Chemosensitization as a means to augment commercial antifungal agents

20 Pages·2012·0.5 MB·English

by  Bruce C. Campbell

REVIEWARTICLE
published:29February2012
doi:10.3389/fmicb.2012.00079
Chemosensitization as a means to augment commercial
antifungal agents
BruceC.Campbell*,KathleenL.Chan andJongH.Kim
PlantMycotoxinResearchUnit,WesternRegionalResearchCenter,AgriculturalResearchService,UnitedStatesDepartmentofAgriculture,Albany,CA,USA
Editedby: Antimycoticchemosensitizationanditsmodeofactionareofgrowinginterest.Currently,
JulianneTeresaDjordjevic,University useofantifungalagentsinagricultureandmedicinehasanumberofobstacles.Foremost
ofSydney,Australia
oftheseisdevelopmentofresistanceorcross-resistancetooneormoreantifungalagents.
Reviewedby:
Thegenerallyhighexpenseandnegativeimpact,orsideeffects,associatedwithantifungal
PeterR.Williamson,National
InstitutesofHealth,USA agentsaretwofurtherissuesofconcern.Collectively,theseproblemsareexacerbatedby
AmeetaAgarwal,Universityof effortstocontrolresistantstrains,whichcanevolveintoatreadmillofhigherdosagesfor
Mississippi,USA longerperiods.Thiscycleinturn,inflatescostoftreatment,dramatically.Afurtherproblem
SharonChen,WestmeadHospital,
is stagnation in development of new and effective antifungal agents, especially for treat-
Australia
mentofhumanmycoses.Effortstoovercomesomeoftheseissueshaveinvolvedusing
*Correspondence:
BruceC.Campbell,PlantMycotoxin combinations of available antimycotics (e.g., combination therapy for invasive mycoses).
ResearchUnit,WesternRegional However,thisapproachhashadinconsistentsuccessandisoftenassociatedwithamarked
ResearchCenter,Agricultural increase in negative side effects. Chemosensitization by natural compounds to increase
ResearchService,UnitedStates
effectiveness of commercial antimycotics is a somewhat new approach to dealing with
DepartmentofAgriculture,800
BuchananStreet,Albany,CA94710, theaforementionedproblems.Thepotentialforsafenaturalproductstoimproveantifun-
USA. galactivityhasbeenobservedforoverthreedecades.Chemosensitizingagentspossess
e-mail:[email protected] antifungal activity, but at insufficient levels to serve as antimycotics, alone.Their main
functionistodisruptfungalstressresponse,destabilizethestructuralintegrityofcellular
andvacuolarmembranesorstimulateproductionofreactiveoxygenspecies,augmenting
oxidativestressandapoptosis.Useofsafechemosensitizingagentshaspotentialbenefit
tobothagricultureandmedicine.Whenco-appliedwithacommercialantifungalagent,an
additiveorsynergisticinteractionmayoccur,augmentingantifungalefficacy.Thisaugmen-
tation,inturn,lowerseffectivedosages,costs,negativesideeffectsand,insomecases,
countermandsresistance.
Keywords:mycoses,azoles,naturalproducts,antimycoticresistance,MAPK,cellwall/membraneintegrity
INTRODUCTION antimycotics,withoutincreasingrisksofnegativesideeffects.Such
The term “chemosensitization” was originally introduced as a chemosensitizers could function by debilitating,or stressing the
strategytocounterthedevelopmentofresistanceintumorcellsto fungussothatitbecomesmorevulnerabletocommercialagents.
anticancerchemotherapeuticagents.Simplyput,chemosensitiza- Afundamentalcharacteristicofachemosensitizer,however,isthat
tionincancertherapyinvolvesuseofachemicalthatrenderscan- while it contributes to augmenting the efficacy of drug activity,
cercellsmoresensitivetoachemotherapeuticagent.Development whetherfortreatmentof cancerormycoses,itdoesnotpresent,
ofresistanceincancercellstoanticancerdrugsinvolvedmutations oronlyminimallypresents,additionaltoxicsideeffects.
intargetgenes,up-regulationorover-expressionofgenescontrol- Chemosensitizingagents,alone,mayhavesomenominalanti-
ling efflux pumps, production of enzymes that“detoxified”the fungal activity,but most often an order of magnitude less than
drugs,andDNArepair(Shabbitsetal.,2003).Employmentof a thatofcommercialantimycotics;whichgenerallyfunctionatthe
chemosensitizerresultsinloweringdosagesofcytotoxicanticancer micromolar level, or less. The chemosensitizing agents, on the
drugs,overcomingchemo-resistancebycancercellstothesedrugs, other hand, function mainly as synergists that affect the tar-
avoiding toxicity to non-target cells and lowering the extreme get pathogen in such a way that it becomes more vulnerable
negativesideeffectstothepatientassociatedwithanticancerdrugs. (chemosensitized) to the commercial antimycotic agent. With
Themechanismsofresistancetoanticancerchemotherapeutic chemosensitization, effective dosage levels of the commercial
agentsarealmostcompletelyparalleltothosedevelopedbyfungal product can be significantly lowered, perhaps 10-fold or more.
pathogens against antifungal agents. As in the advancement for Additionally,chemosensitizationmaycausestrainsthatareresis-
cancer treatment, chemosensitizers may be especially useful for tanttoanantimycotictoreverttobecomingsusceptible,oratleast
improvingantimycoticchemotherapyagainsthumanmycosesor moresensitivetotreatment.
strategiestocontrolplantfungalpathogens.Useof chemosensi- There are laboratories, worldwide, performing research on
tizerscouldfacilitateovercomingresistancetocurrentlyavailable chemosensitizing agents to boost antifungal activity. Yet, there
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Campbelletal. Antifungalchemosensitization
has been a general unawareness of each other’s activities. This centuries (Martin and Jones, 1940). However, it has only been
was,andstillisevident,bythefactthatmanyofthepublications within the last two decades that a sense of urgency developed
onchemosensitizationofantimycoticshavenotbeencitingeach in combating human mycoses. This urgency stems from a vast
other’spapersinthefield.Weincludeourselvesinthisoversight. increase in incidence of opportunistic invasive fungal infections
Recent reviews on synergism of antibiotics by natural products (IFIs).MajorIFIsincludedisseminatedcandidiasis,cryptococcal
mainlyentailedantibacterialresearch(Hemaiswaryaetal.,2008; meningitis,andinvasiveaspergillosis.Theincreaseinincidenceof
WagnerandUlrich-Merzenich,2009).Thesereviewsdidciteeight these diseases had its inception in the early 1990s as a result of
papers,however,onsynergismofantimycotics,specifically.There prodigiousgrowthinthenumberofimmunocompromisedindi-
arenowwellover70papersonchemosensitizationofantimycotics viduals, mainly arising from the AIDS epidemic, in addition to
cited in this review. Many of these papers have been published anincreaseinimmunosuppressivetreatmentsrequiredfororgan
onlywithinthepastfewyears.Interestinthisstrategyisgrowing transplants,andanoverallgeneraldebilitation(e.g.,cancermalig-
immenselyasgenetictoolshavefacilitatedthediscoveryofstress nancies)associatedwithanagingpopulation(LowandRotstein,
responsenetworkstoserveastargetstoweakenfungaldefenses, 2011).TheincreasedincidenceofIFIshasrequiredmassivelevels
whetherstructuralorbiochemical. of antimicrobialchemotherapyaccompaniedbytheoverarching
This review begins with an encapsulated overview of fungal problem in the progressive emergence of resistance to available
diseasesanddevelopmentof antimycoticagents.Then,apicture antimycotic agents (Loeffler and Stevens, 2003). This resistance
of the emerging problem of resistance is presented as it applies hassetoff aviciouscycleof intensiveantimicrobialchemother-
tocurrent-daycircumstancesintowhichchemosensitizationmay apy,emergenceofmoreresistantstrains;which,inturn,promotes
playaroleforfungalcontrol.Thesubjectofchemosensitization, emergence of more therapeutically intractable IFIs (Tseng and
itself, is presented in-depth in the Section“Chemosensitization Perfect, 2011; refs. therein). Compounding this problem is the
of Antifungal Agents.” Therein, research on various facets of sluggishpaceatwhichnewantimycoticsarebeingdiscoveredthat
chemosensitization,includingthechemicalnatureof theagents, havetherapeuticpracticality(Messeretal.,2009).
their interaction with various commercial products, and their
modeofactionorhowtheysensitizeafungalpathogen,ifknown, FUNGICIDESANDANTIMYCOTICS
iscomprehensivelycovered.Asfungaldiseasesconfrontbothagri- Following is a brief overview on the history in development of
cultureandmedicine,thisreviewwilladdressfungalcontroland antifungalagentsforuseinagricultureandmedicine.Includedis
useof chemosensitizingagentsagainstfungalpathogensinboth anintroductiontoantifungalagentsusedandoversightcommit-
fields.However,mostof theresearchonchemosensitizationhas tees that monitor emergence of resistant strains and which also
involved antifungal agents against human mycoses; which will, provideadviceontheircontrolortreatment.
thus,befeatured.
AGRICULTURALFUNGICIDES
FUNGALDISEASES Development and use of commercial antifungal agents in agri-
AGRICULTURALFUNGALDISEASES culture predated that for human medicine. One of the main
Forcenturies,successfultreatmentoffungaldiseasesofcrops,ani- concerns in development of plant fungicides was phytotoxicity,
mals,andhumanswasrelativelyintermittent.Fungaldiseasesof characteristic to many early fungicides. The earliest fungicides
crops have had historic impacts on human civilization. Notable used saltwater or lime to control wheat bunt in the seventeenth
examplesincludetherelativelyrecentGreatPotatoFamineofIre- century.Aboutacenturylatercoppersulfate,thenknownasthe
landandGreatBengalFamine(rice)duringthemidnineteenth Bordeauxmixture,wasusedtocontrolpowderymildewofgrapes.
andtwentiethcenturies,respectively.Thesefamineseachresulted Mostearlyfungicideswerebasedoninorganicminerals,includ-
inmillionsofhumandeaths.Moreover,70%ofallmajorcropdis- ingarsenicandmercury.Thesehadtobeappliedfrequentlyand
eases,tothisday,areduetofungi.Thelonglistofphytopathogenic in large amounts to be effective, presenting obvious hazards to
fungiandrelatedcropdiseases,numberinginthe100s,isbeyond both human health and to the environment. In the early 1940s,
thescopeofthisreview.Overviewsoncropfungalpathogensand new classes of fungicides, the dithiocarbamates and phthalides,
fungicidesemployedareavailable(LevetinandMcMahon,2003; werevastimprovementsovertheirpriorinorganiccounterparts.
Russell,2005;StrangeandScott,2005).However,specialmention These newer fungicides had higher fungicidal activity,were eas-
shouldbegiventothecurrentcropfungaldiseasethreateningthe ilyformulatedforapplication,andwerelessphytotoxic(Horsfall,
World.ThisisthefungalstrainUg99(TTSK)ofPucciniagraminis 1975).
tritici,thefungusthatcauseswheatstemrust.Ug99hasthepoten- The modern panoply of agricultural fungicides began to be
tialofinfecting90%oftheWorld’swheatvarieties.Ithasalready developed in the 1970s. The main ones included the ergosterol
had devastating effect on the wheat crop of EastAfrica and has biosyntheticinhibitorsbenzimidazoles,morpholines,piperazines,
spreadtoAsiaandtheMiddleEast(Singhetal.,2011). imidazoles, pyrimidines, and triazoles; the mitochondrial res-
piration inhibitors, the anilides and strobilurins (Krämer and
FUNGALDISEASESOFMEDICALIMPORTANCE Schirmer,2008;refs.therein)andosmoregulationdisruptors,the
Theimpactofhumanmycoseshasnothadthesamelevelofhis- phenylpyrroles(Kanetisetal.,2008).
toricalinfamyasthoseaffectingcrops,untilrecently.Fatalcases Incontrasttohumanpatients,cropshaveanadvantageinthat
of thrush, later identified as being caused by the yeast Candida theycanbegeneticallymodifiedforresistanceagainstfungaldis-
albicans fromthepatient’sownmicroflora,hadbeenknownfor eases (Cornelissen and Melchers,1993). But,as with fungicides,
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Campbelletal. Antifungalchemosensitization
fungal pathogens eventually can produce resistant strains, as Shortcomings of imidazole agents for systemic mycoses led
poignantly demonstrated, recently, with the Ug99 pandemic of to discovery of a second group of azoles, the triazoles. How-
wheat(Singhetal.,2008). ever, triazoles have a greater affinity for 14α-sterol demethylase
enzymethandotheimidazoles.Anadditionalcontributingfactor
ANTIMYCOTICCHEMOTHERAPY to the antifungal effect of the triazoles is aggravation of oxida-
Development of antimycotics against human mycoses lagged tivestressinthefungusthroughgreateraccumulationofmethyl
behindthatforagriculturalfungicidesandeventothatofantibi- sterols.Accumulationofthesesterolssensitizesfungalcellstothe
oticstargetingbacteria.Thisdelaywasbornebythefactthat,unlike oxidativeburstfromphagocyticcells(ShimokawaandNakayama,
bacteria,fungiareeukaryotesandhaveacloserevolutionaryaffil- 1992).
iationtohumans.Assuch,theypossessbiochemical,genetic,and However, toxicity of triazoles to the patient can result from
cellularbiologicaltraitsmorecommontohumansthandobacte- cross-reactivitywithseveralhumancytochromeP450-dependent
ria.Hence,findingsuitabletargetsforfungalcontrolthatdidnot enzymes (Thompson et al., 2009). There is a long list of other
present a risk to the human host has been a more complicated drugs posing a risk of exacerbating negative interactions, if co-
endeavorthanthatforantibacterialagents. administered with triazoles. The current clinically approved tri-
Itwasnotuntilthe1950sthatthefirstfungus-specificantimi- azoles include itraconazole, the first of this class approved for
crobials were discovered, some two decades after the discovery clinical use, followed by fluconazole (FLU), perhaps the most
of penicillin. These first antimycotics included griseofulvin and widelyused,andthree“secondgeneration”triazoles,voriconazole,
nystatin (Odds,2003a). Griseofulvin,a polyketide isolated from posaconazole,andravuconazole(Odds,2003a).
Penicillium griseofulvum, has limited application and is mainly Themostrecentclassofcommerciallyavailablesystemicanti-
usedagainstdermatophytosis.Itsmodeofactionisbydisrupting fungal agents is the echinocandins. This class of antimycotics
mitoticspindlemicrotubulesinfungi,preventingcellulardivision inhibitssynthesisof β-1,3-glucan,anessentialconstituentof the
(Pandaetal.,2005). fungalcellwallandnotfoundinhumans.Lackofthispolysaccha-
Thevastmajorityofantimycoticdrugsdiscoveredsincegrise- rideunderminescellwallintegrityrenderingthecellvulnerableto
ofulvin disrupt fungal cell membrane integrity, in one way or osmoticstressandcelllysis.Thefirstechinocandinapprovedfor
another.Thefirstof thesetobediscoveredwasnystatin,apoly- clinical use was caspofungin, discovered in 2000 (Onishi et al.,
eneantimycoticisolatedfromaStreptomycesbacterium.Nystatin 2000). Additional echinocandins approved for therapeutic use
forms hydrogen bonds with ergosterol, a fungus-specific sterol, includemicafunginandanidulafungin.Thisclassofantimycotics
creatingporesintheplasmamembranewithconcomitantcellular hasreportedlyfewersideeffectsthanthepolyenesandazoles,and
leakage(Cassetal.,1970).Nystatinhasabroaderrangeof anti- areconsideredtobewelltoleratedbypatients.Whileshowingeffi-
fungalactivitythangriseofulvin,butbecauseofitsnephrotoxicity cacyagainstCandidaspp.andAspergillusspp.,theechinocandins
it is limited to topical or oral administration, for treatment of havelowefficacyagainstcryptococci(Eschenaueretal.,2007).
dermatophyticandoropharyngealinfections.
Within a few years another antimycotic polyene was isolated RESISTANCE
fromStreptomycesnodosus,amphotericinB(AMB),havingmuch Developmentofresistancetocurrentlyavailable,commercialanti-
greater antifungal activity than nystatin,which is a mainstay of fungalagentsis,perhaps,themajorproblemconfrontingtheiruse
antifungal chemotherapy to this day. Like nystatin, AMB binds inbothagricultureandmedicine.Asecondcommonissueisthe
to ergosterol (Dutcher,1968). However,it can,to a much lesser inherenttoxicitythatantifungalagentspresenttohumanhealth,
degree, bind to other sterols, such as cholesterol, a component andothernon-targetorganismsintheenvironment.Ofparticular
of mammalian cell membranes,and its use has been associated concernisthepossibilitythatfungicidesappliedintheenviron-
with significant side effects,including kidney failure and death. mentforcontrolofagriculturalfungalpathogensmaycontribute
New formulations of AMB as a lipid or a cholesteryl sulfate to development of cross-resistance by human infectious fungi.
complexorinliposomalform(L-AMB)havebeensuccessfulin Thismayespeciallybethecasewithtriazoles,aclassofantifungal
reducing these side effects and improving patient tolerance to agentsusedbothinagricultureandinmedicine(Sneldersetal.,
this drug (Thompson et al., 2009). AMB was the primary drug 2009).
used against IFIs until the advent of the triazoles (Walsh et al., Despitesuccessindiscoveryof newtherapeuticantimycotics,
2008). development of resistance remains the main clinical problem in
Theazoleswerethenextclassofclinicallyacceptableantifungal treatment of IFIs. Resistant strains of yeast and filamentous IFI
agents,firstappearingin1969.Theprimarymodeofactionofall agents have been found for all major classes of antimycotics,
azolesispreventionofergosterolbiosynthesis.Azolesinhibitafun- includingthemostrecentechinocandins,withtheirnovelmode
galenzyme,14α-steroldemethylase(cytochromeP450 ),thatis ofaction.Asaresult,clinicalfailuresinantimycoticchemotherapy
DM
requiredfordemethylationoflanosteroltoergosterol(Maertens, areanincreasinglyvexingandworrisomeproblem.
2004).Theimidazoles(e.g.,miconazole,bifonazole,clotrimazole, Chemosensitization has the potential to fundamentally aug-
ketoconazole,etc.),werethefirstazolestobeused,mainlyintopi- ment efficacy of antifungal agents. But, perhaps its greatest
calointments.Oralorsystemicuseofimidazoleswasunacceptable promise is in subverting resistance. In order to understand this
inviewofhepatotoxicityanderraticeffectiveness.Althoughketo- capability,abriefoverviewisprovidedonthemechanismsofresis-
conazole was eventually approved for systemic use in 1981,it is tanceandhowsignificantofaconcernresistancehasbecomefor
noteffectiveagainstdeep-seatedmycoses. thecontrolofbothagriculturalandmedicalpathogenicfungi.
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Campbelletal. Antifungalchemosensitization
RESISTANCETOAGRICULTURALFUNGICIDES echinocandindrugsavailable,developmentofresistantstrainsto
Currently, there are over 150 different agricultural fungicides at least any one of these drugs has resulted in clinical failures
in use, worldwide (Phillips McDougall, 2006). Development of incryptococcosis,candidiasisoraspergillosis(TsengandPerfect,
widespreadfungicideresistanceincroppathogenshasbeenacon- 2011). Resistance to these drugs may reflect the dearth of tar-
tinuous,fundamental problem,especially over the past 35years. gets,suchasergosterol-bindingorinhibitionofsingularenzymes
Recognition of this problem reached such significance that an involved in ergosterol or β-1,3-glucan biosynthesis,respectively.
international consortium of fungicide manufacturers,academic, With the current set of clinical antimycotics the overall target
and government scientists and crop advisors, was organized in involvesmembrane/cellwallintegrity.Anotherclass,represented
1994astheFungicideResistanceActionCommittee(FRAC).Pub- byflucytosine,disruptstranscription/translationprocessesinfun-
lication of an overview of fungicide resistance in crop systems, galcellsbyinterruptinguracilproduction(Vermesetal.,2000).
worldwide,shortlyfollowed(Brent,1995).FRAChassincepub- However, because of the propensity for rapid development of
lished a number of monographs,the latest update published in resistance against flucytosine,when used alone,it is often com-
2007(BrentandHollomon,2007).Fungicideresistancemanage- binedwithotherantimycotics,suchasAMBagainstcryptococcal
menthasbecomeamainstayinpre-andpost-harvestagriculture meningitis(Hopeetal.,2004;Paponetal.,2007).
(Försteretal.,2007). The mechanisms of resistance of human and crop fungal
Today,resistantstrainsofcropfungalpathogenscanbefound pathogensaresimilar.Antifungaldrugresistanceisdividedinto
for just about all classes of agricultural fungicides. The type of twocategories,“microbiological”and“clinical”(KanafaniandPer-
resistanceandthedegreetowhichithasspreadgreatlydepends fect, 2008). Microbiological resistance is when a strain is not
onseveralfactorsincludingmodeofaction,exclusivityofusage, susceptible to a dosage above an established breakpoint level,
geographicexpanseof application,singlegene-target,reproduc- based on standardized laboratory protocols, e.g., CLSI (2008a)
tiverateofthepathogen,andcross-resistancewithintheclassof andisdividedintocategories,“primary”and“secondary.”These
fungicide.Thepotentialinteractionofthesefactorsinpromoting categories are analogous to“quantitative”and“qualitative”used
resistancerequiresavigilanceandcommunication;whichisgen- todescriberesistanceincropfungalpathogens.Inprimaryresis-
erally coalesced into an“action”compendium by FRAC. FRAC tance,thepathogenisinnatelyresistantwithoutanypriorexposure
has classified the major fungicides into various risk categories tothedrug.AnexampleisresistanceofCryptococcusneoformans
with the benzimidazoles,dicarboximides,phenylamides and the toechinocandins.Incontrast,secondaryresistancedevelopsafter
strobilurins,afairlynewclassoffungicides,relegatedtothe“high- repeatedexposuretoadrug,creatingselection-pressureforgenetic
risk”category(MortonandStaub,2008).Cross-resistancebetween changesinthepathogen,suchasup-regulationof effluxpumps
fungicidesmaynowextendintodevelopmentofresistanceinfungi (Niimietal.,2004;Cannonetal.,2009),sitemutationsintarget
of medical importance from exposure to agricultural triazoles genes, gene duplication, etc. Secondary resistance can be found
(Deisingetal.,2008;Sneldersetal.,2009). extensivelyamongclinicalstrainsandspeciesofCandida.Exam-
A prevalent mechanism by which fungi develop resistance is plesincludeFLUresistanceowingtoenhancedexpressionofgenes
calledalteredtargetsiteorqualitativeresistance.Thiscanbeasin- codingeffluxpumps(CDR1andCDR2),orlanosteroldemethy-
glepointmutationinoneaminoacidinafungaltargetprotein. lation to ergosterol (ERG11; Shen et al., 2010) or resistance to
Generally,this type of resistance renders the fungus fundamen- echinocandinsbymutationintheβ-1,3-glucanbiosyntheticgene
tally resistant, reflected by ineffectiveness no matter the rate of FKS1(Ben-Amietal.,2011).Insomecases,mechanismsofdrug
application of the fungicide. Qualitative resistance has played a resistance can involve multiple genes,as in certain strains of A.
significantroleinlimitingefficacyoffungicidestargetingvarious fumigatusshowingpan-resistancetoazoles(Campsetal.,2012).
components of mitochondrial respiratory complexes, known as Clinicalresistancebasicallyentailscaseswhere invitro assays
quinoneoutsideinhibitors(Q Is),suchasthestrobilurins.Resis- failtomatchinvivoresults.Thisoccurswhenthereisadiscrep-
O
tance to QoIs may have developed rapidly because their target ancybetweenlaboratorydeterminationspredictingdrugefficacy,
proteinsareencodedonmitochondrialDNA,and,thus,maynot based on minimum inhibitory concentrations (MICs) accord-
havetheDNArepaircapabilityequaltothatfoundinnuclearDNA ing to standardized protocols (e.g., CLSI, 2008a) and failure of
(Gisietal.,2002).Othertypesofresistanceincludeincreasedefflux drugtreatmentunderclinicalconditions.Thefactorsinvolvedin
transporteractivity,alteredmembranestructurereducingfungi- clinical resistance are responsible for the majority of drug fail-
cideabsorption,fungicidedegradationorover-expressionofthe uresinIFItherapy(Anderson,2005;KanafaniandPerfect,2008).
targetgene(DelSorboetal.,2000).Theselattermechanismscan These factors may entail misdiagnosis, drug pharmacokinetics,
bepossiblyovercomebyincreasedfungicideapplicationrates,and host/pathogen interactions, the infection site, fungal burden,
arereferredtoasquantitativeresistance(Deisingetal.,2008). interactionswithotherinfectingagents,immunocompetence,etc.
RESISTANCETOCHEMOTHERAPEUTICANTIMYCOTICS REMEDIATIONSTRATEGIES:MANAGINGRESISTANCETOANTIFUNGAL
The rapid development of resistance to clinical antimycotics is AGENTS
a worrisome predicament for effective treatment of IFIs (Nucci There are similarities in strategies in agricultural and medicine
and Perfect, 2008). There have already been over a half million tocircumventemergingresistancetoantifungalagents.However,
deathsfromcryptococcosisinsub-SaharanAfrica,alone,withan therearealsosomevastdifferences,mainlyaresultofconstraints
estimated >600,000 deaths per year from cryptococcal menin- placedonthelatitudeinriskinghumanhealth;althoughenviron-
gitis, worldwide (Park et al., 2009). Of the polyene, triazole, or mentalrisksassociatedwithfungicidalusageareaconcern.Infact,
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Campbelletal. Antifungalchemosensitization
theoriginalinorganicagriculturalfungicides(copperandsulfur drugs.ArecentexampleiswhereMICsweredeterminedforthe
based) are still used. However,overcoming antifungal resistance azoles,FLU,voriconazoleandposaconazoleforalmost1000strains
by chemosensitizing agents could benefit both agriculture and fromaglobalcollectionof C.neoformans (Pfalleretal.,2011b).
medicine, discussed further in the Section“Chemosensitization This survey provided a means of detecting emerging resistance
ofAntifungalAgents.” in several global regions. Establishment of clinical susceptibil-
ity breakpoints provides reasonable predictors of failure of any
Managingresistancetoagriculturalfungicides particularantimycotic.Inthisstudy,noclinicalbreakpointshad
Management strategies and guidelines employed for resistance been established for any of these drugs against C. neoformans,
to agricultural fungicides are evaluated regularly and published yetthesurveyprovidedepidemiologiccut-off valuesforeachof
in monograph form under the aegis of FRAC (Brent and Hol- the drugs based on strains having MICs departing greatly from
lomon,2007).Successfulmanagementof resistanceincropfun- overallmodalMICsofwild-typestrains.Forexample,theoverall
galpathogensgenerallynecessitateslarge-scalecooperationover modalMICofFLUagainstC.neoformans,inworldwidesurveys,
broadareas;whichrequiresagreementandcommitmentbyboth is4μgmL−1.Whereas,MICs>64μgmL−1 of strainsinSouth-
fungicidemanufacturersandfarmers.Strategiesincludethefol- eastAsiaandAfrica(Chandenieretal.,2004;Bicanicetal.,2007)
lowing:(1)Combineapplicationofanddiversifyfungicides(thisis reflectedclinicalfailuresusingFLU.
analogoustocombinationtherapyinclinicalpractice,seebelow), Combinationtherapyinvolvesuseoftwoormoreantimycotics,
or rotate use, so that more than one target in the fungus is concurrentlyorsequentially(BaddleyandPappas,2005).Suchan
beingaffected;(2)Restrictapplicationtocriticalperiodstoreduce approachmaybeeffectiveintwoways.Oneisthroughsynergis-
selection-pressurefordevelopingresistance;(3)Donotexceedrec- ticinteraction,whereantifungalefficacyisincreasedwhendrugs
ommendedrates;(4)Iftheresistantfungalpathogenisalreadywell arecombined.Theotherisimpedingdevelopmentof resistance
established,avoidapplyingtoeradicate,butuseprophylacticlevels because multiple genetic targets in the fungus are being over-
tokeeppopulationslow;(5)Withdrawineffectivefungicidesfrom whelmed. However,the practicality of combination therapy has
themarket;and(6)Developnewfungicides.Itisthislatterfactor hadmixedresults.Laboratoryassays(invitro)haveshownpos-
thatagriculturehasanadvantageoverthatofmedicineinamelio- itive or synergistic interactions between combinations of many
rating fungicide resistance. The multitude of available fungicide commercialantimycotics(Cuenca-Estrella,2004;Chaturvedietal.,
classes, and actual number of agricultural/horticultural fungi- 2011).Combinationsofallthreerepresentativesofthe“modern”
cides,eclipsethenumberofdrugsavailableforhumanantimycotic classesofsystemicantimycotics,AMB,voriconazoleandcaspofun-
chemotherapy.Thus,thereisagreaterprobabilityformaintain- gin,showedhighlysynergisticantifungalactivity,invitro,against
ingandimprovingtheefficacyoffungicidesusedforagriculture, aspergillosisagents,A.fumigatus,A.terreus andA.flavus.How-
through altering chemical structure, without the limitations of ever,theinteractionbecameantagonisticathigherconcentrations
causingserioussideeffects,asinthemedicalarena.Moreover,in of AMB or voriconazole (O’Shaughnessy et al., 2006). Another
agriculture,the fungal pathogen only need be“controlled”dur- example is where combinations of azole and one or two of the
ingcropgrowthorstorage,andnotcompletely“eradicated,”asis caspofunginantimycoticsaresynergistic,invitro,againstclinical
generallyrequiredincasesofhumanmycoses. strainsofCandidaandCryptococcus(Rolingetal.,2002).However,
whenadministeredclinically(invivo),thebenefitsofcombination
Managingresistancetoantimycoticdrugs therapy may not always be realized as there may not be greater
Managing antimycotic resistance involves a number of factors. antimycoticefficacyandtheremaybeaworseningofnegativeside
Theseincludeimprovinghospitalsanitation(Benetetal.,2007), effects(Steinbachetal.,2011).
surveillance for resistance, changing antimycotic, combination New antifungal agents, beyond the more recently available
therapyanddevelopmentofnewantimycoticdrugsortherapeutic azolesandechinocandins,aredesperatelyneededasthereisemerg-
strategies(e.g.,chemosensitization). ingresistanceandsomestrainsshowinginnaterefractorinessto
Vigilancewithregardtoemergingresistanceinbothfilamen- all of them. Optimally, such new agents should target different
tous fungi andYeast is,today,performed by either of two stan- systems within the fungus than those being targeted currently.
dardized laboratory protocols for determining respective MICs, Such new targets could include the calcineurin pathway, β-1,6-
as proposed by CLSI (2008a,b) and the European Committee glucanbiosynthesis,mitochondrialrespiration,biofilmformation
on Antimicrobial Susceptibility Testing (EUCAST, 2008a,b). A orgenesspecificallycontrollingfungalphysiologicalevents,such
recent side-by-side comparison of each committee’s respective assporeformation,filamentation,etc.(Espinel-Ingroff,2009).
assaymethodsforMICsofselectedtriazolesforstrainsandspecies However, it is within the concept of “combination therapy”
ofCandida(>1000clinicalisolates)andforAspergillus(245clini- or“newantifungalagents”thatchemosensitizationmayhaveits
calisolates)found>97%(Pfalleretal.,2011c)and>98%(Pfaller greatest utility. The remainder of this review is devoted to the
etal.,2011a)concordancebetweenthetwoprotocols,respectively. research on safe, chemosensitizing agents that could improve
Surveysofglobalclinicalcollectionstodetectpotentiallyresis- antifungal chemotherapy (medicine) or phytopathogenic fungal
tantstrainsandspeciescanprovideinsightsonhowtoapproach control(agriculture).
antimycoticchemotherapyinagivenglobalregion.Thisapproach
iscurrentlyperformedbytheARTEMISAntifungalSurveillance CHEMOSENSITIZATIONOFANTIFUNGALAGENTS
Program(seePfalleretal.,2005).Suchglobalsurveysprovideuse- Agrowingnumberofpapershavebeguntoappearoverthepast
ful information with regard to potential efficacy of antimycotic decadeshowingthatcertainnaturalproducts,relativelynon-toxic
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Campbelletal. Antifungalchemosensitization
to humans, increase antifungal activity when co-administered are phytotoxic and, thus, are of limited utility in crop systems
with a commercial antifungal agent. Many of these products other than, perhaps, as promising candidates as chemosensitiz-
(“chemosensitizers”)areknownconstituentsof commonlyused ersforantimycoticdrugs(seebelow).Fewhavebeenassessedas
herbsandspices.Becausetheseherbsandspiceshavebeenpartof chemosensitizingagentstoagriculturalfungicides.
thehumanculinaryregimenforcenturies,thereisapresumption Cinnamaldehyde is perhaps the most widely known of plant
that the constituents of these plants are probably relatively safe. natural products used as a fungicide against plant, or plant-
Moreover, various synthetic products have been identified hav- productfungalpathogens(CoppingandDuke,2007).Itsmodeof
ingasynergisticinteractionwithcommercialantimycotics,while actionwasproposedtobesimilartothatoftheechinocandins,in
notpossessingsignificantantifungalactivityalone.Themodeof thatitinhibitedβ-1,3-glucansynthase(Bangetal.,2000).Alone,
actionofthesenaturalandsyntheticchemosensitizerstoincrease cinnamaldehyde has only nominal antifungal activity. However,
antimycoticactivityisnotalwaysunderstood.However,research cinnamaldehyde in combination with other plant natural prod-
isbeginningtoshowthatmanyofthesecompoundsinterferewith ucts,the phenolics eugenol,quercetin,and catechin,resulted,in
theabilityoffungitorespondtostress.Suchstressmaybefroman somecases,ina>100-foldsynergisticantifungalactivityagainst
environmentalsource(e.g.,uv,heat,drought,etc.)orfromacom- wood-decayingfungi(YenandChang,2008).Itwasconcludedthat
mercialantifungalagent.Othermodesof actionof chemosensi- thecombinationoffungalcellwalldegradation,andtheassociated
tizersincludedisruptionoffungalmembraneintegrity,inhibition osmoticstress(cinnamaldehyde),withtheadditionofanoxidative
of effluxpumps,orinductionof oxidativestress.Acomprehen- stressagent(thephenolics)resultedinsynergisticantifungalactiv-
sivelistingofresearchpapersonchemosensitizationofantifungal ity.Combinationofcinnamaldehydeandanotherplantphenolic,
agents,typesofcompoundstestedandmodesofaction,ifknown, octylgallate,also showed a promising level of antifungal activity
isavailableinTable1. againstwood-decayingfungi(Hsuetal.,2007);themodeofaction
Interactions between antifungal compounds are usually first ofoctylgallateproposedtoresultfromcellmembranesurfactant
determined,invitro,bymicrodilutioncheckerboardassayseither properties(Kuboetal.,2001).
using CLSI (2008a,b) or EUCAST (2008a,b) protocols. Syner- With regard to actual synergism of commercial fungicides,
gistic, additive or antagonistic interactions are defined by com- octylgallate was found to synergize antifungal activity of flu-
paring the lowest concentration of agent needed to inhibit fun- dioxonil and a strobilurin, kresoxim-methyl, against the apple
gal growth, MICs, after test agents are combined, to MICs of pathogenPenicilliumexpansum,invitro.However,basedonhyper-
the agents, alone. These interactions are recorded as Fractional sensitivity of singular gene deletion mutants of Saccharomyces
Inhibitory Concentration Indices (FICI), where: FICI=MIC of cerevisiae,asamodelfungus,itwasconcludedthatoctylgallatedis-
compound A in combination with compound B/MIC of com- ruptedtheoxidativestressresponsesystem,debilitatingthefungus
pound A, alone)+(MIC of compound B in combination with furthertotheoxidativestressintroducedbythefungicides(Kim
compound A/MIC of compound B, alone). Compound inter- et al., 2010b). In addition, the combination of octylgallate and
actions are defined as follows: synergistic (FICI≤0.5), additive fludioxonilrenderedP.expansum fludioxonil-resistantstrainsto
(0.5<FICI≤1),neutral(1<FICI≤2)orantagonistic(FICI>2; becomesusceptible.
Isenberg,1992),orthemoreguardedinterpretationof FICIval- Further research on chemosensitization of crop fungal
uesof≤0.5assynergy,and>0.5–4asindifference(Odds,2003b). pathogenstocommercialfungicideshasmainlyinvolvedA.flavus.
Interactionscanalsobecalculatedaccordingtofungicidalactivity A. flavus is not a plant pathogen, per se, but an opportunistic
asMinimumFungicidalConcentration(MFC)asthelowestcon- fungus, generally infecting a variety of crops or crop-products
centration of agent where ≥99.9% fungal death is achieved and under some form of environmental stress (e.g., drought, heat,
fractionalfungicidalinteractionindices(FFCIs)arecalculatedas insect damage). The main concern of infections of crops by A.
forFICIs. flavusiscontaminationbyaflatoxin,ahighlycarcinogenicmyco-
toxin (Yu et al., 2007). In an effort to develop methods for
CHEMOSENSITIZATIONOFAGRICULTURALFUNGICIDES controllingA. flavus,focus was placed on various natural prod-
Compared to the amount of research on chemosensitization of ucts as chemosensitizing agents to commercial fungicides. One
antifungaldrugs,therehasbeenrelativelylittleresearchontheir ofthefirsteffortsfoundthatthephenolic,2,5-dihydroxybenzoic
usewithagriculturalfungicides.Thisisquiteincontrasttothat acid,disruptedglutathionehomeostasis,significantlyenhancing,
oftheresearchondiscoveryofnaturalfungicides,ofwhichthere by almost 100-fold, the antifungal activity of fludioxonil (Kim
are many. However, the source of almost all of the widely used etal.,2007b).Additionalnaturalcompoundswerealsofoundto
commercialfungicidesforagricultureismainlybacterial,partic- enhance activity of commercial fungicides. These included the
ularly species of Streptomyces (Copping and Duke, 2007). Such alkaloid,berberine,andseveralphenoliccompounds,enhancing
fungicides include the polyoxins, kasugamycin, and more. The antifungalactivityoffludioxonilandstrobilurinagainstA.flavus
strobilurins,alreadydiscussed,arenaturalproductsfromthefun- by further disrupting its oxidative stress response system (Kim
gus, Strobilurus tenacellus. While these compounds have potent etal.,2007a).Alsotestedwere2,3-dihydroxybenzaldehyde,salicy-
antifungalactivitytheyalsopossesshighmammaliantoxicity. laldehydeandotherbenzoanalogs.Thesecompoundsenhanced
Plant natural products have also been used for controlling activity of strobilurins and antimycinA against A. flavus and P.
plant fungal pathogens. However, these compounds, in general, expansumbydisruptingtheHOG1signalingpathway,whichcon-
donotexhibitantifungalactivitiessufficientforwidecommercial trolsosmoticandoxidativestressresponsesinfungi(Kimetal.,
acceptance. Moreover, many of these plant-derived compounds 2008b,2011).
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Campbelletal. Antifungalchemosensitization
Bidgoli ntinued)
1) 1) n Co
9 9 a (
oticfungi. Reference YenandChang(2008) Hsuetal.(2007) Kimetal.(2010b) Kimetal.(2007b) Kimetal.(2007a) Kimetal.(2008b,2011) Raoetal.(2010) KuboandHimejima(19 KuboandHimejima(19 LeeandKim(1999) ShinandKang(2003) ShinandPyun(2004) MahboubiandGhazi (2010) Sureshetal.(1997) Giordanietal.(2001) Shin(2003) Rosatoetal.(2009) Rosatoetal.(2009) Rosatoetal.(2009) Giordanietal.(2004) Giordanietal.(2006) Zoreetal.(2011) Shreazetal.(2011b) Shreazetal.(2011a) Fariaetal.(2011)
c
y
ulturalphytopathogenicand/orhumanm Interaction/target Synergistic/cellmembrane Synergistic/cellmembrane Synergistic,overcomeresistance/oxidative stress Synergistic/glutathionehomeostasis Synergistic/oxidativestress Synergistic/HOG1signalingpathway, oxidative/osmoticstress Synergistic/ionhomeostasis Synergistic Antagonistic Synergistic Synergistic Antagonistic Synergistic Synergistic Synergistic Additiveandsynergistic Synergistic Synergistic Neutral Synergistic Synergistic Synergistic+Synergistic,overcomeresistance/H-efflux Synergistic,overcomeresistance/ATPase protonpump Synergistic
c
gri s
a u
ntifungalagentsagainst Targetfungus Wood-decaying Wood-decaying Penicilliumexpansum, Saccharomycescerevisiae Aspergillusflavus A.flavus A.flavus,P.expansum Candidaalbicans C.albicans C.albicans C.albicans Blastoschizomycescapitat C.albicans C.albicans,A.niger C.albicans C.albicans Aspergillus C.albicans C.albicans C.albicans C.albicans C.albicans C.albicans Candida Candida Cryptococcusneoformans
a
al
ci
er
m
Table1|Chemosensitizingagentstestedincombinationwithcom ChemosensitizerAntifungalagent AGRICULTURE CinnamaldehydeEugenol,quercetin,catechin CinnamaldehydeOctylgallate OctylgallateStrobilurin,fludioxonil 2,5-dihydroxybenzoicacidFludioxonil BerberineFludioxonil,strobilurin 2,3-dihydroxybenzaldehyde,Strobilurin,antimycinA salicylaldehyde,otherbenzoanalogs MEDICINE Plantessentialoils,extracts,andconstituents Carvacrol,essentialoilofOriganumAzoles(theorized) vulgare AnetholePolygodial AnetholeAmphotericinB AnetholeMiconazole,amphotericinB EstragoleAgastacherugosaessentialKetoconazole oil EstragoleAmphotericinBα-Pinene,1,8-cineole,essentialoilofAmphotericinB myrtle EssentialoilSantolinaClotrimazole chamaecyparissus/mainlyterpenoids LatexofEuphorbiacharaciasKetoconazole EssentialoilofPelargoniumAmphotericinB,ketoconazole graveolens,mainlygeraniolcitronellol EssentialoilofPelargoniumgraveolensNystatin EssentialoilofOriganumvulgareNystatin EssentialoilofMelaleucaalternifoliaNystatin EssentialoilofThymusvulgarisAmphotericinB EssentialoilofCinnamomumcassia,AmphotericinB cinnamaldehyde Linalool,benzylbenzoate,eugenolFluconazole CinnamaldehydeanalogsFluconazole AnisaldehydeanalogsFluconazole CinnamicacidItraconazole
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Campbelletal. Antifungalchemosensitization
Reference Ahmadetal.(2010a,b) Bragaetal.(2007) Kimetal.(2008a,b) Guoetal.(2009),Bietal.(2010), Fariaetal.(2011) Amberetal.(2010) Hornbyetal.(2001),Ohetal. (2001),HornbyandNickerson (2004),Jabra-Rizketal.(2006), Shirtliffetal.(2009),Yuetal.(2012) Sunetal.(2009a,b),Changetal. (2011) Jacobetal.(2003),Lietal.(2006), Digirolamoetal.(2009) Johannetal.(2010) Andrewsetal.(1977),Beggsetal. (1978a,b),Beggs(1983) Andrewsetal.(1979) Strippolietal.(2000),D’Auriaetal. (2001) Xuetal.(2006) Kimetal.(2010a) Parketal.(2006,2011) HirasawaandTakada(2004) Navarro-Martinezetal.(2006),Han (2007a) Han(2007b) Sharmaetal.(2010a,b)
g B) g
n M n
Interaction/target Synergistic/ATPaseprotonpump Synergistic/plasmamembrane Synergistic,additive,antagonisticdependi uponstrains/MAPKsignalingpathway; oxidative/osmoticstressresponse Synergistic/drugefflux,ironuptake, ribosomebiogenesis Synergistic/chitinsynthase Synergistic/biofilmformation,oxidative stress,ergosterolbiosynthesis Synergistic/effluxpump,ergosterol biosynthesis,oxidativestress Synergistic/drugefflux Synergistic(itraconazole),antagonistic(A Enhancement,synergistic/stabilizationof polyenes Synergistic Enhancement/cellmembrane,ergosterol biosynthesis Synergistic,additive,antagonisticdependi ondrugandstrain Synergistic/oxidativestresssystem Synergistic,overcomeresistance Synergistic Synergistic,prolongedsurvivalofBALB/c mice/folicacidmetabolism Synergistic,prolongedsurvivalofBALB/c mice/folicacidmetabolism Highlysynergistic/oxidativestresssystem
Targetfungus Candida C.albicans A.fumigatus,A.terreus, A.flavus C.albicans,C.neoformans, S.cerevisiae Candida C.albicans,C.dubliniensis C.albicans S.cerevisiae(overexpressing C.albicansCDR1orMDR1) Paracoccidioidesbrasiliensis Candida Candida C.albicans Fusariumandother filamentousfungi A.flavus,A.fumigatus, A.terreus Trichophyton,Candida C.albicans C.albicans C.albicans C.albicans
Antifungalagent Fluconazole Combinedtogether Ketoconazole,fluconazole, amphotericinB Fluconazole,itraconazole, amphotericinB Fluconazole,ketoconazole Fluconazole Fluconazole,ketoconazole, itraconazole Fluconazole Itraconazole,amphotericinB2s(EGCG),andpolyphenols AmphotericinB AmphotericinB imidazoles,triazoles AmphotericinB,terbinafine, butenafine,ketoconazole AmphotericinB,fluconazole, ketoconazole Fluconazole,flucytosine AmphotericinB,fluconazole AmphotericinB AmphotericinB Fluconazole,ketoconazole, miconazole,itraconazole, voriconazole,nystatin, amphotericinB
e
at
ed all
Table1|Continued Chemosensitizer Simpleterpenoids Thymol,eugenol,methyleugenol Thymol,eugenol Thymol Thymol EssentialoilofOcimumsanctum Sesqui-andtriterpenoids Farnesol RetigericacidB 1ECTA,capisteroneAandB,sulfat triterpenoids Schinol Teaextracts,epigallocatechins,g Propylgallate 3L-DOPA,dopamine Propylgallate Propylgallate Octylgallate EGCG EGCG EGCG Grapeseedextract(polyphenols) Curcumin
FrontiersinMicrobiology|FungiandTheirInteractions February2012|Volume3|Article79|8
Campbelletal. Antifungalchemosensitization
Shenetal.(1996) PyunandShin(2006) Ogitaetal.(2006,2010),Borjihanetal. (2009),Yuetal.(2010) Guoetal.(2010) HanandLee(2005) Quanetal.(2006) Kimetal.(2007a),Xuetal.(2009), Iwasakietal.(2010),Leietal.(2011) Afeltraetal.(2004) Guoetal.(2008),Gamarraetal.(2010) Cernickaetal.(2007),Batovaetal. (2010),Culakovaetal.(2012) BulatovaandDarwish(2008) Zaremberetal.(2009) Zaremberetal.(2009) Yuetal.(2011) Cowen(2009),Cowenetal.(2009), LaFayetteetal.(2010),Singhetal. (2009) Ogitaetal.(2007),Yutanietal.(2011b) HarrisandCoote(2010) Chabrier-Roselloetal.(2008),Mizuno etal.(2011),Snelletal.(2011) Dr.MaurizioDelPoeta(personalcom- munication)
y
a
Synergistic Synergistic Synergistic/vacuolarmembrane,sulfur aminoacidmetabolism,oxidativestress Synergistic,invitroandinvivo(BALB/c mice) Synergistic,invitroandinvivo(mouse) Synergistic Synergistic(exceptitraconazole, antagonistic)/Mn-SOD Synergistic/sodiumiontransport Highlysynergisticagainstresistant+2strains,only/Cahomeostasis Synergistic/oxidativestress Synergistic Synergistic/iron-chelation Synergistic/iron-chelation Synergistic Synergistic/Hsp90inhibition,PKCpathw Synergistic/plasmamembrane,vacuolar membrane,oxidativestress Synergisticinvitroonly Synergistic/oxidativestress Synergistic/fungalsphingolipidpathway
nt,
a
C.neoformans Trichophyton S.cerevisiae,C.albicans C.albicans Candida,C.albicans C.albicans A.fumigatus,C.albicans A.fumigatus C.albicans S.cerevisiaepdr3-9mut C.albicans,A.niger C.albicans A.fumigatus A.fumigatus C.albicans A.fumigatus,A.terreus, C.albicans S.cerevisiae,C.albicans C.albicans,C.glabrata C.albicans C.neoformans ormationisavailable.
nf
i
ole, here
z w
a
Non-phenolicnaturalproducts DiallyltrisulfideAmphotericinB EssentialoilofAlliumsativum(garlic)Ketoconazole AllicinAmphotericinB AllicinFluconazole BerberineAmphotericinB BerberineFluconazole BerberineStrobilurin,fludioxonil,flucon itraconazole Syntheticcompounds AmiodaroneItraconazole AmiodaroneFluconazole,itraconazole, voriconazole4Fluconazole,itraconazoleCTBT Gemfibrozil,quinine,chlorpromazineFluconazole Ciclopirox,deferiproneKetoconazole LactoferrinAmphotericinB TriclosanFluconazole GeldanamycinandsyntheticanalogsFluconazole AlkylguanidineanalogsAmphotericinB DermaseptinS3(1–16),ranalexinCaspofungin,anidulafungin PorphyrinTMP-1363andotherAzoleantimycotics photodynamictherapeuticagents5BHBMFluconazole Typeofinteractionandtargetofchemosensitizerinthefungusareprovided,ααβ19,11-epoxycholest-7-ene-3,5,6,19-tetrol6-acetate. 2epigallocatechingallate.β3L--3,4-dihydroxyphenylalanine. 47-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine. (cid:4)5N-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide.
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Campbelletal. Antifungalchemosensitization
Interestingly, gallic and caffeic acids are potent inhibitors of Moreover,many of the individual compounds of these extracts,
aflatoxinbiosynthesis(MahoneyandMolyneux,2004;Kimetal., suchasthymol,eugenol,andtheirO-methylderivatives,possess
2008c).Basedonmicroarrayanalysisitwasfoundthatonlyafew nominal antifungal activity (Fontenelle et al., 2011). The steam
genesofA.flavuswereupregulatedwhentreatedwithcaffeicacid; distillateofThymuspulegioides,alsoamemberofthemintfamily,
themostprominentonesbeingperoxiredoxingenes.Thesegenes containing mainly carvacrol and thymol,had considerable anti-
encode enzymes that reduce endogenous peroxides,but also act fungalactivityagainstavarietyofstrainsofdermatophyticfungi,
inthesignalingpathwayoftheoxidativestressresponse.Further CandidaandAspergillus.AswiththesteamdistillateofL.viridis,
studiesonphenotypicresponsesofgenedeletionmutantsshowed thisoilcreatedlesionsinthefungalcellmembraneandreduced
thatantioxidants,suchasgallicorcaffeicacid,specificallytarget ergosterolcontent(Pintoetal.,2006).Ithasbeenproposedthat
theantioxidativestressresponsesystemofA.flavus,shuttingdown antifungalactivityofcarvacrolfollowedasimilarpatternasthatfor
theaflatoxinbiosyntheticpathway(Kimetal.,2005,2008c).Col- thymol,creationofcellularmembranelesions(Pintoetal.,2009).
lectively,theseresultsindicatethatthechemosensitizingmodeof However,othermodesofactionhavebeenproposed.Antifungal
actionofredox-activephenoliccompoundsisassociatedwiththe activityofcarvacrol,thymolandeugenolwereshowntobedirectly
fungalantioxidationsystem. correlatedtoamplitudeofCa2+burstscoupledwithup-regulation
All of the aforementioned studies were conducted in vitro. of stress response pathways and drug efflux. Carvacrol triggers
Many compounds have been found to show some promise as ionicdisruptionandcellularstressrelatedtoaninabilitytoreg-
chemosensitizers to commercial fungicides. However, so far, no ulatevacuolaracidification.Suchactivityofcarvacrol,andother
field studies on use of these chemosensitizers co-applied with a phenolic compounds found in essential oils,may synergistically
+
commercialfungicidehavebeenreported. interactwithazoledrugs,alsoknowntoinhibitH -efflux,affect-
ingvacuolaracidification(Raoetal.,2010).Thechemosensitizing
CHEMOSENSITIZATIONOFANTIMYCOTICDRUGS
mode of action of these types of compounds with commercial
Useofnaturalfungicidalagentstotreathumanmycoses,especially
antimycoticsisdiscussedfurther,below.
dermatophytosis,isanancientpractice,notablyinChineseherbal
Combinationsof plantessentialoilsandcommercialantimy-
medicine(Hanetal.,2007).Naturalproductsaschemosensitizers
cotics have resulted in synergistic antifungal activity. Combina-
were first used as a means of“pharmacologic circumvention of
tion of two plant terpenoids,anethole,a natural phenylpropene
multidrugresistance”usingVinca alkaloidsasinhibitorsof drug
imparting flavor to anise and fennel,and polygodial had syner-
efflux pumps overexpressed in malignant cells resistant to anti-
gisticantifungalactivityagainstC.albicans,atclinicallypractical
cancerdrugs(FordandHait,1993).Itis,perhaps,forthesetwo MICs (<0.1μgmL−1). But,combination of anethole andAMB
reasons there has been far more research on chemosensitization
wereantagonistic,loweringantifungalactivity(KuboandHime-
of antifungaldrugsinhumanmedicine,thanwithfungicidesin
jima,1991).AnetholecombinedwithbothmiconazoleandAMB
agriculture. The higher level of chemosensitization research on
resultedinsynergisticactivityagainstC.albicans (LeeandKim,
antimycotic drugs may also result from the much more limited
1999).Estragole,anotherphenylpropeneandthemainconstituent
numberof antifungalagentsavailable,thanthatforagriculture.
oftheessentialoilsofaKoreanmedicinalherb,Agastacherugosa,
Thesefactors,plustheadditionalproblemofemergingresistance
combinedwithketoconazolealsoresultedinsynergisticantifungal
toantifungaldrugs,haveallspurredonanefforttoinvestigatenew
activityagainstBlastoschizomycescapitatus,ararefungalpathogen
approachesinantimycoticchemotherapy,especiallywithregardto
oftenresultinginfatalmycosesinimmunocompromisedindivid-
phytopharmaceuticals(WagnerandUlrich-Merzenich,2009).
uals (Shin and Kang,2003). However,combination of estragole
withAMBwasantagonistic,reducingantifungalactivityagainstC.
Plantandmarinenaturalproducts
albicans (ShinandPyun,2004).Itwasrecentlyshownthatanet-
Forcenturies,crudeinfusionsofleavesandflowershavebeenused
holeisaninhibitoroffungalchitinsynthase(Yutanietal.,2011a).
infolkmedicinetotreatsuperficialcutaneousfungalinfections,
Hence,theantifungalsynergismbetweenthephenylpropenesand
especiallyhistoricwithregardtoChineseandAyurvedicmedicinal
theazolesisprobablyrelatedtoeffectivelyreducingoverallfungal
herbs.Theseinfusionscontainnaturalproductsofplantessential
cellwall/membraneintegrity.Themodeofphenylpropeneantag-
oils (Kalemba and Kunicka, 2003) and many of those found in
onismwithAMBisunknown.Perhapsthephenylpropenesinhibit
spices,inparticular,havebeenfoundtobeantimycotic(Beuchat,
bindingofpolyenedrugstoergosterol.However,theessentialoil
1994; Arora and Kaur, 1999). Moreover, plant natural products
ofmyrtle(Myrtuscommunis),consistingmainlyofthemonoter-
thatshowantimicrobialactivityhavesomepotentialastherapeu-
penesα-pineneand1,8-cineole,hasbeenshowntohavesynergistic
ticagentsincombinationorcanserveasprecursorsforstructural
activitywithAMB(FICI0.26)againststrainsofC.albicansandA.
modificationtoaugmentactivity(Wink,2008).
niger (MahboubiandGhazianBidgoli,2010).Nomodeofaction
Plant extracts and essential oils. In general, it has long been forthissynergismwasprovided.
shown that the essential oils of “spices” have antifungal activ- One of the earliest investigations of the effects of combining
ityagainstcutaneousandsystematicmycoticagents.Onerecent plant essential oils and a clinical antimycotic involves a small
example shows that antifungal activity of the essential oil of medicinalherb,Santolinachamaecyparissus,cultivatedinEurope,
Lavandula viridis, a member of the mint family, is due to its Asia and Africa. The essential oil from this plant was found to
monoterpenes.Thecompositeextractofthisoilshowedantifungal haveasynergisticantifungaleffectagainstC.albicanswhencom-
activityagainstvariousdermatophytes,C.albicansandC.neofor- bined with clotrimazole, in both in vitro and in vivo (murine)
mansbydisruptingcellmembraneintegrity(Zuzarteetal.,2011). assays(Sureshetal.,1997).TheessentialoilofS.chamaecyparissus
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