtaeirne as,
Clostridium spp. [25], studies on bioactivity of 4-methylcinnamic acid are very limited.
except for few reports on metabolism of 4-methylcinnamic acid by certain Clostridium spp. [25],
Noteworthy is that methyl-replaced anti-tuberculosis drug possessed higher antibacterial
studiesonbioactivityof4-methylcinnamicacidareverylimited.
activity when compared to the methoxyl-containing drug [26]. For instance, moxifloxacin (containing
Noteworthyisthatmethyl-replacedanti-tuberculosisdrugpossessedhigherantibacterialactivity
“8-methoxyl” moiety) is one of the fluoroquinolones for treating tuberculosis, especially multidrug-
whencomparedtothemethoxyl-containingdrug[26].Forinstance,moxifloxacin(containing“8-methoxyl”
resistant infections [26]. Moxifloxacin interacts with the DNA gyrase, the DNA strand-breaking
moiety) is one of the fluoroquinolones for treating tuberculosis, especially multidrug-resistant
enzyme [26]. Certain mutations in DNA gyrase trigger bacterial drug resistance, which is due to the
infections[26]. MoxifloxacininteractswiththeDNAgyrase,theDNAstrand-breakingenzyme[26].
disruption of bridge—enzyme interaction (thus, decreasing drug affinity). However, introduction of
Certain mutations in DNA gyrase trigger bacterial drug resistance, which is due to the disruption
“8-methyl” moiety into moxifloxacin made the modified drug more potent compared to the 8-
ofbridge—enzymeinteraction(thus,decreasingdrugaffinity). However,introductionof“8-methyl”
methoxy-moxifloxacin against gyrase, especially the mutant enzyme causing drug resistance [26].
moietyiTnhtoermefooxrief,l oitx aisc isnumrmaidseedth tehamt,o adsi foiebdsedrrvuegd mino rmeopxoitfelonxtaccoinm, p4a-mreedthtoyltchienn8a-mmeicth aocxidy -cmoouxldif laolxsoac in
agapinosstsegsys rhaisgeh,eers pafefciniaitlyly toth tehem cueltlaunlatre tnazrgyemt(es)c wauhseinn gcodmrupgarreeds itsot a4n-mceet[h2o6x].ycinnamic acid, resulting
inT bhetetreerf coornet,riotl iosf sfuunrmgails pedaththoagte,nass. Porbesciesrev deedteirnmminoaxtiioflno oxfa tchine ,m4e-cmheatnhisymlc ians ntoa moviecracocimdincgo ugllrd1Δal so
poshsyespserh-itgohleerranafcfien (titoy 4t-omtehtehocxeyllcuilnanratmarigc eatc(sid))w bhye 4n-mcoemthpyalcriendnatom4ic-m aceitdh owxayrcrainnntsa mfuitcuraec isdtu,rdeys.u ltingin
bettercontroloffungalpathogens. Precisedeterminationofthemechanismastoovercomingglr1∆
hyp2e.4r.- tOovleerrcaonmcien(gt oFl4u-dmioextohnoilx Tyoclienrannacme oifc Aascpider)gbilylu4s -Fmumetihgyatlucisn Annatmioixcidaacnitd MwAaPrrKa nMtsutfaunttus rbey s4t-uCdhylo.ro-
α-methylcinnamic Acid
2.4. OvercomingFludioxonilToleranceofAspergillusFumigatusAntioxidantMAPKMutantsby
Fludioxonil is a commercial phenylpyrrole fungicide, which triggers abnormal and excessive
4-Chloro-α-methylcinnamicAcid
stimulation of the “antioxidant” MAPK signaling system [27]. This abnormal activation of
anFtilouxdiidoaxnot nMilAisPKa cpoamthmwaeyrc rieasluplthse inny cleplylurlraorl eenfeurnggyi dciedper,ivwathioicnh vtirai gmgeetrasboalbicn oshrmiftas lfraonmd neoxrcmesasli ve
stimfuunlagtailo ngroofwththe “toa netixohxaiudsatnivt”e MoxAidPaKtivsieg nstarleinssg rseysspteomnse[2. 7A].cTcohridsianbgnlyo,r mapapllaiccatitvioanti oonf oflfuadnitoixooxnidila nt
MApPrKevpenattsh wthaey grreoswutlths oinf fcuenllgualal rpeantheorggyendse. pHroivwaetivoenr,v aisa ombestearbvoedlic insh cieftlsl fwroamll–dniosrrmupatlinfugn dgraulggsr oowr th
toeaxgheanutss t(isveee oInxtirdoadtuivcetisotnr)e,s fsurnegsip hoanvsien.gA mccuotardtiionngsl yin, acpomplpicoantieonntso offfl uupdsitorexaomni slipgrneavlienngt ssythsteemgr,o vwizt.h, of
antioxidant MAPK signaling pathway, can escape fludioxonil (10 to 100 μg/mL) toxicity [27].
fungalpathogens. However,asobservedincellwall–disruptingdrugsoragents(seeIntroduction),
The sakAΔ and mpkCΔ are antioxidant MAPK mutants of the human pathogen A. fumigatus
fungihavingmutationsincomponentsofupstreamsignalingsystem,viz.,antioxidantMAPKsignaling
[28,29]. As shown in Figure 4, A. fumigatus sakAΔ and mpkCΔ mutants exhibited tolerance to
pathway,canescapefludioxonil(10to100µg/mL)toxicity[27].
fluTdhieoxsaoknAil∆ (5a0n μdMm,p ekqCu∆ivaarleenatn ttoio 1x2i.d5a μngt/MmAL)P, Kthmusu dtaenvtesloopfitnhge rhaudmiala ngrpoawththo g(seankAAΔ.:f u6m6.i7g a±t u2s0.[02%8,;2 9].
AsmshpokCwΔn: i7n2.F3i g±u 3re1.40,%A; .%fu: mRieglaattiuvses gakroAw∆tha ncdommppakrCe∆d mto u“tnaon ttsreeaxthmibenitte”d ctoonlterroaln (c1e00to.0 fl±u d0.i0o%xo))n oiln( 50
potato dextrose agar (PDA), whereas the growth of WT was completely inhibited. However, co-
µM,equivalentto12.5µg/mL),thusdevelopingradialgrowth(sakA∆: 66.7±20.0%;mpkC∆: 72.3
Molecules 2017, 22, 1783 7 of 13
application of sub-fungicidal concentration of 4-chloro-α-methylcinnamic acid (0.5 mM) with
fludioxonil (50 μM) effectively prevented fungal tolerance to fludioxonil, thus achieving complete
growth inhibition of MAPK mutants (Figure 4). (Note: 4-Methyl- or 4-methoxycinnamic acid did not
overcome fludioxonil tolerance of A. fumigatus MAPK mutants at the same test conditions—data not
shown.)
Of note, A. fumigatus MAPK mutants exhibited higher sensitivity to the independent treatment
of 4-chloro-α-methylcinnamic acid, alone, when compared to the WT (Figure 4). For example, A.
fumigatus WT developed radial growth on PDA at 1.0 mM of 4-chloro-α-methylcinnamic acid, while
the growth of MAPK mutants (sakAΔ, mpkCΔ) was completely inhibited at the same condition.
Natural phenolic agents or their structural analogs are potent redox cyclers in cells, thus
inhibiting fungal growth via disruption of cellular redox homeostasis or redox-sensitive cellular
components [30,31]. Therefore, it is speculated that 4-chloro-α-methylcinnamic acid (as a redox
cycler) further targets fungal antioxidant system, such as MAPK pathway. Accordingly, in addition
Molecules2017,22,1783 7of14
to interfering with cell wall integrity, debilitation of cellular antioxidant system could also be one
mechanism of antifungal action of 4-chloro-α-methylcinnamic acid. Fungi with mutated MAPK
± 31.0%; Relative growth compared to “no treatment” control (100.0 ± 0.0%)) on potato dextrose
systems (e.g., A. fumigatus sakAΔ, mpkCΔ) are incapable of initiating a fully operational defense
agar (PDA), whereas the growth of WT was completely inhibited. However, co-application of
against oxidative stress triggered by 4-chloro-α-methylcinnamic acid, thus resulting in increased
sub-fungicidalconcentrationof4-chloro-α-methylcinnamicacid(0.5mM)withfludioxonil(50µM)
growth inhibition.
effectivelypreventedfungaltolerancetofludioxonil,thusachievingcompletegrowthinhibitionof
CMoAllePcKtimveultya,n 4ts-c(Fhilgourroe-α4)-.m(Neothtey:l4c-iMnnetahmyli-co arc4i-dm (e0th.5o mxyMcin)n caomuilcda fcuidndctidionno atso vaenr cinomteervfleundtiiooxno ncialtalyst
to ovetrocleormanec efloufdAio.fxuomniigla (tu5s0 MμAMP)K tomleurtaanntcsea otft hfeunsagmael pteastthcoongdeintiso. n s—datanotshown.)

Figure 4. Overcoming fludioxonil (50 μM) tolerance of A. fumigatus mitogen-activated protein kinase
Figure4.Overcomingfludioxonil(50µM)toleranceofA.fumigatusmitogen-activatedproteinkinase
(MAP(KM)A mPuKt)amnutsta (nstask(AsaΔkA, m∆,pmkCpkΔC)∆ b)yb y4-4c-hchlolorroo--αα--mmeetthhyylclcininnnamamiciacc iadci(d0. 5(0m.5M m).M).
2.5. AntifuOngfanlo Cteh,eAm.fousmenigsaittuizsaMtiAonP Kof m4-uCtahnlotsroe-xαh-imbiteetdhyhli-g ohre r4s-Menseitthivyiltcyintonathmeiicn Adecpide ntdo eCnAttSre oartm OeGnt of
4-chloro-α-methylcinnamicacid,alone,whencomparedtotheWT(Figure4).Forexample,A.fumigatus
Antifungal chemosensitization to enhance antifungal potency of CAS was investigated in
WTdevelopedradialgrowthonPDAat1.0mMof4-chloro-α-methylcinnamicacid,whilethegrowth
filamentous fungus Aspergillus brasiliensis ATCC16404 according to the methods outlined by the
ofMAPKmutants(sakA∆,mpkC∆)wascompletelyinhibitedatthesamecondition.
Clinical Laboratory Standards Institute (CLSI) M38-A [32]. 4-Chloro-α-methyl- and 4-
Naturalphenolicagentsortheirstructuralanalogsarepotentredoxcyclersincells,thusinhibiting
methyfulcnignanlagmrowict hacviidasd i(spruopsstieosnsoinfgce ltlhuela rhriegdhoexsht oamnetiofsutansgisaolr erfefdicoax-csye)n switievreec eelxlualmaricnoemdp foonre nthtse[i3r0 p,3o1]t.ential
as cheTmheorseefonrsei,tiizteisrss p(ie.ecu.,l aintetdertvheantt4i-ocnhl ocraot-aαly-mstest)h (y4lc-Minneathmoicxyacciidnn(aasmaicr eadcoixd cwycalesr a)lfsuor tihnecrlutadrgeedts in the
test fofur ncgoamlapnatiroixsiodna)n.t Bsyostthem m,siuncihmausmM AinPhKibpiattohrwya yc.oAnccceonrtdriantgiolyn,sin (aMddICitiso)n atnodin tmerifneriimnguwmi thfucnelglicidal
concewntarlaltiinotnegsr it(yM,dFeCbsil)i,t atainond ofthceulslu, labroatnht iofxraidcatinotnsayls teimnhciobuitldorayls ocobencoennetmraetcihoann iisnmdoicfeasn ti(fFuInCgIasl) and
action of 4-chloro-α-methylcinnamic acid. Fungi with mutated MAPK systems (e.g., A. fumigatus
fractional fungicidal concentration indices (FFCIs) of fungus were determined (See Experimental
sakA∆,mpkC∆)areincapableofinitiatingafullyoperationaldefenseagainstoxidativestresstriggered
section for calculation). A. brasiliensis is one of the test microorganisms (mold) for evaluating
by4-chloro-α-methylcinnamicacid,thusresultinginincreasedgrowthinhibition.
antifungal efficacy of industrial products [33]. CAS, like other echinocandin drugs, generally cannot
Collectively,4-chloro-α-methylcinnamicacid(0.5mM)couldfunctionasaninterventioncatalyst
achieve complete inhibition of the growth of filamentous fungal pathogens [34], thus resulting in
toovercomefludioxonil(50µM)toleranceoffungalpathogens.
fungal survival during CAS treatment.
2.5. AntifungalChemosensitizationof4-Chloro-α-methyl-or4-MethylcinnamicAcidtoCASorOG
OG, an alkyl derivative of the natural product gallic acid, was also included in this
chemosenAsintitzifautniogna ltecshte maso ase rnesfietirzeanticoen pthoeennohlaicn cceomanptoifuunngda.l OpGot eisn ccylaossfifCieAdS aws aas GinRvAesSt ifgoaotedd aidnditive
filamentous fungus Aspergillus brasiliensis ATCC16404 according to the methods outlined by the
ClinicalLaboratoryStandardsInstitute(CLSI)M38-A[32]. 4-Chloro-α-methyl-and4-methylcinnamic
acids(possessingthehighestantifungalefficacy)wereexaminedfortheirpotentialaschemosensitizers
(i.e.,interventioncatalysts)(4-Methoxycinnamicacidwasalsoincludedinthetestforcomparison).
Bothminimuminhibitoryconcentrations(MICs)andminimumfungicidalconcentrations(MFCs),
andthus,bothfractionalinhibitoryconcentrationindices(FICIs)andfractionalfungicidalconcentration
indices(FFCIs)offungusweredetermined(SeeExperimentalsectionforcalculation). A.brasiliensisis
oneofthetestmicroorganisms(mold)forevaluatingantifungalefficacyofindustrialproducts[33].
Molecules2017,22,1783 8of14
CAS,likeotherechinocandindrugs,generallycannotachievecompleteinhibitionofthegrowthof
filamentousfungalpathogens[34],thusresultinginfungalsurvivalduringCAStreatment.
OG,analkylderivativeofthenaturalproductgallicacid,wasalsoincludedinthischemosensitization
testasareferencephenoliccompound. OGisclassifiedasaGRASfoodadditive[16],andisrecently
determinedasasafer,moreeffectivepreservativeforconsumerproducts[35]. OGwaspreviously
showntonegativelyaffectthegrowthofcellwallMAPKmutants[36].
For FICIs of 4-chloro-α-methylcinnamic acid, despite the absence of calculated synergism,
there was increased antifungal activity of 4-chloro-α-methylcinnamic acid and CAS or OG (viz.,
chemosensitizingeffect;FICIs=1.0),whichwasreflectedinloweredMICsof4-chloro-α-methylcinnamic
acid and CAS or OG when compounds were co-applied (Table 3). For instance, co-application of
4-chloro-α-methylcinnamicacid(1.6mM)withCAS(16µg/mL)orOG(0.05mM)completelyinhibited
the growth of A. brasiliensis, while individual treatment of each compound, alone, at the same
concentrationsallowedthesurvivaloffungus(Table3).
Table 3. Antifungal chemosensitization of 4-chloro-α-methyl- and 4-methylcinnamic acids to
caspofungin(CAS)(µg/mL)orOG(mM),testedagainstAspergillusbrasiliensis:SummaryofClinical
LaboratoryStandardsInstitute(CLSI)bioassays.
Compounds MICAlone MICCombined FICI MFCAlone MFCCombined FFCI
4-Chloro-α-methyl 3.2 1.6 12.8 12.8
1 2
CAS 32 16 32 32
4-Methyl 12.8 6.4 12.8 12.8
0.8 2
CAS 32 8 32 32
4-Methoxy 12.8 12.8 12.8 12.8
2 2
CAS 32 32 32 32
Mean:Chemosensitizers 9.6 6.9 − 12.8 12.8 −
CAS 32 18.7 − 32 32 −
t-test1:Chemosensitizers − p=0.590 − − p=1.000 −
CAS − p=0.132 − − p=1.000 −
4-Chloro-α-methyl 3.2 1.6 12.8 12.8
1 2
OG 0.1 0.05 0.4 0.4
4-Methyl 12.8 6.4 12.8 12.8
1 2
OG 0.1 0.05 0.4 0.4
4-Methoxy 12.8 12.8 12.8 12.8
2 2
OG 0.1 0.1 0.4 0.4
Mean:Chemosensitizers 9.6 6.9 − 12.8 12.8 −
OG 0.1 0.07 − 0.4 0.4 −
t-test1:Chemosensitizers − p=0.590 − − p=1.000 −
OG − p=0.116 − − p=1.000 −
1Student’st-testforpaireddata(combined,i.e.,chemosensitization)wasvs.meanminimuminhibitoryconcentration
(MIC)orminimumfungicidalconcentrations(MFC)ofeachcompound(alone,i.e.,nochemosensitization)determined
instrains.Notethatpvaluesforchemosensitizationweredeterminedas>0.05.
Similar trends were also observed in FICIs of 4-methylcinnamic acid. There was increased
antifungalactivityof4-methylcinnamicacidandCASorOG(viz.,chemosensitizingeffect;FICIs=0.8
to 1.0) in A. brasiliensis, which was reflected in lowered MICs of 4-methylcinnamic acid and CAS
or OG when compounds were co-applied. For example, co-application of 4-methylcinnamic acid
(6.4mM)withCAS(8µg/mL)orOG(0.05mM)completelyinhibitedthegrowthofA.brasiliensis,
whileindividualtreatmentofeachcompound,alone,atthesameconcentrationsallowedthegrowthof
fungus(Table3). Incomparison,nochemosensitizingcapabilitywasfoundwith4-methoxycinnamic
acid(FICIs=2.0;Table3).
For FFCIs, no chemosensitizing capability was determined in any of the combinations of
compoundstested,indicatingchemosensitizingcapabilityof4-chloro-α-methyl-or4-methylcinnamic
acidwasatthelevelofloweringMICs(butnotMFCs)oftestcompounds(Table3).
Molecules2017,22,1783 9of14
Molecules 2017, 22, 1783 9 of 13
structure Aolfl t“oegtehthyelr”, recsinulntsasmugicg eastcitdha td4e-crhivloartoi-vαe-sm egtehnyel-roarll4y-m reetshuyllcteindn aimn iceanchidancocuelmdebnetd eovfe loapnetdifungal
aspotentinterventioncatalysts/chemosensitizersforimprovedfungalcontrol.
activity as follows: (1) w/electron-donating groups (hydroxyl, methoxyl): Order of antifungal activity
Antifungalactivitiesofvariouscinnamicacidderivativeshavebeendocumentedelsewhere[37–39].
was o-substitution > p-substitution > m-substitution, (2) w/electron-withdrawing groups (halogen
Structure-activity relationship revealed that the substitution design of cinnamic phenyl ring
atoms, such as chlorine): Highest activity was observed with p-substitution [39].
significantlyaffectedantifungalactivity[39].Forinstance,introducingsubstituentstotheringstructure
With regard to electron-donating groups, our results from cinnamic acid analogs were not well
of“ethyl”cinnamicacidderivativesgenerallyresultedinenhancementofantifungalactivityasfollows:
aligned with that of “ethyl” cinnamic acid derivatives [39]. For example, in our study, 4-
(1)w/electron-donatinggroups(hydroxyl,methoxyl): Orderofantifungalactivitywaso-substitution>
methopx-syucbisntintuatmioinc >amci-dsu b(ps-tistuutbiostni,tu(2t)iown/)e lbecetlroonng-ws itthod rtahwe inGgrgoruopu p1s,( hpaolosgseenssaitnogm sth,seu chhigahsechstlo arinntei)f:ungal
activiHtyi g(Theasbtlaec t1i)v,i twyhwiales 2ob- saenrvde 3d-wmieththpo-sxuybcsitnitnuatimonic[3 a9c].ids (o- and m-substitution, respectively) belong
to Group W3, isthhorewgianrdg tnooe laencttriofun-ndgoanla aticntgivgirtoyu (pTsa,boluer 1re).s u lts from cinnamic acid analogs were not well
alignedwiththatof“ethyl”cinnamicacidderivatives[39].Forexample,inourstudy,4-methoxycinnamic
Whereas for electron-withdrawing group, like the “ethyl” cinnamic acid [39], enhanced
acid(p-substitution)belongstotheGroup1,possessingthehighestantifungalactivity(Table1),while2-
antifungal activity with para substitution was also observed in our study. For example, while 2-
and3-methoxycinnamicacids(o-andm-substitution,respectively)belongtoGroup3,showingno
chloro-α-methylcinnamic acid (o-chlorine) exhibited only moderate antifungal activity (Group 2;
antifungalactivity(Table1).
Table 1), its analog 4-chloro-α-methylcinnamic acid (p-chlorine) possessed the highest antifungal
Whereasforelectron-withdrawinggroup, likethe“ethyl”cinnamicacid[39], enhancedantifungal
potenaccyti v(iGtyrwoiuthpp a1r;a sTuabbstlietu t1io)n. wThasuasl,s oooubrs errveesduilntso uwrsittuhd yc.hFloorreinxaem-cpolen,twahinilien2g-c hclionron-aαm-miect hayclciidn nwameirce well
aligneadci dw(ioth-c htlhoaritn eo)f e“xehtihbyitle”d coinnnlyammiocd aecraidte daenrtiivfuantgivaelsa c[3ti9v]i.t yR(eGsurolutsp fu2;rtThaebrl ein1d),iciattsedan tahloagt basic
struct4u-rceh loofr oc-iαn-nmaemthiycl caicnindasm, eic.ga.,c icdin(pn-acmhloicr iancei)dp vosss. e“sestehdytlh”e chinignhaemstica natcifiudn (gaasl cpoomtenpcayre(dG raobuopv1e;), also
affectTs adblieffe1)r.enTthiuasl ,aonutrifruensuglatsl wacitthivcithylo wrinhee-nco nsutabinstinitguecinntnsa amriec ianctidrowduerceewd/ealldadleigdn etdo wthiteh pthhaetnoyfl ring.
“ethyl”cinnamicacidderivatives[39]. Resultsfurtherindicatedthatbasicstructureofcinnamicacids,
Elucidation of comprehensive structure-activity relationship of cinnamic acid derivatives warrants
e.g.,cinnamicacidvs. “ethyl”cinnamicacid(ascomparedabove),alsoaffectsdifferentialantifungal
future in-depth investigation.
activitywhensubstituentsareintroduced/addedtothephenylring. Elucidationofcomprehensive
In summary, use of small molecules as intervention catalysts could serve as an alternative
structure-activityrelationshipofcinnamicacidderivativeswarrantsfuturein-depthinvestigation.
strategy for effective control of fungal pathogens. Improvement of the efficacy of CAS or OG was
Insummary,useofsmallmoleculesasinterventioncatalystscouldserveasanalternativestrategy
achieved by 4-chloro-α-methyl- or 4-methylcinnamic acid-mediated chemosensitization. 4-Chloro-α-
foreffectivecontroloffungalpathogens. ImprovementoftheefficacyofCASorOGwasachievedby
methy4-lcchinlonroa-mα-imc etahcyild- ofru4r-mtheethr ylocvinenracmaimcaec ifdl-umdeidoixaotendilc hteomleorsaenncsiet izoaft ioAn.. 4fu-Cmhilgoarotu-αs- manetthioylxciidnnaanmt iMc APK
mutanactsid wfuhretnh ecroo-avperpclaimede (flSuedei oFxiognuirlet o5lae rfaonrc escohfeAm.ef)u. mMigoadtuisficaanttiiooxni doafn cthMemAPicKal msturtuacnttusrwesh, esnuch as
deoxycgoe-anpaptliioend o(Sfe peaFriag suureb5satitfuortesdch memeeth).oMxyodl mificoaiteiotyn ooff ccihnenmaimcalics tarcuicdtu (rSeese, sFuicghuarse d5ebo fxoyrg esnchateiomneo)f, could
parasubstitutedmethoxylmoietyofcinnamicacid(SeeFigure5bforscheme),couldalsoabolishglr1∆
also abolish glr1Δ tolerance to 4-methoxycinnamic acid.
toleranceto4-methoxycinnamicacid.

Figure5.Schemeshowingovercomingfungaltolerancetoantifungalagents.(a)Chemosensitization
Figure 5. Scheme showing overcoming fungal tolerance to antifungal agents. (a) Chemosensitization
to overcome fludioxonil tolerance of A. fumigatus MAPK mutants by small molecule
to overcome fludioxonil tolerance of A. fumigatus MAPK mutants by small molecule chemosensitizer
chemosensitizer4-chloro-α-methylcinnamicacid;(b)Deoxygenationof4-methoxylgrouptoovercome
4-chlo4r-om-eαt-hmoxeytchiynnlcaimnnicaamciidc toalecriadn;c e(obf)S .cDereeovixsiyageegnlra1t∆iomnu taonft . 4-methoxyl group to overcome 4-
methoxycinnamic acid tolerance of S. cerevisiae glr1Δ mutant.
3. Materials and Methods
3.1. Fungal Strains and Culture Conditions
Aspergillus fumigatus AF293 wild type (WT), A. fumigatus MAPK gene deletion mutants (sakAΔ,
mpkCΔ) [28,29] and Aspergillus brasiliensis ATCC16404 were cultured at 35 °C or 28 °C, respectively,
on potato dextrose agar (PDA). Saccharomyces cerevisiae BY4741 wild type (Mat a his3Δ1 leu2Δ0
met15Δ0 ura3Δ0) and twenty-one selected single gene deletion mutants (Cell wall integrity system
mutants: bck1Δ, slt2Δ; Glutathione (GSH) homeostasis mutants: glr1Δ, gsh1Δ, gsh2Δ; Twelve GLR1
interacting gene mutants (Table 2); Four caspofungin (CAS) responsive antioxidant gene mutants
(Table 2)) were procured from Invitrogen (Carlsbad, CA, USA) and Open Biosystems (Huntsville,
Molecules2017,22,1783 10of14
3. MaterialsandMethods
3.1. FungalStrainsandCultureConditions
AspergillusfumigatusAF293wildtype(WT),A.fumigatusMAPKgenedeletionmutants(sakA∆,
mpkC∆)[28,29]andAspergillusbrasiliensisATCC16404wereculturedat35◦Cor28◦C,respectively,
onpotatodextroseagar(PDA).SaccharomycescerevisiaeBY4741wildtype(Matahis3∆1leu2∆0met15∆0
ura3∆0)andtwenty-oneselectedsinglegenedeletionmutants(Cellwallintegritysystemmutants:
bck1∆,slt2∆;Glutathione(GSH)homeostasismutants: glr1∆,gsh1∆,gsh2∆;TwelveGLR1interacting
gene mutants (Table 2); Four caspofungin (CAS) responsive antioxidant gene mutants (Table 2))
were procured from Invitrogen (Carlsbad, CA, USA) and Open Biosystems (Huntsville, AL, USA;
Seealso[22]). YeastswereculturedonSyntheticGlucose(SG;Yeastnitrogenbasewithoutaminoacids
0.67%,glucose2%withappropriatesupplements: Uracil0.02mg/mL,aminoacids0.03mg/mL)or
YeastPeptoneDextrose(YPD;Bactoyeastextract1%,Bactopeptone2%,glucose2%)mediumat30◦C.
AllchemicalsforculturingfungioryeastwereprocuredfromSigmaCo. (St. Louis,MO,USA).
3.2. AntifungalSusceptibilityTesting
3.2.1. YeastDilutionBioassayinSaccharomycescerevisiae
Petriplate-basedyeastdilutionbioassayswereperformedontheWTandgenedeletionmutantsto
determinetheirlevelofsusceptibilitytothirty-fourcinnamicacidderivatives(Testconcentrations: 0.1,
0.2,0.3,0.4,0.5,and1.0mM;Table1).1×106cellsoftheWTormutantsofS.cerevisiae,culturedonYPD
plates,wereseriallydiluted10-foldinSGliquidmediumsupplementedwithaminoacidsanduracil
(Seeabove)fivetimestoyieldcelldilutioncohortsof106,105,104,103,102,and101cells. Cellsfrom
eachdilutionofrespectivestrainswerespottedonSGagarincorporatedwithindividualcompounds,
such as cinnamic acid analogs. Yeast cells were incubated at 30 ◦C (5 to 7 days). Results were
monitored/evaluated based on a designated (log10) value of the highest dilution where colonies
becamevisible(Score“0”—nocolonieswerevisiblefromanyofthedilutionspots,Score“1”—only
coloniesfromthespotwith106 cells,Score“2”onlycoloniesfromthespotswith106 and105 cells
werevisible,etc.,whileScore“6”—colonieswerevisiblefromalldilutionspots). Therefore,eachunit
(1to6)ofnumericaldifferencewasequivalenttoa10-folddifferenceinthesensitivityofteststrainto
thetreatment.
Yeast dilution bioassays were also performed on the WT and glr1∆, as described above,
toassesseffectsofreducedoroxidizedglutathione(GSHorGSSG,respectively;1mM)onreverting
4-methoxycinnamicacidtoleranceofglr1∆.
Compounds were dissolved in dimethylsulfoxide (DMSO; absolute DMSO amount: <2% in
media)beforeincorporationintoculturemedia. Throughoutthisstudy,controlplates(Notreatment)
containedDMSOatlevelsequivalenttothatofcohortsreceivingantifungalagents,withinthesame
setofexperiments.
3.2.2. AgarPlateBioassayinAspergillusfumigatus: SusceptibilityofWTandMAPKMutantsto
CinnamicAcidDerivatives
DeterminationofovercomingfludioxoniltoleranceofA.fumigatussakA∆andmpkC∆mutants
wasbasedoncomparisonoffungalradialgrowthbetweentreatedandcontrolcolonies(SeeFigure4).
Fungalconidia(5 × 103)weredilutedinphosphatebufferedsalineandinoculatedasadroponto
the center of PDA plates containing: (1) No treatment (control); (2) 4-chloro-α-methyl-, 4-methyl-,
or4-methoxycinnamicacid(0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0mM);(3)fludioxonil(50µM);
and(4)4-chloro-α-methyl-,4-methyl-,or4-methoxycinnamicacid+fludioxonil. Growthwasobserved
forfivetosevendaysat35◦C.