Identification of Antifungal H+-ATPase Inhibitors with Effect on Plasma Membrane Potential

14 Pages·2017·1.41 MB·English

MECHANISMS OF ACTION:
PHYSIOLOGICAL EFFECTS
crossm
Identification of Antifungal H(cid:2)-ATPase
Inhibitors with Effect on Plasma
Membrane Potential
LasseKjellerup,a,bSandraGordon,aKarenO’HanlonCohrt,aWilliamDalbyBrown,a D
o
AnjaThoeFuglsang,bAnne-MarieL.Winthera w
Pcovery,Copenhagen,Denmarka;DepartmentofPlantandEnvironmentalSciences,Universityof n
Copenhagen,Frederiksberg,Denmarkb lo
a
d
ABSTRACT The plasma membrane H(cid:2)-ATPase (Pma1) is an essential fungal protein ed
and a proposed target for new antifungal medications. The compounds in a small- Received6January2017 Returnedfor f
r
molecule library containing (cid:3)191,000 commercially available compounds were modification5February2017 Accepted15 o
April2017 m
screened for their ability to inhibit Saccharomyces cerevisiae plasma membranes con-
Acceptedmanuscriptpostedonline24 h
taining Pma1. The overall hit rate was 0.2%, corresponding to 407 compounds. April2017 tt
p
These hit compounds were further evaluated for ATPase selectivity and broad- CitationKjellerupL,GordonS,CohrtKO, :
/
spectrum antifungal activity. Following this work, one Pma1 inhibitor series based BrownWD,FuglsangAT,WintherA-ML.2017. /a
IdentificationofantifungalH+-ATPase a
on compound 14 and analogs was selected for further evaluation. This compound c
inhibitorswitheffectonplasmamembrane .
series was able to depolarize the membrane and inhibit extracellular acidification in potential.AntimicrobAgentsChemother a
s
intact fungal cells concomitantly with a significant increase in intracellular ATP lev- 61:e00032-17.https://doi.org/10.1128/AAC m
.00032-17. .
els. Collectively, we suggest that these effects may be a common feature of Pma1 o
Copyright©2017Kjellerupetal.Thisisan r
inhibitors. Additionally, the work uncovered a dual mechanism for the previously g
open-accessarticledistributedundertheterms /

identified cationic peptide BM2, revealing fungal membrane disruption, in addition oftheCreativeCommonsAttribution4.0 o
to Pma1 inhibition. The methods presented here provide a solid platform for the Internationallicense. n
N
AddresscorrespondencetoLasseKjellerup,
evaluation of Pma1-specific inhibitors in a drug development setting. The present in- o
[email protected],orAnne-MarieL.Winther, v
hibitors could serve as a starting point for the development of new antifungal [email protected]. e
m
agentswithanovelmodeofaction. A.T.F.andA.-M.L.W.areco-seniorauthors.
b
e
KEYWORDS ATPase,antifungalagents,drugdevelopment,membraneproteins r

1
6
,
Fungalinfectionsaffect25%ofthepopulation,withsuperficialinfectionsoftheskin 2
0
andnailsrepresentingthemostcommontype.Inasurveyofwomen(age16years 1
and older, n (cid:4) 6,000), up to 49% had been diagnosed with vulvovaginal candidiasis 8
b
(VVC), depending upon their ethnic origin, and approximately 20% of those women y

experienced recurrent VVC in a 12-month follow-up period (1), with a pronounced g
u
impact on quality of life. Invasive fungal infections are less common but of much e
s
greaterconcernbecausetheyareassociatedwithextremelyhighmortalityrates(20to t
90%)(2).ThemostcommoninvasivefungalinfectionsarecausedbytheyeastsCandida
andCryptococcusspp.,followedbythemoldsAspergillusandMucorspp.Keyareasof
concern in the treatment of invasive fungal infections with the current antifungal
medications include delays in diagnosis and the identification of the specific patho-
genicspecies,intrinsicandacquireddrugresistance,inconvenientdrugadministration,
safety, and tolerability issues with prolonged use. For these reasons, there is a major
unmetneedfornewantifungalagents(3).
The fungal plasma membrane H(cid:2)-ATPase has long been recognized to be a
promisingantifungaltarget(4–6).Theprotonpumpisessentialforfungalgrowth,as
shownbyknockoutstudies(7).ThePMA1geneencodestheH(cid:2)-ATPase,andthepump
isreferredtoasPma1.ThefungalcellisdependentonPma1creatinganelectrochem-
ical gradient across the plasma membrane, which is used by other transporters to
energizetheuptakeofionsandnutrients.Pma1pumpsprotonsfromthecytosoltothe
July2017 Volume61 Issue7 e00032-17 AntimicrobialAgentsandChemotherapy aac.asm.org 1
Kjellerupetal. AntimicrobialAgentsandChemotherapy
exterior of the cell, energized by ATP hydrolysis. In this regard, fungal cells are
fundamentally different from human cells, where the plasma membrane potential is
createdbytheNa(cid:2),K(cid:2)-ATPase(8).
Pma1belongstothetypeIIIfamilyofP-typeATPases.TherelatedhumanATPases,
Na(cid:2),K(cid:2)-ATPase, Ca2(cid:2)-ATPase (sarcoplasmic endoplasmic reticulum Ca2(cid:2)-ATPase
[SERCA]), and H(cid:2),K(cid:2)-ATPase, belong to the type II family. All mammalian ATPases
share less than 30% amino acid sequence identity with Pma1. In contrast, the fungal
H(cid:2)-ATPase appears to be relatively conserved across the fungal kingdom (the amino
acidsequencesimilarityisgenerally70to90%).Thehighlevelofconservationseenfor
Pma1 warrants efforts to identify a specific Pma1 inhibitor with broad-spectrum anti-
fungalactivity.
Withinthelast35years,anumberofnonspecificcompoundshavebeenevaluated D
as Pma1 inhibitors. To date, only a few Pma1 inhibitors, such as ebselen and the o
w
peptide BM2, have been demonstrated to inhibit the growth of living fungal cells at n
concentrationsinthelowmicromolarrange(4,5,9–12).Omeprazoleisaninhibitorof lo
a
the human H(cid:2),K(cid:2)-ATPase and has also been evaluated as an inhibitor of Pma1 (4). d
e
StudieshaveshownthatsinglemutationsintheproposedbindingsiteinPma1greatly d

alter the growth-inhibitory effects of omeprazole (4). Omeprazole requires an acid fr
o
activationsteptoinhibitPma1,andfungalgrowthinhibitionispHdependent.Forfull m
growthinhibitionofSaccharomycescerevisiae,430(cid:2)MomeprazoleisrequiredatpH3 h
t
(4),makingitalow-potencyantifungalcompound. tp
:
TheclassicalP-typeATPaseinhibitor,vanadate,doesnotinhibitlivingcellsdueto //
a
alackofmembranepenetrability.EbselenisknowntoinhibitthemammalianH(cid:2),K(cid:2)- a
c
ATPase(13)andtheCa2(cid:2)-ATPaseATP6ofPlasmodiumfalciparum(PfATP6),inaddition .
a
toPma1(14).Duetothereactivityofebselenwithproteinthiols,itisbelievedtotarget s
m
severalenzymesandmodifyarangeofbiologicalactivities(15). .
o
Naturalproducts,suchastellimagrandinII(16)andtheimmunoproteinlactoferrin r
g
(17),inhibitPma1.TellimagrandinIIpotentlyinhibitsthegrowthofS.cerevisiaebutnot /
o
thatofCandidaalbicans.LactoferrinexhibitsantifungalactivityagainstbothS.cerevi- n

siaeandC.albicansatconcentrationsinthelowmicromolarrange. N
o
BM2isacationicpeptidewhichpotentlyinhibitsPma1inanATPhydrolysisassay v
(50%inhibitoryconcentration[IC50](cid:4)0.5(cid:2)M)(10).Itdisplaysgreaterthan90%fungal em
growth inhibition at 0.63 (cid:2)M and pH 7.3 and at 5 (cid:2)M and pH 7.0. At concentrations b
e
above the MIC for S. cerevisiae, BM2 also inhibits the Na(cid:2),K(cid:2)-ATPase and HEp-2 cell r

1
growthandcausesbloodcelllysis.BM2,ebselen,andvanadatehavebeenselectedas 6
comparatorstothecompoundscharacterizedinthiswork. ,
2
Inthepresentstudy,selectedcompoundswerescreenedfortheirabilitytoinhibit 0
1
ATP hydrolysis by measuring the formation of free phosphate ions, corresponding to 8

inhibition of Pma1 under the assay conditions used. The identified inhibitors were b
y
testedfortheireffectsonthemembranepotential,theintracellularATPconcentration, g
u
extracellularmediumacidification,andfungalgrowthinhibition. e
s
t
RESULTS
Screeningandevaluationof191,000compoundsforPma1inhibition.Alarge
library of small-molecule compounds was employed with the aim of finding novel
Pma1 inhibitors. In total, 191,000 library compounds were screened using the ATP
hydrolysis assay. An overview of the screening cascade is presented in Fig. 1. Com-
pounds (at a final concentration of 20 (cid:2)M) inhibiting Pma1 enzymatic activity by
greaterthan50%wereregardedashitsinthisstudy.All407hitswerethencounter-
screened for their activity against Na(cid:2),K(cid:2)-ATPase and SERCA in order to distinguish
Pma1-specificcompoundsfromgeneralP-typeATPaseinhibitors.Allhitswerescreened
forpotentialantifungalactivityagainstS.cerevisiaeandC.albicans.
After evaluating the 407 hits, a selection of compounds was repurchased and
reevaluatedforPma1inhibitionandantifungalactivity.Thesestepswereundertakento
confirm the structural identity of the library compounds and to ensure that no
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H(cid:2)-ATPaseInhibitorswithEffectonMembranePotential AntimicrobialAgentsandChemotherapy
FIG1Overviewoflibraryscreening.HitsweretestedforselectivityforthetwomammalianATPasesSERCAandNa(cid:2),K(cid:2)-ATPase
as well as antifungal activity against Candida albicans and Saccharomyces cerevisiae. From this, several promising Pma1
inhibitorswereidentified,withcompound14beingthemostpromisingcandidate.
D
compounddegradationhadoccurredduringthelibrarystorageperiod.Thestructures o
w
ofaselectednumberofthesehitsarepresentedinFig.2.
n
Evaluation of Pma1 inhibitors. Several interesting compounds with Pma1- lo
inhibitoryactivity(IC (cid:5)25(cid:2)M)wereidentifiedthroughthelibraryscreening(Table a
50 d
1).However,anumberofthesecompounds,compounds1,2,3,6,9,11,and12,were e
d
poor antifungal compounds, with their MICs being above 150 (cid:2)M for several of the f
r
Candida spp. tested (Table 1). Compounds 4 and 8 were also poor antifungal com- o
m
pounds, with their MICs being (cid:6)38 (cid:2)M, which was the highest concentration tested
h
due to poor compound solubility in dimethyl sulfoxide (DMSO). The precipitation of t
t
p
several of these compounds observed in the fungal growth medium may partially :
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FIG2 StructuresofselectedPma1inhibitorsidentifiedinthelibraryscreening.
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Kjellerupetal. AntimicrobialAgentsandChemotherapy
TABLE1ATPhydrolysisdataandgrowthinhibitionforidentifiedPma1inhibitorsfromthelibraryscreeninga
IC ((cid:3)M) MIC((cid:3)M)
50
Compound Pma1 Na(cid:2),K(cid:2)-ATPase SERCA C.albicans C.parapsilosis C.tropicalis C.glabrata A.fumigatus
1b 0.040(cid:7)0.035 13.2(cid:7)0.91 17.5(cid:7)4.7 (cid:6)150c (cid:6)150 (cid:6)150 (cid:6)150 (cid:6)150
2 2.1(cid:7)1.3 (cid:6)105 (cid:6)105 (cid:6)150d NA NA NA NA
3 2.5(cid:7)2.5 (cid:6)105 (cid:6)105 (cid:6)150e (cid:6)150 (cid:6)150 (cid:6)150 (cid:6)150
4b 3.7(cid:7)0.7 15.0(cid:7)7.2 4.1(cid:7)0.2 13d (cid:6)38 12 (cid:6)38 (cid:6)38
5b 4.4(cid:7)0.6 1.3(cid:7)0.3 0.46(cid:7)0.04 6.5f 7.5 7.5 5.8 (cid:6)75
6b 5.0(cid:7)3.0 3.5(cid:7)3.7 3.4(cid:7)0.6 (cid:6)150d (cid:6)150 (cid:6)150 (cid:6)150 (cid:6)150
7b 5.5(cid:7)0.3 5.8(cid:7)0.8 3.8(cid:7)0.6 7.3d 8.2 26 4.7 (cid:6)150
8 6.9(cid:7)2.0 (cid:6)105 (cid:6)105 (cid:6)38d (cid:6)38 (cid:6)38 (cid:6)38 (cid:6)38
9 9.2(cid:7)1.0 (cid:6)105 (cid:6)105 (cid:6)150d (cid:6)150 (cid:6)150 (cid:6)150 (cid:6)150
10b 9.4(cid:7)1.7 (cid:6)105 3.4(cid:7)1.8 88 116 15 12 99
11 12.8(cid:7)5.0 30.3(cid:7)2.4 15.7(cid:7)2.0 (cid:6)150d (cid:6)150 (cid:6)150 (cid:6)150 (cid:6)150 D
12 15.5(cid:7)8.1 58.7(cid:7)40.2 (cid:6)105 12e (cid:6)150 (cid:6)150 (cid:6)150 (cid:6)150 o
w
13 22.6(cid:7)12.4 30.6(cid:7)11.1 22.0(cid:7)3.0 23 47 (cid:6)150 31 NA
n
14 13.7(cid:7)2.0 18.4(cid:7)2.1 42.0(cid:7)8.0 36 24 24 41 75 lo
a
aStructuresareshowninFig.2.ForIC ,dataarefrom2to3experiments.ForMICs,dataarefrom2to5experiments.NA,notavailable.
50 d
bTheATPhydrolysisassaywasperformedatpH6.5forPma1andpH7.4forNa(cid:2),K(cid:2)-ATPaseandSERCA.Forallothercompounds,theATPhydrolysisassaywas e
performedatpH7forallATPases. d
cCompoundprecipitationwasobservedinthegrowthmediumatconcentrationsdownto15(cid:2)M. fr
dCompoundprecipitationwasobservedinthegrowthmediumatconcentrationsdownto48(cid:2)M. o
m
eCompoundprecipitationwasobservedinthegrowthmediumatconcentrationsdownto150(cid:2)M.
fCompoundprecipitationwasobservedinthegrowthmediumatconcentrationsdownto75(cid:2)M. h
t
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explain their lack of antifungal activity (Table 1). Unfortunately, several of these a
a
compoundsthatlackedantifungalactivitywerethecompoundsthatwereselectivefor c
.
Pma1 rather than SERCA and Na(cid:2),K(cid:2)-ATPase. Compounds 5, 7, 10, and 13 were all a
s
potentPma1inhibitors(IC (cid:5)25(cid:2)M)withantifungalactivitybutweregenerallymore m
50 .
potent against the mammalian ATPases than against Pma1 or equally potent against o
r
boththemammalianATPasesandPma1.Themostpromisinghitfromthescreenwas g
/

compound14,apyrido-thieno-pyrimidine,whosestructureisshowninFig.3,together o
n
with the structures of selected analogs of compound 14. Compound 14 was the
N
only compound more potent against Pma1 than against the mammalian ATPases o
v
and which displayed a broad spectrum of antifungal activity against both yeasts e
m
and molds (Table 1).
b
Additionally,48commerciallyavailableanalogswerepurchasedforthisstudy,and e
r
alloftheseanalogssharedthecommonmotifshowninFig.3.Eightoftheseanalogs 1
hadanIC oflessthan20(cid:2)MforPma1.Compound14andfourofthemostpotent 6
50 ,
orselectivecommercialanalogswerechosenforfurthercharacterization(Fig.3;Tables 2
0
2 to 5) in parallel with BM2, vanadate, and ebselen. Compound 17 was chosen as a 1
8
negative control because it had a scaffold similar to that of compound 14 but weak
b
Pma1inhibition,probablyduetothelackofanimportantlipophilicinteractioninthe y

g
R1position(ethylversusn-butylincompounds17and14,respectively). u
The four most potent analogs had a low level of Pma1 selectivity (Table 2), with e
s
compound 18 being the most selective, exhibiting approximately 3- to 4-fold greater t
selectivitytowardPma1thantowardSERCAorNa(cid:2),K(cid:2)-ATPase.Compounds15,16,and
19 were more potent than compound 14 but had no selectivity for Pma1. All of the
compounds exhibited antifungal activity against both Candida and Aspergillus spp.
(Table 3), as well as a reduction in their activity on the viability/proliferation of
mammalian Hep-G2 cells (Table 4). In our hands, BM2 was 8-fold more selective for
Pma1 over Na(cid:2),K(cid:2)-ATPase inhibition but had no selectivity toward SERCA. Further-
more, BM2 inhibited the growth of S. cerevisiae and C. albicans but had no effect on
mammalianHep-G2cells.
TodetermineiftheobservedantifungalactivitywascausedbyPma1inhibitionor
another mechanism, a range of additional assays was performed. Following glucose
activation of Pma1, subsequent proton extrusion leads to acidification of the buffer
surroundingthecells.Inhibitorswhichpreventthisextracellularacidificationlikelyact
through Pma1 inhibition, although these observations alone cannot discriminate be-
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H(cid:2)-ATPaseInhibitorswithEffectonMembranePotential AntimicrobialAgentsandChemotherapy
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FIG3 Structureofcompound14andanalogs. 1
6
,

2
0
tweendirectorindirectPma1inhibition(e.g.,viamembranedisruption).Theacidifica- 1
8
tion of the external medium is measured within a few minutes after compound
b
addition. Thus, compounds which cause fast plasma membrane disruption indirectly y

affect proton transport and appear to be inhibitors of the acidification process. How- g
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TABLE2Effectsofcompound14andanalogsonATPaseactivitya
IC ((cid:3)M)forATPhydrolysis
50
S.cerevisiae C.albicans
Compound Pma1 Pma1 Na(cid:2),K(cid:2)-ATPase SERCA
14 13.7(cid:7)2.0 17.0(cid:7)0.9 18.4(cid:7)2.1 42.0(cid:7)8.0
15 5.9(cid:7)0.2 6.3(cid:7)0.5 4.1(cid:7)0.6 4.5(cid:7)0.0
16 7.8(cid:7)0.6 9.5(cid:7)1.0 9.1(cid:7)1.0 11.1(cid:7)0.0
17 106.7(cid:7)18.2 150.7(cid:7)6.2 (cid:6)333 (cid:6)333
18 18.5(cid:7)4.3 22.9(cid:7)2.0 85.7(cid:7)26.6 69.5(cid:7)6.9
19 7.3(cid:7)1.0 8.7(cid:7)0.5 5.5(cid:7)1.8 4.4(cid:7)0.3
BM2 1.2(cid:7)0.1 0.9(cid:7)0.2 10.4(cid:7)2.7 1.1(cid:7)0.0
Vanadate 0.25(cid:7)0.01 0.23(cid:7)0.05 0.006(cid:7)0.001 10.4(cid:7)2.4
Ebselen 0.92(cid:7)0.15 0.51(cid:7)0.04 0.16(cid:7)0.01 0.12(cid:7)0.00
aExperimentswereperformedatpH7,andstandarddeviations(n(cid:4)2)aregiven.RabbitCa2(cid:2)-ATPase
(SERCA1a)andporcinekidneyNa(cid:2),K(cid:2)-ATPasewereused.
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Kjellerupetal. AntimicrobialAgentsandChemotherapy
TABLE3Antifungalactivitiesofcompound14andanalogsagainstyeastandmoldspecies
MIC((cid:3)M)forgrowthinhibition
Compound S.cerevisiae C.albicans C.parapsilosis C.tropicalis C.glabrata A.fumigatus A.flavus
14 7.5 36 24 24 41 75 75
15 1.5 4.8 15 47 15 15 24
16 3.2 15 15 28 11 15 47
17 47 150 150 150 150 (cid:6)150 (cid:6)150
18 24 75 24 58 41 75 (cid:6)75
19 3.7 6 3.7 3.7 3.7 12 12
BM2 4.7 15 26 4.7 150 (cid:6)150 (cid:6)150
Vanadate (cid:6)750 (cid:6)750 (cid:6)750 (cid:6)750 (cid:6)750 (cid:6)750 (cid:6)750
Ebselen 7.5 7.5 24 11 7.5 24 24
D
o
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ever, amphotericin B, a marketed antifungal which forms pores in the fungal mem- n
brane,didnotinhibitextracellularacidificationintheassaytimeframeof12min(Table lo
a
5).Compound14anditsanalogsinhibitedtheacidificationprocessatconcentrations d
e
similartotheIC forPma1intheATPhydrolysisassay(Tables2and5),whichsupports d
50

the direct inhibition of Pma1. BM2 was slightly more potent in inhibiting the acidifi- fr
o
cation process than inhibiting the purified Pma1 enzyme in the ATP hydrolysis assay m
(Tables 2 and 5). The IC50 of ebselen for C. albicans was found to be 11 (cid:2)M, in h
agreementwiththepreviouslyreportedvalueof14(cid:2)M(18). tt
p
ThemembranepotentialdecreasesuponPma1inhibitionwithoutchangesin :/
/
membraneintegrity.Astrikingfeatureofthefungalcellmembraneistheveryhigh a
a
membranepotentialgeneratedbyPma1.Ithasbeenmeasuredtobe(cid:8)175mVwithin c
.
a
the fungal species Neurospora crassa (19). This value is significantly higher than that s
m
observedinmammaliancells,wherethemembranepotentialistypicallymeasuredto
.
be(cid:8)65to(cid:8)85mV.TheN.crassameasurementswereperformedonthelargehypha o
r
g
ofthismoldspecieswithpatchclampelectrodes,asetupwhichistechnicallydifficult /

to reproduce on smaller cells, such as those of S. cerevisiae. To further demonstrate o
n
Pma1 inhibition in living fungal cells, image cytometry was employed to investigate N
membrane potential changes and membrane integrity upon compound exposure. o
v
Membrane depolarization was assessed with the fluorescent probe bis-(1,3-dibutyl- e
m
barbituric acid) trimethine oxonol [DiBAC (3)], which is only able to enter cells with a
4 b
decreasedmembranepotential.Simultaneously,propidiumiodide(PI)wasusedtodistin- e
r
guishbetweenmembranedepolarizationandgeneralmembranepermeabilization,sincePI 1
6
onlyenterscellswithpermeablecellmembranes. ,

2
S. cerevisiae cells exposed to compound 14 yielded an elevated DiBAC (3) signal
4 0
after 5 min of exposure, while only a minimal increase of the PI signal was observed 1
8
(Fig.4AtoCandFig.S1).After15or30minofexposure,thesizesofbothDiBAC4(3)- b
positivecellpopulationsandDiBAC (3)-andPI-positivecellpopulationshadincreased. y
4 g
These observations suggest that cells were first depolarized prior to the membrane u
e
integrity becoming compromised, thus allowing PI to enter the cells. After 30 min of s
t
exposure to compound 14, only 16% of the cells remained both PI and DiBAC (3)
4
negative, 44% were DiBAC (3) positive and PI negative, and 39% were both PI and
4
TABLE4EffectsofPma1inhibitorsonHep-G2cells
Compound EC ((cid:3)M)forHep-G2cellsat24h
50
14 2.3(cid:7)0.0
15 2.8(cid:7)0.1
16 2.6(cid:7)0.9
17 5.8(cid:7)1.1
18 3.6(cid:7)1.1
19 24.2(cid:7)0.5
BM2 (cid:6)100
Vanadate (cid:6)500
Ebselen 10.7(cid:7)7.0
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H(cid:2)-ATPaseInhibitorswithEffectonMembranePotential AntimicrobialAgentsandChemotherapy
TABLE5EffectsofPma1inhibitorsonmediumacidificationinS.cerevisiaeand
C.albicansa
IC ((cid:3)M)foracidification
50
Compound S.cerevisiae C.albicans
14 11.2(cid:7)2.7 6.6(cid:7)2.5
15 1.7(cid:7)0.1 1.6(cid:7)0.0
16 7.6(cid:7)3.4 3.4(cid:7)1.6
17 39.6(cid:7)10.3 19.1(cid:7)1.7
18 12.5(cid:7)1.1 8.8(cid:7)0.4
19 2.3(cid:7)0.1 2.3(cid:7)0.5
BM2 0.2(cid:7)0.0 0.5(cid:7)0.1
Vanadate (cid:6)40 (cid:6)40
Ebselen 7.6(cid:7)0.0 9.4(cid:7)2.1
AMB (cid:6)75 (cid:6)75 D
o
aTheIC wasdeterminedtobetheconcentrationwhichresultedin50%inhibitionofmediumacidification w
50
normalizedtotheresponsefromglucose-activatedversusnon-glucose-activatedcells.AMB,amphotericinB. n
IC withstandarddeviations(n(cid:4)2to3)areindicated. lo
50 a
d
e
d
DiBAC (3)positive.Only0.7%ofthecellswerePIpositivebutDiBAC (3)negative.Inthe
4 4 f
r
DMSOcontrolsample,92%ofthecellswerebothPIandDiBAC (3)negative. o
4 m
The most potent antifungal analog of compound 14 was compound 19, and this

h
compoundwasalsoevaluatedwithimagingcytometryatconcentrationsof5and15 t
t
(cid:2)M.Similarlytocompound14,exposureofcellstocompound19alsoresultedintwo p
:
/
large populations of DiBAC4(3)-positive and PI-negative cells and DiBAC4(3)- and PI- /a
a
positivecellsafter30minofexposure.
c
BM2exposureledtoamarkedincreaseinthePI-positivepopulationwithin5min, .a
s
indicatingrapideffectsonmembraneintegrity(Fig.4AandFig.S1).However,wealso m
observedanincreaseintheDiBAC4(3)signalafter15or30mininBM2-treatedcells.Given .o
r
g
/

o
n

N
o
v
e
m
b
e
r

1
6
,

2
0
1
8

b
y

g
u
e
s
t
FIG4 EffectsofinhibitorsonmembranepotentialandmembraneintegrityinS.cerevisiae.(A)Barchartofthecumulativepercentage
ofcellsthatwereDiBAC(3)positiveornegativeandPIpositiveornegative,asdefinedbythequadrantsinpanelB.Cellsweretreated
4
withcompound14,17,19,orBM2orCCCPat5,15,or150(cid:2)Mfor5,15,or30min(asindicated).ErrorbarsindicatingSEMsareshown
inonlyonedirectionforclarity.(B)Scatterplotofcellstreatedwith1%DMSOfor30min(control).(C)Scatterplotofcellstreatedwith
15(cid:2)Mcompound14for30min.RepresentativescatterplotsforallconditionsareshowninFig.S1inthesupplementalmaterial.RFU,
relativefluorescentunits.
July2017 Volume61 Issue7 e00032-17 aac.asm.org 7
Kjellerupetal. AntimicrobialAgentsandChemotherapy
D
o
w
n
FIG5Pma1inhibitorsincreaseiATPlevelsinS.cerevisiae(A)andC.albicans(BandC).ThetotalcellularATPconcentrationsweredeterminedafter lo
a
30minoftreatment.Themeandata(n(cid:4)3)withtheSDareindicatedasthepercentchangecomparedtothatfortheuntreatedcontrolsample d
(1%DMSO)(AandB)orastheconcentration(innanomolar)ofiATP(C).olig.,oligomycinA(at40(cid:2)Minallpanels).*,P(cid:5)0.05comparedtothe e
untreatedcontrolbyunpairedttest;**,P(cid:5)0.01comparedtotheuntreatedcontrolbyunpairedttest. d

f
r
o
m
theeffectsonmembraneintegrity,itisdifficulttoattributetheincreasedDiBAC (3)signal
4 h
todirectPma1inhibition.Theprotonophorecarbonylcyanidem-chlorophenylhydrazone tt
p
(CCCP)hadaneffectonthePIandDiBAC4(3)signalssimilartothatofcompounds14and ://
19.Compound17,whichhadanIC of107(cid:2)MforpurifiedS.cerevisiaePma1,didnot a
50 a
increase the DiBAC (3) signal in S. cerevisiae cells, and the PI signal was not significantly c
4 .a
increased. s
m
iATPlevelsincreaseuponPma1inhibition.Pma1requiresATPtoenergizeproton
.
transport out of the fungal cell. A decrease in the intracellular ATP (iATP) levels o
r
g
indirectly affects the activity of Pma1. To rule out the possibility that the Pma1 /

inhibitionobservedwasindirectlycausedbydecreasedATPlevels,wedeterminediATP o
n
levelsaftercompoundexposure(Fig.5AtoC).TreatmentofS.cerevisiaeandC.albicans N
cellswithcompound14ledtoasignificantincreaseiniATPlevels(200%and1,500%, o
v
respectively)comparedtothosefortheuntreatedcontrol.WhenS.cerevisiaecellswere e
m
exposed to compound 14 in the presence of oligomycin, an ATP synthase inhibitor,
b
iATPlevelsremainedcomparabletothoseforcontrolcells.Compound19behavedin e
r
a manner similar to that for compound 14, leading to a significant increase in iATP 1
6
levelsinS.cerevisiaeandC.albicans.TreatmentwithBM2alsoledtoalargeincrease ,

inATPlevels((cid:3)1,100%)comparedtothatforthecontrolcells.WhenS.cerevisiaecells 2
0
weretreatedwithBM2andoligomycintogether,theincreaseiniATPlevelswaslimited 1
8
to300%.However,theadditionofoligomycintoBM2-treatedC.albicanscellsdidnot b
altertheiATPlevelcomparedtothatincellstreatedwithBM2alone.Treatmentwith y

g
oligomycinalone,CCCP,vanadate,andthelow-potencyPma1inhibitorcompound17 u
e
had minimal effects on iATP levels compared to those found after treatment with s
t
compounds14,19,andBM2.Ebselencouldnotbetested,asitinhibitedtheluciferase
enzymeusedintheiATPassay.TheincreaseiniATPlevelsinC.albicansforcompounds
14, 19, and BM2 was shown to be dose responsive, with all compounds producing a
maximum iATP level of about 500 nM (per well) at the highest concentrations of
inhibitorstested(Fig.5C).Thehalf-maximal(50%)effectiveconcentrations(EC )were
50
calculated to 13, 7, and 2 (cid:2)M for compounds 14, 19, and BM2, respectively. Interest-
ingly,thesevalueswerefairlyclosetotheIC forthePma1enzymefromC.albicans
50
of17,9,and0.9(cid:2)M,respectively.
DISCUSSION
In the initial screening process, we identified a number of selective and relatively
potent Pma1 inhibitors; however, many lacked antifungal activity. One interesting
compoundserieswhichexhibitedbothantifungalactivityandanaffinityforPma1over
themammalianATPaseswasidentifiedandfurthercharacterized.Aninitialunderstand-
July2017 Volume61 Issue7 e00032-17 aac.asm.org 8
H(cid:2)-ATPaseInhibitorswithEffectonMembranePotential AntimicrobialAgentsandChemotherapy
ingofthestructure-activityrelationshipforthisserieswasestablishedonthebasisof
suchrelationshipsforcommerciallyavailablecompounds.
Thecompound14series,containingapyrido-thieno-pyrimidinegroup,wasconsid-
ered especially interesting since small changes to the scaffold yielded significant
changesintheinhibitorypropertiestowardthethreeATPasestested.Ourdatasuggest
adirectinteractionwiththeATPasesratherthananinteractionviacompoundaggre-
gates, which is a common false-positive mechanism (20, 21). 4-Aminoalkylamino de-
rivativesofpyrido[3=,2=:4,5]thieno[3,2-d]pyrimidinehavebeenshowntointeractwith
anumberofmoleculartargets,includingphosphodiesteraseIV,andtohavepotential
useinthetreatmentofasthmaandchronicobstructivepulmonarydisease(22;S.L.M.
PagesandM.J.Taltavull,U.S.patentapplication,June2006)andinthecontroloftumor
necrosis factor alpha (TNF-(cid:3)) release (23) and to have beta2 adrenoreceptor agonist D
activity (24). Additionally, diverse biological activities, such as anticonvulsant (25–27) o
w
andneurotropic(28,29)activities,havebeenascribedtocompoundssimilartothose n
presented here. To our knowledge, this is the first description of pyrido-thieno- lo
a
pyrimidinesasinhibitorsofP-typeATPases.ThePma1-inhibitoryactivitywasconfirmed d
e
both through the inhibition of acidification of the extracellular medium and through d
thedemonstrationofmembranedepolarizationbutnotanimmediatelossofintegrity f
r
o
bytheDiBAC4(3)assay. m
DiBAC (3) and PI are frequently used as stains for dead or dying cells. However,
4 h
DiBAC4(3)entersthecellsduetoalossofmembranepotential,whereasPIentersthe ttp
cellsduetoalossofmembraneintegrity.Byusingashorttimeframe(5to30min)and :
/
/
observing individual cells, DiBAC (3) can be used to estimate changes in membrane a
4 a
potential, while costaining with PI reveals when the membrane loses its integrity. c
.
a
Previously, DiBAC (3) (30) and other fluorescent probes used to measure membrane
4 s
potential,suchas3,3-dipropylthiacarbocyanideiodide)[diS-C3(3)](31),havebeenused m
.
toestimatechangesinmembranepotential.Toourknowledge,DMM-11,acompetitive o
r
inhibitor of Mg2(cid:2)-ATP, is the only other Pma1 inhibitor which has previously been g
/

showntodepolarizethefungalplasmamembrane(12). o
n
TheprotonophoreCCCPwasusedasapositivecontrolinthemembranepotential
N
study; however, it required high concentrations to overcome the proton excretion o
v
inducedbyPma1anddepolarizethemembrane.CCCPhaspreviouslybeenshownto e
m
affectthemitochondrialmembraneand,thereby,thesupplyofATP(32).Inthisstudy,
b
treatmentwithCCCPresultedinlimitedchangestotheiATPlevelsinS.cerevisiaeand e
r
C.albicans(Fig.5).TreatmentwiththeATPsynthaseinhibitoroligomycindidnotresult 1
6
inalargesignificantchangeiniATPlevels,inagreementwithearlierobservationsby
,

Andrésetal.(17).ThesedatasuggestthatfungalcellscancompensateforATPsynthase 2
0
inhibitionforshortperiodsoftimeundertheseconditions. 1
8
Compounds14,19,andBM2gaverisetoalargesignificantincreaseiniATPlevels,
b
whilecompound17(alow-potencyPma1inhibitor)gaverisetoasubstantiallylower y

g
increase. Increases in iATP levels have previously been reported with exposure to u
omeprazole (36% increase) in S. cerevisiae (33) and lactoferrin ((cid:6)600% increase) in C. e
s
albicans (17). Both omeprazole and lactoferrin are described to be Pma1 inhibitors, t
although omeprazole is not fully capable of inhibiting extracellular acidification (33).
WesuggestthatanincreasediATPlevelisattributabletoPma1inhibition,considering
thatPma1isthemajorconsumerofATPinthecellandisthoughttoconsume20to
50%ofthecellularATP(34).Therefore,unusedATPaccumulatesinthecellwhenPma1
isinhibited.IncreasediATPlevelshavealsobeenobservedinPma1mutantswithpartial
defectsinpumpingactivity(35),supportingthehypothesisthatincreasediATPlevels
areaconsequenceofPma1inhibition.
EbselenwasfoundtoinhibitnotonlyPma1butalsothemammalianATPasesSERCA
andNa(cid:2),K(cid:2)-ATPase.GiventhatebselenhaspreviouslybeenreportedtoinhibitPfATP6
and the H(cid:2),K(cid:2)-ATPase (13, 14), this compound is likely an unspecific P-type ATPase
inhibitor.
Inourhands,BM2exhibitedlessselectivityforS.cerevisiae(ATCC9763)Pma1over
porcineNa(cid:2),K(cid:2)-ATPasethanthepreviouslyreported50-foldselectivityofS.cerevisiae
July2017 Volume61 Issue7 e00032-17 aac.asm.org 9
Kjellerupetal. AntimicrobialAgentsandChemotherapy
(T48)Pma1overcanineNa(cid:2),K(cid:2)-ATPase,determinedwhentheIC ofS.cerevisiae(T48)
50
Pma1 and canine Na(cid:2),K(cid:2)-ATPase were compared (10). Furthermore, we observed no
selectivity of BM2 for Pma1 over SERCA. BM2 affected the membrane potential, but
given the rapid and severe effects on overall membrane integrity, it is unclear if the
depolarization was directly caused by Pma1 inhibition. Increased cell permeation of
rhodamine6G(Rh6G)wasalsopreviouslyobservedinanRh6Geffluxassaywith(cid:6)10
(cid:2)MBM2,whichfurthersupportsourobservationsthatBM2compromisesmembrane
integrity(10).Despitethedecreasedmembraneintegrity,ourresultssuggestthatPma1
wasinhibited,asindicatedbytheobservedlargeincreaseiniATPlevels.
Oligomycin did not completely prevent the rise in iATP levels of S. cerevisiae cells
treated with BM2. Furthermore, iATP levels in C. albicans cells were significantly
changed in response to BM2 exposure, regardless of the presence of oligomycin. We D
thereforespeculatethattheinhibitionofATPsynthasebyoligomycinisslowerthanthe o
w
accumulationofiATPresultingfromtheactionsofBM2.Wespeculatethattheincrease n
in iATP levels due to Pma1 inhibition, together with BM2’s additional membrane- lo
a
disruptiveeffects,facilitatesthekillingoffungalcells. d
e
Together,ourdatasupportthesuggestionthatBM2operatesviaadualmechanism d
whereboththefungalmembraneandthefungalprotonATPaseareaffected,leading f
r
o
to fungal growth inhibition and cell death. Greater selectivity toward SERCA and the m
Na(cid:2),K(cid:2)-ATPase is likely required. Special attention should be paid to possible mem-
h
braneinteractions,asBM2affectsfungalmembraneintegrityandhaspreviouslybeen tt
p
linkedtolow-levelhemolysisanddetectabletoxicityinHEp-2cells(10).Onthebasisof :
/
/
themembranepotentialandintegrityresults,thecompound14serieshasamodeof a
a
actiondifferentfromthatofBM2.Theantifungaleffectofcompound14appearstobe c
.
a
moreadirectconsequenceofPma1inhibitionascomparedtoBM2,asthelatteraffects
s
both Pma1 activity and the membrane integrity. As expected for an inhibitor of the m
.
essentialandconservedPma1protein,thecompound14serieshasabroadspectrum o
r
ofantifungalactivity,inhibitingthegrowthofallCandidaandAspergillusspp.tested. g
/

This is in contrast to BM2, which has poor activity against Candida glabrata and the o
n
Aspergillusspp.
N
SomeofthecompoundsidentifiedduringthelibraryscreeninhibitedPma1onan o
v
enzymaticlevel;however,thisdidnotalwaystranslateintoantifungalactivity.Thereare e
m
severalplausibleexplanationsforthislackoftranslation.Fungalcellscontainacomplex
b
cell wall, which may serve as an impermeable barrier to compound entry. The com- e
r
pounds might not be able to cross the cell membrane due to aggregate formation, 1
6
leadingtofalse-positivehitsintheenzymaticassay.False-positivehitsmayalsoresult
,

from unspecific protein inhibition activity in a homogeneous assay setup (20, 21). 2
0
Finally,thechemicalpropertiesofthecompoundsmustbetakenintoconsideration,as 1
8
the possibility that some of the compounds tested precipitated when approaching
b
concentrationsrequiredfortheireffectcannotberuledout. y

g
Inthisstudy,novelcompoundswithPma1-inhibitorypropertieswereidentifiedand u
direct inhibition of Pma1 in fungal cells was confirmed by a decrease in the plasma e
s
membrane potential, while membrane integrity was preserved. Encouragingly, com- t
pounds 14 and 18 displayed selectivity against the mammalian ATPases, with both
showing3-foldselectivitytowardSERCAandcompound18showing4-foldselectivity
toward the Na(cid:2),K(cid:2)-ATPase. Compound 19 was interesting, as it displayed a 3-fold
toxicitywindowbetweenC.albicansandHep-G2cells,despiteitbeinganunselective
ATPaseinhibitor.However,thefungalgrowthandHep-G2cellassayconditionsdiffered
significantlybythepresenceof10%fetalbovineserum(FBS)intheHep-G2cellassay
mixture.InC.albicansgrowthassays,a10-foldincreaseintheMICvalueofcompound
19 occurred when 10% FBS was added to the assay mixtures (see Table S2 in the
supplementalmaterial).Thissuggestssignificantproteinbindingofcompound19.The
reducedtoxicitywindowobservedwhenfungalandHep-G2cellassayswererununder
comparable conditions also highlights that selectivity for Pma1 over mammalian
ATPasesshouldhavehighpriorityinanyfuturePma1drugdevelopment.
The collection of methods presented herein is especially useful in antifungal drug
July2017 Volume61 Issue7 e00032-17 aac.asm.org 10