Antifungal Potential of Host Defense Peptide Mimetics in a Mouse Model of Disseminated

15 Pages·2017·1.42 MB·English

Journal of
Fungi
Article
Antifungal Potential of Host Defense Peptide
Mimetics in a Mouse Model of
Disseminated Candidiasis
MobaswarHossainChowdhury1,†,LisaKathleenRyan2,KartikeyaCherabuddi2 ID,
KatieB.Freeman3,DamianG.Weaver3,JeffryC.Pelletier3,RichardW.Scott3
andGillDiamond1,*
1 DepartmentofOralBiology,UniversityofFloridaCollegeofDentistry,1600SWArcherRoad,Gainesville,
FL32610,USA;[email protected]
2 DivisionofInfectiousDiseasesandGlobalMedicine,DepartmentofMedicine,UniversityofFloridaCollege
ofMedicine,Gainesville,FL32610,USA;[email protected]fl.edu(L.K.R.),
[email protected]fl.edu(K.C.)
3 FoxChaseChemicalDiversityCenter,Inc.,PennsylvaniaBiotechnologyCenter,Doylestown,PA18902,USA;
[email protected](K.B.F.),[email protected](D.G.W.),[email protected](J.C.P.),
[email protected](R.W.S.)
* Correspondence:[email protected]fl.edu;Tel.:[phone]
† CurrentAddress:BengalSchoolofTechnology—CollegeofPharmacy,Chinsurah,Hooghly712102,
WestBengal,India.
Received:6February2018;Accepted:24February2018;Published:27February2018
Abstract: InvasivecandidiasiscausedbyCandidaalbicansandnon-albicansCandida(NAC)presenta
seriousdiseasethreat. Althoughtheechinocandinsarerecommendedasthefirstlineofantifungal
drug class, resistance to these agents is beginning to emerge, demonstrating the need for new
antifungal agents. Host defense peptides (HDP) exhibit potent antifungal activity, but as drugs
they are difficult to manufacture efficiently, and they are often inactivated by serum proteins.
HDPmimeticsarelowmolecularweightnon-peptidecompoundsthatcanalleviatetheseproblems
andwereshowntobemembrane-activeagainstC.albicansandNAC.Here,weexpanduponour
previousworkstodescribetheinvitroandinvivoactivityof11newHDPmimeticsthatareactive
againstC.albicansandNACthatarebothsensitiveandresistanttostandardantifungaldrugs. These
compoundsexhibitminimuminhibitory/fungicidalconcentration(MIC/MFC)intheµg/mLrange
inthepresenceofserumandareinhibitedbydivalentcations.Rapidpropidiumiodideinfluxintothe
yeastcellsfollowinginvitroexposuresuggestedthattheseHDPmimeticswerealsomembraneactive.
TheleadcompoundswereabletokillC.albicansinaninvasivecandidiasisCD-1mousemodelwith
somemimeticcandidatesdecreasingkidneyburdenby3–4logsafter24hinadose-dependentmanner.
Thedataencouragedfurtherdevelopmentofthisnewanti-fungaldrugclassforinvasivecandidiasis.
Keywords: defensin;membrane-activity;Candida;peptidemimetics
1. Introduction
Invasivecandidiasis,includingcandidemia,isaseriousfungalinfectioncausedbyCandidaalbicans
andnon-albicansCandida(NAC)speciesthatcanleadtomortalityratesashighas70%,depending
onthepopulationsampled[1]. Themostcommonformofinvasivecandidiasisisbloodinfection,or
candidemia,leadingtodisseminatedcandidiasisthatcanresultintheinfectionofvarioustissuesas
well,resultingininfectionofboneandliver,endocarditis,meningitis,pulmonaryandsplenicabscesses
andendophthalmitis[2].
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Despite the emergence of new antifungal therapies, particularly the echinocandins, the overall
incidence of invasive candidiasis remains high, especially in populations susceptible to opportunistic
Despitetheemergenceofnewantifungaltherapies,particularlytheechinocandins,theoverall
infections such as elderly or immunosuppressed patients. However, due to underdetection and the
incidenceofinvasivecandidiasisremainshigh,especiallyinpopulationssusceptibletoopportunistic
difficulty of diagnosing non-candidemia candidiasis, the exact rate of invasive candidiasis is difficult
infectionssuchaselderlyorimmunosuppressedpatients. However,duetounderdetectionandthe
to ascertain, and the incidence of candidemia varies depending on when and where the information
difficultyofdiagnosingnon-candidemiacandidiasis,theexactrateofinvasivecandidiasisisdifficult
was collected and the incidence denominator used in each study or surveillance [3]. Treatment
toascertain,andtheincidenceofcandidemiavariesdependingonwhenandwheretheinformation
options are complicated by the emergence of resistance to fluconazole and to echinocandins, the
wascollectedandtheincidencedenominatorusedineachstudyorsurveillance[3]. Treatmentoptions
difficulty in diagnosis of invasive candidiasis, and the shift in infection from C. albicans to NAC spp.
arecomplicatedbytheemergenceofresistancetofluconazoleandtoechinocandins, thedifficulty
which are frequently more resistant. Many of these NAC spp. are preferentially selected using
indiagnosisofinvasivecandidiasis, andtheshiftininfectionfromC.albicanstoNACspp. which
prophylactic anti-fungal therapy with fluconazole in the intensive care unit and in
arefrequentlymoreresistant. ManyoftheseNACspp. arepreferentiallyselectedusingprophylactic
immunocompromised patients [4]. Fluconazole resistance in C. albicans is uncommon in the clinical
anti-fungaltherapywithfluconazoleintheintensivecareunitandinimmunocompromisedpatients[4].
setting, but common in NAC spp. especially with C. glabrata [5]. Echinocandins cannot be used for
FluconazoleresistanceinC.albicansisuncommonintheclinicalsetting,butcommoninNACspp.
infections of the eye or urinary tract. For those infections, patients intolerant of fluconazole are only
especiallywithC.glabrata[5]. Echinocandinscannotbeusedforinfectionsoftheeyeorurinarytract.
left with usage of Amphotericin with significantly higher toxicity. In addition, the relatively recent
Forthoseinfections,patientsintolerantoffluconazoleareonlyleftwithusageofAmphotericinwith
emergence of the multi-drug resistant C. auris is a concern. Thus, there is a great need for the
significantlyhighertoxicity. Inaddition,therelativelyrecentemergenceofthemulti-drugresistant
development of new anti-fungal agents [3] which have activity in C. albicans, fluconazole sensitive
C.aurisisaconcern. Thus,thereisagreatneedforthedevelopmentofnewanti-fungalagents[3]
and resistant NAC isolates.
whichhaveactivityinC.albicans,fluconazolesensitiveandresistantNACisolates.
One potential avenue to address this need is the development of host defense peptides (HDP).
One potential avenue to address this need is the development of host defense peptides
HDPs have broad-spectrum anti-microbial and immunomodulatory properties and develop
(HDP).HDPshavebroad-spectrumanti-microbialandimmunomodulatorypropertiesanddevelop
antimicrobial resistance infrequently [6]. Examples of HDPs include magainin, α- and β-defensins,
antimicrobialresistanceinfrequently[6]. ExamplesofHDPsincludemagainin,α-andβ-defensins,
and cathelicidins such as LL-37. Attempts to exploit these desirable properties of HDPs as drugs have
andcathelicidinssuchasLL-37. AttemptstoexploitthesedesirablepropertiesofHDPsasdrugshave
been thwarted by the cost of producing these peptides and mostly by the susceptibility to protease
beenthwartedbythecostofproducingthesepeptidesandmostlybythesusceptibilitytoprotease
digestion and serum-binding properties in vivo, greatly diminishing their activity [7]. To circumvent
digestionandserum-bindingpropertiesinvivo,greatlydiminishingtheiractivity[7]. Tocircumvent
these problems, the small synthetic, nonpeptidic oligomers were designed to adopt the amphiphilic
theseproblems,thesmallsynthetic,nonpeptidicoligomersweredesignedtoadopttheamphiphilic
secondary structures of HDPs and to exhibit potent and selective antimicrobial activity [8,9].
secondarystructuresofHDPsandtoexhibitpotentandselectiveantimicrobialactivity[8,9].
We have previously described two new, potent anti-fungal HDP mimetics that are active in vitro
Wehavepreviouslydescribedtwonew,potentanti-fungalHDPmimeticsthatareactiveinvitro
against both Candida albicans and NAC, even in the presence of 50% human serum, and are active
againstbothCandidaalbicansandNAC,eveninthepresenceof50%humanserum, andareactive
against both the planktonic form and biofilms [10–12]. These HDP mimetics were prime candidates
againstboththeplanktonicformandbiofilms[10–12]. TheseHDPmimeticswereprimecandidates
for anti-fungal drug development, for C. albicans grown long-term at sub-minimum inhibitory
for anti-fungal drug development, for C. albicans grown long-term at sub-minimum inhibitory
concentration (MIC) did not develop resistance to these mimetics [11] and they appeared to not affect
concentration (MIC) did not develop resistance to these mimetics [11] and they appeared to not
commensal oral bacteria or biofilms [10]. These drugs also are active in vivo, both in an invasive
affectcommensaloralbacteriaorbiofilms[10]. Thesedrugsalsoareactiveinvivo,bothinaninvasive
candidiasis model and in an oral candidiasis model [12,13].
candidiasismodelandinanoralcandidiasismodel[12,13].
In this study, we have described several new HDP mimetics with activity against Candida
Inthisstudy,wehavedescribedseveralnewHDPmimeticswithactivityagainstCandidaspecies.
species. The compounds are analogs of Compounds 1, 2 and 4 (see Figure 1 for structures) and have
ThecompoundsareanalogsofCompounds1,2and4(seeFigure1forstructures)andhavemolecular
molecular weights ranging from 431 to 558 D. These are based on three different scaffolds, whose
weights ranging from 431 to 558 D. These are based on three different scaffolds, whose structure
structure and activities have been previously described [14]. Several of these new HDP mimetics have
andactivitieshavebeenpreviouslydescribed[14]. SeveralofthesenewHDPmimeticshavepotent
potent anti-fungal activity against Candida both in vitro and in vivo.
anti-fungalactivityagainstCandidabothinvitroandinvivo.
N N
N N
N N
H2N NH2
NH NH
(a)
Figure1.Cont.
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HN
NH2
NH NH
H2N
HN
O HN
N O
H
NH
NH2
HN HN
H2N
NH
(b)
NH2
H2N
NH2
O
O
O
H2N O
N
NH2
(c)
Figure 1. Representative compound structures. Structures of Compounds 1 (a), 2 (b) and 4 (c),
Figure 1. Representative compound structures. Structures of Compounds 1 (a), 2 (b) and 4 (c),
correspondingtotriacyl,bis-amideandtricyclicstructuresareshown.
corresponding to triacyl, bis-amide and tricyclic structures are shown.
2. MaterialsandMethods
2. Materials and Methods
2.1. Materials
2.1. Materials
Peptide mimetic compounds were received from Fox Chase Chemical Diversity Center,
Peptide mimetic compounds were received from Fox Chase Chemical Diversity Center,
PennsylvaniaBiotechnologyCenter,Doylestown,PA,USA.Compoundsweredissolvedindimethyl
Pennsylvania Biotechnology Center, Doylestown, PA, USA. Compounds were dissolved in dimethyl
sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) at the stock concentration of 10 mg/mL
sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) at the stock concentration of 10 mg/mL and
and stored at −20 ◦C. Cyclophosphamide and fluconazole was purchased from Sigma-Aldrich,
stored at −20°C. Cyclophosphamide and fluconazole was purchased from Sigma-Aldrich, St. Louis,
St. Louis, MO, USA. RPMI 1640 containing L-glutamine without bicarbonate, buffered to pH 7.0
MO, USA. RPMI 1640 containing L-glutamine without bicarbonate, buffered to pH 7.0 with 0.165
with0.165morpholinepropanesulfonicacid(MOPS)buffer(Sigma-Aldrich,St.Louis,MO,USA).
morpholinepropanesulfonic acid (MOPS) buffer (Sigma-Aldrich, St. Louis, MO, USA).
2.1.1. YeastStrains
2.1.1. Yeast Strains
A standard invasive candidiasis strain of C. albicans SC5314 was used for peptide mimetic
A standard invasive candidiasis strain of C. albicans SC5314 was used for peptide mimetic
compoundsscreening. Non-albicansCandidaspecies(NAC)C.dubliniensis(NCPF3949), C.glabrata
compounds screening. Non-albicans Candida species (NAC) C. dubliniensis (NCPF3949), C. glabrata
(ATCC90030),C.krusei(ATCC6258),C.parapsilosis(ATCC22019)andC.tropicalis(ATCC750)(obtained
(ATCC 90030), C. krusei (ATCC 6258), C. parapsilosis (ATCC 22019) and C. tropicalis (ATCC 750)
from the laboratory of David Perlin, PHRI/Rutgers), were cultured on YPD (1% yeast extract, 2%
p(oebpttaoinneed,2 f%rodme xtthroe slea,bpoHra5to.7r)ya ogfa rDaatv3i7d◦ CPearnlidn,w PeHreRuI/sRedutfgoerrsa)l,l ewxepreer icmueltnutraelds tound iYesP.DFl u(1c%on ayzeoalset
extract, 2% peptone, 2% dextrose, pH 5.7) agar at 37°C and were used for all experimental studies.
resistantclinicalisolatesofC.glabratafromfivedifferentpatientsandC.tropicalisfromonepatientwere
Fluconazole resistant clinical isolates of C. glabrata from five different patients and C. tropicalis from
collectedfromtheclinicalmicrobiologylaboratory,ShandsHospital,UniversityofFlorida,Gainesville,
one patient were collected from the clinical microbiology laboratory, Shands Hospital, University of
FL,USA.
Florida, Gainesville, FL, USA.
2.1.2. MICandMFCStudies
2.1.2. MIC and MFC Studies
Candida albicans SC5315, NAC species and fluconazole-resistant clinical isolates of invasive
candiCdainasdiisdast raalibnicsaonfsC S.Cgl5a3b1ra5t,a NanAdCC .stproepciiceasl isanwde rfeluucsoednafzoorlme-irneismisutamnti nchliibniitcoarly icsoonlacteenst roaft ioinnv(aMsiIvCe)
caannddmidiinaismisu smtrafiunns goifc iCd.a lglcaobnractean atrnadti oCn. (tMropFiCca)lisst uwdeirees uofseleda fdorc ommipniomuundms .inMhIiCbistowrye rceodnecteenrtmraitnioedn
(uMsiInCg)t haenCd limniicnailmanudmL afubonrgaitcoidryalS tcaonndcaerndtsraIntisotnit u(tMe(FCCL)S Is)tsutdaniedsa rodf blreoatdh mcoicmropdoiuluntdiosn. mMeItChso dw[1e2re].
determined using the Clinical and Laboratory Standards Institute (CLSI) standard broth
microdilution method [12]. Suspensions of C. albicans were prepared by measuring optical density at
J.Fungi 2018,4,30 4of15
SuspensionsofC.albicanswerepreparedbymeasuringopticaldensityat595nm(OD )of1.0in
595
phosphatebufferedsaline(PBS).HDPmimeticcompoundsweredilutedin50µLRPMI/MOPScell
culturemediumina96-wellplate(tissueculturetreated;Costar,CorningIncorporated,Corning,NY,
USA).Toeachwell,asuspension(50µL)ofCandidawasadded,andthenplatewasincubatedat37◦C
inahumidifiedchamberforaperiodof24h. TodeterminetheMFC,asample(100µL)fromthewell
definedashavingtheMICandthewellswithotherthreehigherconcentrationswereplatedontoYPD
agar. Candidacolonieswereobservedafter24h. TheMFCisdefinedasthelowestconcentrationof
compoundatwhichnocoloniesareobserved. AllMICandMFCassayswereperformedinduplicate.
Toassesstheeffectofadivalentcationsontheelectrostaticinteractionbetweenmimeticsand
cellmembraneofyeast,weusedcalciumchlorideatconcentrationsof2,5,7.5and10µM.Thestudy
wasconductedusingbothRPMI/MOPScellculturemediumandMullerHinton(MH)broth. Ateach
concentrationofthiscation,theMICofmimeticsweredeterminedbythemethoddescribedabove.
2.1.3. MammalianCellCytotoxicityAssay
Cytotoxicity(50%effectiveconcentration(EC ))wasdeterminedagainstmouse3T3fibroblasts
50
(ATCCCRL-1658),OKF6/TERT-1cells(oralkeratinocytes)[12],andhumantransformedliverHepG2
cells(ATCCHB-8065),usinganMTSviabilityassay(Promega,Fitchburg,WI,USA).Growthmedium
was replaced with medium without serum, and eight 2-fold dilutions of compound were added.
Followingincubationfor1hat37◦C,compoundswereremoved,andmediumcontainingserumwas
returned. ViabilitywasdeterminedusinganMTSviabilityassay(CellTiter96aqueousnonradioactive
cellproliferationassay,Promega).
2.1.4. HemolysisAssay
Peptide mimetic compounds were tested in duplicate using a hemolysis assay previously
published[15,16]. Briefly,two-foldserialdilutionsstartingat2mg/mLin150mMNaCl(saline),20%
DMA(AlfaAesar,WardHill,MA,US),or10%kleptoseinsaline. 3%saponin(Fluka)insalinewas
usedasapositivecontrol. Eachdilutionofeachcompound(25µL)wasplatedina96-wellround
bottomedplateand25µLofwholehumanblood(BIORECLAMATION,Westbury, NY,USA)was
addedtoeachsampleandmixedfor10second. ThebloodwasTypeO,from2malesand2females,
collected with potassium EDTA to prevent clotting. Plates were incubated on a shaker for 30 min
at37◦C,thencentrifugedat600×gfor5min. 5µLofthesupernatantwastransferredtoa96-well
flat-bottomedplatewith200µLH Oineachwellandmixedfor5min. Absorbancewasreadat405nm
2
(Spectramax250,MolecularDevices,SanJose,CA,USA).Percenthemolysiswasthencalculatedas:
P=(OD (PMXcompound)–OD (control))/(OD (Saponin)–OD (control))
405 405 405 405
CurveswerefittedtoreportanHC (50%hemolysis)valueforeachcompound:
50
P(C )=1/(1+(K/C )n)
p p
2.1.5. YeastMembranePermeabilityAssessment
C. albicans was suspendedin RPMI/MOPS mediato a totalconcentration of 2 × 106 cfu/mL.
One ml aliquots of the C. albicans suspension were treated with each mimetic compound at a
concentration of 32 µg/mL, using 95% ethanol as positive control. Samples were incubated with
themimeticsat30◦Cfor0,5,15and30min. Aftertreatment,sampleswerecentrifugedat5000rpm
for 5 min. Supernatants were removed and pellets were washed twice with 1 mL of PBS. Lastly,
afterthesupernatantwasremoved,50µLofPI(0.5µg/mL)wasaddedandmixedviainversionfor
15min,andthepelletwasresuspendedin500µLofPBS.Sampleswereanalyzedbyflowcytometry
(FACSCalibur,BectonDickinson,ImmunocytometrySystems,SanJose,CA,USA)toassesspercent
uptakeofPI.
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2.2. EfficacyofHDPMimeticsinvivo
2.2.1. Mice
FemaleCD-1miceagedfrom4to6weeks(weight,20–22g)wereusedinthisstudy. Micewere
purchasedfromCharlesRiver,Wilmington,MA,USA.Allmicewerehousedinabarrierfacilityin
theBiomedicalScienceBuildingAnimalFacility,UniversityofFlorida. Animalswerefedasterilized
standardrodentdietanddrinkingsterilizedwateradlibitum. Micewereacclimatizedtotheanimal
houseenvironmentfor7dayspriortouseinexperiments.
2.2.2. CandidaStrainsandInoculumPreparation
C.albicansSC5314weregrowninYPDbroth(Difcolaboratories, Detroit, MI,USA)andstock
cultureswerestoredat−80◦C.ThefrozenstockswerefirstgrownonYPDplatesandkeptat4◦C.
Priortoeachexperiment,astockinoculumsuspensionofCandidawaspreparedinYPDbrothand
incubatedat30◦Cwithshakingat200rpmfor12h. Theculturewaswashedtwicewithsterile1X
phosphate buffered saline (PBS), counted with a hemocytometer, and diluted with PBS to get the
desiredconcentrationof3.5×105cfu/mL.100µLwasinjectedintravenously(IV)intoeachmousevia
thetailvein. Thedesiredinfectiondosewasconfirmedbyplating10-folddilutionsoftheinoculumon
YPDplates,whichwereincubatedat37◦Cfor24htoyieldviablecounts(cfu)ofyeastcolonies[12].
2.2.3. DrugPreparation
Fortheanimalstudies,mimeticsweredissolvedin20%kleptoseata2mg/mLconcentration
andsonicatedfor10min. Cyclophosphamide(150mg/kgin10mL/kg)wasdissolvedinsterilePBS.
Fluconazole(5mg/mL)wasdissolvedin10%DMSOinsterilePBS.
2.2.4. DeterminationoftheMaximumToleratedDose(MTD)
FemaleCD-1mice(CharlesRiverLaboratories),8–10weeksold,>20gwereusedtodetermine
theMTD.Micewerehousedindividuallyinstainlesssteel,wire-bottomcagestominimizeaccessto
urineandfecesandfedHarlanTekladCertifiedGlobalDiet® withtapwateradlibitum. Micewere
acclimatedtothestudyroomatleast3dayspriortodosing. Mice(3pergroup)weredosedwitha
subcutaneous(SC)bolusinjectionbeginningwith10mg/kgofthetestcompoundataconcentration
of2mg/mL.Doseswereloweredtwo-foldforsubsequentgroupstofindtheMTD.Thedosevolume
was5mg/kg,calculatedusingtheweightoftheanimalonthedayofinjection. Therewere3dose
events,with2daysbetweeneachevent. Micewereobservedforchangesinappearanceandbehavior
at2and24hfollowingeachinjectionandmiceshowingpronouncedimmediateeffectswereremoved
fromthestudy. BodyweightswereobtaineddailyuntilthedayofeuthanizationwithaCO overdose.
2
Anymortalityduringthestudywasalsorecorded. MTDdatawereobtainedbyRicercaBiosciences,
LLC,Concord,OH,USA.MTDofeachHDPmimeticinCD-1micewasdeterminedandourstudies
utilizeddosesbeginningwith0.5XMTD.
2.2.5. ImmunosuppressiveMouseModelofInvasiveCandidiasisandComparisonofHDPMimetic
Efficacyat50%MTD
MaleCD-1mice,6–8weeksold(20–22g),weremadeneutropenicwithtwointraperitoneal(IP)
injectionsof150mg/kgin10mL/kgcyclophosphamidefourdaysandonedaybeforeinoculation[17].
Eachanimalwastheninoculatedbyinjecting0.1mLof3.5×105cfu/mLC.albicansinthetailvein.
Animalsweredividedintodifferentgroups(n=5pergroup). 2hafterC.albicansinoculation,mimetic
compounds were injected subcutaneously (SC) at 0.5X of the MTD (determined by Charles River
Laboratoriesasdescribed). Fluconazole(20mg/kg)wasadministeredtoanothergroupbyoralgavage
at2hpostinoculation,asapositivecontrolfortheexperiments,toconfirmtheabilitytoquantifyan
effectonkidneyburden. TheanimalswerehumanelykilledbyCO asphyxiationandbothkidneys
2
werecollectedfromfivemiceofinfectedcontrolgroup2hafterinfectionandfromtheremaining
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controlanddrug-treatedmiceinthestudyat24hafterinfection. Twokidneyswereweighedand
combined in a sterile homogenizer tube/mouse. Ten mL sterile PBS was added to each tube and
thecontentsuniformlyhomogenizedwithatissuehomogenizer(Ultra-TURRAXTubeDrive,IKA
Works,Inc.,Wilmington,NC,USA).Serialdilutionsofthetissuehomogenateswereconducted,0.1mL
aliquotswerespreadonYPDagarplates,andtheplatesincubatedat35◦Covernight. cfu/gkidneys
weredeterminedfromcolonycounts.
2.2.6. SingleInjectionDoseResponseStudyofHDPMimetics
Various doses of each compound were administered as a single SC injection of compound at
2hafterinfectionandcfu/gkidneyweredeterminedat24hpostinfection. Thecyclophosphamide
inducedCD-1neutropenicmousewasusedasdescribedabove. Micewereintravenouslyinoculated
with3.5×105cfu/mLC.albicansviathelateraltailvein. After2hofinfection,asingleSCinjection
ofvariousdoses(40,20and10mg/kg,bodyweight)ofCompound6weregiventomice(n=5per
group)at2hpostinfection. Afluconazoletreatedgroupwasalsotestedforcomparison. At24hpost
infection,allanimalsweresacrificedandkidneyswereharvested,andcfu/gkidneywasdetermined.
2.2.7. ComparisonofaSingleDosewithaSplitDoseofHDPMimetic
Groups of 5 mice were given 0.5 MTD of Compound 6, either as a single bolus SC dose, or
twice half of the 0.5 MTD dose 2 or 4 h apart. The compound was initially given 2 h following
infectionandkidneycfuwasassessed24hfollowinginfection. Infectionwasgivenasabove,with
3.4×104cfu/mouseinjectedIVintothedorsaltailvein.
2.3. StatisticalAnalysis
All graphic data are expressed as mean ± SD and were statistically analyzed by analysis of
variance(ANOVA)usingcomputerPrismsoftware(Prism5;GraphPadSoftware,Inc.,SanDiego,CA,
USA).Burdendifferencebetweentestingandcontrolgroupswasassessedbyposthocanalyses,using
Dunnett’smultiplecomparisontest. p<0.05wasconsideredstatisticallysignificant. TheEC was
50
calculatedusingGraphPadPrismsoftware(nonlinearfit).
2.4. EthicalStatement
All animals were maintained in accordance with American Association for Accreditation of
LaboratoryCarecriteria. Theanimalexperimentsweredesignedandconducteduponapprovalofthe
InstitutionalAnimalCareandUseCommittees(IACUC)(protocolnumber201408371,29May2014),
UniversityofFlorida,USAandstrictlyinaccordancewiththeguidelinesofIACUC.
3. Results
3.1. InVitroEfficacyofHDPMimeticsagainstCandidaSpecies
To screen for potentially effective compounds, MIC/MFC assays were performed, with and
without50%humanserum. Table1comparestheinvitroactivityofHDPmimeticsregardingMIC,
with and without 50% human serum, and MFC (without human serum) against Candida albicans,
strainSC5314. Mostcompoundswerealsotestedforactivityagainstfivenon-albicansspecies(NAC):
C. tropicalis, C. parapsilosis, C. dublinensis, C. glabrata, and C. krusei. All compounds showed broad
activityagainstalltheCandidaspecies,withtheMICofCompounds3,5,7and6risingfrom2or4
to16µg/mLwiththeadditionof50%humanserumtotheassaywithC.albicans. Wethentested
Compound6againstfluconazole-resistantNACderivedfromclinicalisolatesfromdifferentpatients
with invasivecandidiasis. Infive patientisolates offluconazole-resistant C. glabrata, the MICwas
>64µg/mL.However,withoneisolateoffluconazole-resistantC.tropicalis,Compound6hadanMIC
of4µg/mLandanMFCof32µg/mL(Table2).
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Table1.MICstudyofelevenHDPmimetics.
Compound 1 2 3 4 5 6 7 8 9 10 11 Fluconazole Caspofungin
Series Tri-aryl Bis-amide Tri-cyclic Tri-cyclic Bis-amide Bis-amide Bi-aryl Bis-amide Bis-amide Bi-aryl Bi-aryl
ActivityagainstC.albicans(µg/mL)
MIC 4 4 2 4 4 4 4 8 >8 0.3 8 0.25 0.25
MFC 8 16 8 8 8 8 8 16 >8 0.78 ND 0.5
MIC+50%
4 4 16 4 16 16 16 32 >8 ND >8
Humanserum
NACspeciesMIC(µg/mL)
C.tropicalis 4–8 8 4 2–4 2 4 8 4 2 ND 1
C.parapsilosis 4–8 1 1 2 1 2 2 4 0.5 ND 0.25
C.dublinensis 8 32 16 4 2 2 4 4 ND ND ND
C.glabrata 4 8 2 2 4 4 4 8 >8 ND 1
C.krusei 32 8 0.50 16 2 2 4 4 1 ND 4
ND,notdetermined.
Table2.MIC/MFCofCompound6againstfluconazole-resistantNACclinicalisolatescomparedwithnon-resistantCandidaalbicans.
Fluconazole-resistantNACDerivedfromClinicalIsolates
MIC(µg/mL) MFC(µg/mL)
fromDifferentPatients:TG-Candidaglabrata,CT-Candidatropicalis
TG-1 >64 >64
TG-3 >64 >64
TG-4 >64 >64
TG-5 >64 >64
TG-6 >64 >64
CT-2 8 32
CandidaalbicansSC5314 4 8
J.Fungi 2018,4,30 8of15
Table3showsinvitroeffectsonmammaliancells,assayingforcytotoxicityandhemolysis. These
parameterswerevariableamongtheHDPmimetics,butthecytotoxicityandhemolyticcapacitywas
low. Compound6waschosenforfurthertestingduetoitslackofcytotoxicityandhemolysis(Table3).
The cytotoxicity EC on both 3T3 and HepG2 cells was >1398.6 µM and for hemolysis, the EC
50 50
was1331µM.Furthermore,Compound6hadahighmaximumtolerateddose(MTD)of≥40mg/kg.
Table4showsallMTDinCD-1femalemiceforallcompounds. Thebesttoleratedcompoundswere
Compounds6and8. However,Compound8hadanEC of390µMfor3T3cellsand249µMfor
50
HepG2cells,whichwasmuchlowerthanCompound6.
Table3.CytotoxicityandhemolyticactivityofHDPmimetics.
Compounds Cytotoxicity3T3EC (µM) CytotoxicityHepG2EC (µM) HemolysisEC (µM)
50 50 50
1 311 ND >1792
2 358 ND >1164
3 478 137 396.2
4 436 ND >1170
5 891 315.1 1453.5
6 >1398.6 >1398.6 1331
7 >788.6 >788.6 >1577.3
8 390 249 >1272
9 725 1456 1173
10 >1445 >1445 >1445
11 >1518 >1518 >1517
Table4.Maximumtolerateddose(MTD)ofHDPmimeticsinfemaleSwissCD.1mice.
Compounds 1 2 3 4 5 6 7 8 9 10 11
MTD(mg/kgfreebase) 10 10 10 20 5 ≥40 20 ≥40 ≥40 10 10
3.2. MembraneEffectsofHDPMimeticsonCandidaalbicans
We previously demonstrated that Compound 4 acts on Candida albicans by leading to rapid
poreformation[13]. Todeterminewhethertheseothercompoundsexhibitasimilarmechanism,we
quantifiedthepropidiumiodide(PI)incorporationafterdrugtreatment. When32 µg/mLPIwas
addedtocellsfollowingtheadditionofeachHDPmimetictoC.albicans,influxofPIoccurredwithin
5 min and maximum incorporation of PI with all compounds occurred by 30 min, although each
reachedthemaximumatdifferentrates(Figure2a). TreatmentofCandidawithCompounds1,2,7
and8resultedinreachingmaximumincorporationofPIin30min(thelongest),whilethemostrapid
membraneactivitywasobservedwithCompounds3,4,5and6.
A second aspect of the antifungal mechanism of Compound 4 is the initial binding to of the
cationiccompoundwiththeanionicmembrane. Todeterminewhetherthecompoundsassayedhere
exhibitasimilarelectrostaticinteraction, weinhibitedtheactivitywithCaCl asadivalentcation.
2
Figure2bshowstheeffectofaddingCaCl ontheactivityofeightHDPmimeticsintheMICassay
2
using C. albicans. The divalent cation was able to inhibit the activity of all eight compounds on
C.albicansataCaCl concentrationof5.0mM,raisingtheMICtoover60µg/mL.AddingCaCl to
2 2
Compounds2and3,raisedtheMICto>60µg/mLatthelowerconcentrationof2.5mMCaCl .
2
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J. Fungi 2018, 4, x FOR PEER REVIEW 9 of 15
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(b)
Figure 2. Membrane activity of potent HDP mimetics. (a) Propidium iodide (PI) incorporation in
Figure2. MembraneactivityofpotentHDPmimetics. (a)Propidiumiodide(PI)incorporationin
Candida albicans exposed to HDP mimetics. PI incorporation after 0–30 min treatments with 32 µg/mL
CandidaalbicansexposedtoHDPmimetics.PIincorporationafter0–30mintreatmentswith32µg/mL
of each HDP mimetic. 95% ethanol was used as a positive control, in which 100% PI entered the cell
ofeachHDPmimetic.95%ethanolwasusedasapositivecontrol,inwhich100%PIenteredthecell
within 7 min. No PI was taken up in untreated yeast cells. (b) Calcium chloride inhibition of MIC of
within7min. NoPIwastakenupinuntreatedyeastcells. (b)CalciumchlorideinhibitionofMIC
potent HDP mimetics. MIC assays were carried out in the presence of increasing concentrations of
ofpotentHDPmimetics. MICassayswerecarriedoutinthepresenceofincreasingconcentrations
oCfaCCal2C. l .
2
3.3. Efficacy of HDP Mimetics in a Mouse Model of Invasive Candidiasis
3.3. EfficacyofHDPMimeticsinaMouseModelofInvasiveCandidiasis
IInn aann iimmmmuunnooccoommpprroommisiseeddf efemmaaleleC CDD-1-1m moouusesem mooddeleol focf acnadniddiidaisaissi,sw, withith3. 43.×4 ×1 10044c cffuu//mmoouussee
iinnjjeecctteedd IIVV,, aallll HHDDPP mmimimeetitcicss wwereer ecocmompapraerde dbyb ycaclcaulcluatliaotnio onf olofgl1o0 gredurecdtiuocnt iionn kiidnnkeiyd ncfeuy 2c4f uh a2f4tehr
10
adfotesirndgo SsCin wgiStCh hwailtfh ofh tahlfe oMfTthDe (MFiTguDre(F 3i)g. uRreed3u)c.tiRoend iunc ctifoun wienrecf duewteermreindeedte brmy icnaelcdublayticnagl cthuela ltoingg10
reduction in cfu for each treated mouse from the average of the 24 h untreated mice (n = 5). Data are
shown in Figure 3 as the average of log10 reduction in cfu for each group (n = 5) ± SEM. The data for
the fluconazole treatment (via gavage) were an average of the log10 reduction in cfu of four
J.Fungi 2018,4,30 10of15
thelog reductionincfuforeachtreatedmousefromtheaverageofthe24huntreatedmice(n=5).
10
DJa. Ftaunagri e20s1h8o, 4w, xn FiOnRF PigEEuRr eR3EVaIsEWth e averageoflog10reductionincfuforeachgroup(n=5)±SEM1.0T ohf e15
dataforthefluconazoletreatment(viagavage)wereanaverageofthelog reductionincfuoffour
10
ininddepepenenddenetnet xepxpereirmimenetnstsa nadndth tehreerew wasasa a3 -3l-olgogr erdeduuctcitoinono foCf C.a. lablibciacnasnsc fcufuin inb booththk kididnneyeyssu usisninggt hthisis
stsatnanddaradrdt rteraetamtmenent.t.T TwwooH HDDPPm mimimeteitcisc,sC, Coommppoouunnddss6 6a anndd8 8,,a appppeeaarerdedt otoh haavveet htheeg grereaatetestste effiffciacacycy
wwhhenenm miciecew wereered odsoesdedw withith4 040m mg/gk/kgg( a(sassusummininggt htheeM MTTDDo fo>f 4>040m mg/g/kkggi sisa attl eleaassttd doouubblele,,s oso0 .05.X5X
MMTTDDis is4 040m mg/gk/kgg),),g giviviningga a3 3-l-ologgr erdeduuctcitoionni nink ikdidnenyeyb ubrudrednen(c (fcuf/ug/g).).O OththeerrH HDDPPm mimimeetitciscsr erdeduucecded
ththeek kididnneyeyb uburdrdenenb ybytw twoolo lgosg:sC: Comomppouounnddss3 ,39, 9a nandd1 11,1e, aecahchg igvievnenS CSCa ta0t .05.X5XM MTDTD. .
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Figure3.ComparisonofHDPmimeticefficacyat50%MTD.Meanlog reductionofcolonyforming
Figure 3. Comparison of HDP mimetic efficacy at 50% MTD. Mean lo1g010 reduction of colony forming
units (cfu) of Candida albicans following SC treatment with different HDP mimetics at 50% of the
units (cfu) of Candida albicans following SC treatment with different HDP mimetics at 50% of the
maximum tolerated dose (MTD) 2 h following IV infection with 3.5 × 104 cfu/mouse for 24 h.
maximum tolerated dose (MTD) 2 h following IV infection with 3.5 × 104 cfu/mouse for 24 h. For
Forcomparison,femaleSwissCD.1miceweregavaged2hafterinfectionwith20mg/kgfluconazole
comparison, female Swiss CD.1 mice were gavaged 2 h after infection with 20 mg/kg fluconazole for
for24h,whichcauseda3-logreductionincfu.Groupswerecomparedtoacontrolgroupdosedwith
24 h, which caused a 3-log reduction in cfu. Groups were compared to a control group dosed with
kleptoseandinfectedwith3×104cfuC.albicanspermouse.
kleptose and infected with 3 × 104 cfu C. albicans per mouse.
Considering toxicity and efficacy, two promising HDP mimetics were tested in vivo for dose-
Considering toxicity and efficacy, two promising HDP mimetics were tested invivo for
response. Figure 4 shows both the kidney burden (2 kidneys, cfu/g kidney) and the log reduction of
dose-response. Figure4showsboththekidneyburden(2kidneys,cfu/gkidney)andthelogreduction
C. albicans using three doses of Compounds 3 and 6, each showing a dose-response. Compound 6
ofC.albicansusingthreedosesofCompounds3and6,eachshowingadose-response. Compound6
reduced cfu by two logs at the highest dose of 40 mg/kg to 0.5 log at the lowest dose of 10 mg/kg.
reducedcfubytwologsatthehighestdoseof40mg/kgto0.5logatthelowestdoseof10mg/kg.
Compound 3 was not as effective; it only reduced cfu by one log at the highest dose of 5 mg/kg (0.5X
Compound 3 was not as effective; it only reduced cfu by one log at the highest dose of 5 mg/kg
MTD) and 0.5 log at the next dose of 2.5 mg/kg, whereas the lowest dose of 1 mg/kg of 3 had almost
(0.5XMTD)and0.5logatthenextdoseof2.5mg/kg,whereasthelowestdoseof1mg/kgof3had
no effect.
almostnoeffect.