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Jou nal o Enzyme Inhibi ion and Medicinal Chemis y
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β-CA-speci ic inhibi o di hioca bama e Fc14–584B:
a no el an imycobac e ial agen wi h po en ial o
ea d ug- esis an ube culosis
Ashok Aspa wa , Milka Hamma én, Sanni Koskinen, B uno Luukinen, Ha lan
Ba ke , Fab izio Ca a, Claudiu T. Supu an, Ma aleena Pa ikka & Seppo
Pa kkila
To ci e his a icle: Ashok Aspa wa , Milka Hamma én, Sanni Koskinen, B uno Luukinen, Ha lan
Ba ke , Fab izio Ca a, Claudiu T. Supu an, Ma aleena Pa ikka & Seppo Pa kkila (2017) β-CA-
speci ic inhibi o di hioca bama e Fc14–584B: a no el an imycobac e ial agen wi h po en ial o ea
d ug- esis an ube culosis, Jou nal o Enzyme Inhibi ion and Medicinal Chemis y, 32:1, 832-840,
DOI: 10.1080/14756366.2017.1332056
To link o his a icle: h p://dx.doi.o g/10.1080/14756366.2017.1332056
© 2017 The Au ho (s). Published by In o ma
UK Limi ed, ading as Taylo & F ancis
G oup.
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Published online: 20 Jun 2017. Submi you a icle o his jou nal
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ORIGINAL ARTICLE
b-CA-speci ic inhibi o di hioca bama e Fc14–584B: a no el an imycobac e ial
agen wi h po en ial o ea d ug- esis an ube culosis
Ashok Aspa wa
a
, Milka Hamma 
en
a
, Sanni Koskinen
a
, B uno Luukinen
a
, Ha lan Ba ke
a
, Fab izio Ca a
b
,
Claudiu T. Supu an
b
, Ma aleena Pa ikka
a
†and Seppo Pa kkila
a,c
†
a
Facul y o Medicine and Li e Sciences, Uni e si y o Tampe e, Tampe e, Finland;
b
Neu o a ba Depa men , Sezione di Chimica Fa maceu ica e
Nu aceu ica, Uni e si 
a degli S udi di Fi enze, Ses o Fio en ino (Fi enze), I aly;
c
Fimlab L d. and Tampe e Uni e si y Hospi al, Tampe e, Finland
ABSTRACT
Inhibi ion o no el biological pa hways in Mycobac e ium ube culosis (M b) c ea es he po en ial o al e -
na i e app oaches o ea ing d ug- esis an ube culosis. In i o s udies ha e shown ha di hioca ba-
ma e-de i ed b-ca bonic anhyd ase (b-CA) inhibi o s Fc14–594 A and Fc14–584B e ec i ely inhibi he
ac i i y o M b b-CA enzymes. We sc eened he di hioca bama es o oxici y, and s udied he in i o
inhibi o y e ec o he leas oxic inhibi o on M. ma inum in a zeb a ish model. In ou oxici y sc eening,
Fc14–584B eme ged as he leas oxic and showed minimal oxici y in 5-day-old la ae a 300 mM concen-
a ion. In i o inhibi ion o M. ma inum showed ha bo h compounds inhibi ed g ow h a a concen a-
ion o 75 mM. In i o inhibi ion s udies using 300 mM Fc14–584B showed signi ican (p>.05) impai men
o bac e ial g ow h in zeb a ish la ae a 6 days pos in ec ion. Ou s udies highligh he he apeu ic po en-
ial o Fc14–584B as a b-CA inhibi o agains M b, and ha di hioca bama e compounds may be de eloped
in o po en an i- ube culosis d ugs.
ARTICLE HISTORY
Recei ed 4 May 2017
Accep ed 15 May 2017
KEYWORDS
Di hioca bama es;
Mycobac e ium ma inum;
b-ca bonic anhyd ase; in
i o inhibi ion; zeb a ish
emb yos
In oduc ion
Tube culosis (TB) caused by M b is highly con agious and easily
sp eads h ough ai bo ne d ople s.
1
The la es es ima es show ha
2 billion people wo ldwide a e cu en ly in ec ed wi h he la en
o m o TB. In 2015, 10.4 million people de eloped ac i e TB, and
1.8 million people died o he disease.
2
An i-TB d ugs we e in o-
duced 40 yea s ago, bu hese ha e become less e ec i e due o
he de elopmen o d ug esis ance. The e is an u gen need o
sa e and po en new d ugs o he ea men o mul i-d ug esis -
an (MDR)-TB. In addi ion, i would be highly desi able o hese
new d ugs o be e ec i e agains he la en o m o TB.
Using sequenced mycobac e ial genomes and p o eome analy-
ses, i is possible o iden i y pa hways ha a e essen ial o he li e
cycle o M b.
3,4
Ca bonic anhyd ase (CA) enzymes o pa hogenic
mic oo ganisms a e possible no el d ug a ge s.
5–7
CA enzymes
ca alyze he e e sible hyd a ion o ca bon dioxide (CO
2
) o bica -
bona e (HCO
3
–) and p o ons (H
þ
), and a e essen ial o many
physiological p ocesses, such as a y acid biosyn hesis, egula ion
o pH homeos asis, and su i al o cells unde hypoxia.
6
Se e al
s udies ha e shown ha he enzyma ic ac i i y o a- and b-CAs
can be success ully inhibi ed bo h in i o and in i o using a ious
inhibi o s, including sul onamides and phenolic acids.
8,9
In he
pas , esea ch has shown ha e hoxzolamide, a sul onamide CA
inhibi o , a enua es i ulence o M b by inhibi ing he exp ession
o i ulence ac o s ha a e c ucial o pa hogenesis.
10
In addi ion,
ecen esea ch showed ha CA inhibi o e hoxzolamide signi i-
can ly educed ex acellula DNA (eDNA) expo as bica bona e
posi i ely in luences eDNA expo in a pH-dependen manne in
M. a ium, M. abscessus, and M. chelonae.
5
The eDNA is an in eg al
pa o bio ilm ma ix o many pa hogens, including M b, and bac-
e ia wi hin bio ilm a e mo e ole an o an ibio ics han mic oo -
ganisms g own plank onically.
11
These s udies sugges ha b-CAs
a e in ol ed in exp ession o i ulence ac o s and he expo o
eDNA in mycobac e ial species, and ha inhibi ion o mycobac e -
ial CAs using chemical inhibi o s could a enua e he i ulence
and educe bio ilm o ma ion.
5,10
S udies ha e shown ha he b-CAs a e essen ial o g ow h
and su i al o M b in he hos o ganism.
12
M b is capable o su -
i al and g ow h in ad e se hos en i onmen s, and has h ee
b-CAs. Impo an ly, humans lack b-CAs, sugges ing ha he d ugs
a ge ed agains he b-CAs o M b would be less ha m ul wi h
ewe side e ec s. Thus, he b-CAs o M b could se e as excellen
a ge s o d ug de elopmen .
Supu an’s g oup has p e iously iden i ied a no el class o an i-
mycobac e ial agen s ha a ge he b-CAs o M b.
13,14
These
di hioca bama es (DTCs) inhibi bo h M b CA1 and CA3 in i o by
binding o he ac i e si e o he enzymes.
13
Howe e , o da e,
none o hese agen s ha e been sc eened o oxici y and sa e y
in animals and no in i o inhibi ion s udies ha e been conduc ed
using model o ganisms.
M. ma inum is a close ela i e o M b and a na u al pa hogen
o zeb a ish (Danio e io).
4
The zeb a ish model has been success-
ully used o modeling di e en aspec s o human ube culosis
du ing he las 15 yea s.
15,16
In he adul zeb a ish model, he ole
o adap i e immune esponses and he wide spec um o disease
CONTACT Ashok Aspa wa [email p o ec ed] Facul y o Medicine and Li e Sciences, Uni e si y o Tampe e, Tampe e, Finland
These au ho s con ibu ed equally o his wo k.
†These au ho s con ibu ed equally o his wo k as las au ho s.
Supplemen al da a o his a icle can be accessed he e.
ß2017 The Au ho (s). Published by In o ma UK Limi ed, ading as Taylo & F ancis G oup.
This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed use,
dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2017
VOL. 32, NO. 1, 832–840
h ps://doi.o g/10.1080/14756366.2017.1332056
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ou comes including la en and eac i a ed in ec ion ha e been
assessed.
17,18
The anspa en zeb a ish la ae, on he o he hand
is well-es ablished as he pe ec model o dissec ing he ea ly
s ages o ac i e mycobac e ial in ec ion
19,20
and as a pla o m o
apid disco e y and oxici y es ing o new an ibio ics.
21
In his s udy, we e alua ed he sa e y and oxici y o he wo
DTCs, Fc14–594 A and Fc14–584B and s udied he inhibi o y p op-
e ies o hese d ugs in i o and in i o using M. ma inum and
zeb a ish as model o ganisms. The s uc u es o he compounds
ha we e used in he p esen s udy a e shown in Figu e 1.
Ma e ials and me hods
Inhibi o s
The wo DTCs Fc14–594 A and Fc14–584B (Figu e 1) used in he
s udy we e p epa ed om he co esponding amine by eac ing
wi h ca bon disul ide in he p esence o a base as epo ed ea -
lie .
16
In i o, he DTCs we e in es iga ed as speci ic inhibi o s o
wo M b b-CA enzymes (M b CA1 and M b CA3).
13
The DTC com-
pounds we e dissol ed in deionized and dis illed wa e (ddH
2
O) o
p epa e 100 mM s ock solu ions. Se ies o dilu ions o each com-
pound we e ca ied ou in ddH
2
O be o e oxicological expe imen s.
Main enance o zeb a ish and e hical s a emen
Wild ype zeb a ish o he AB s ains we e main ained a 28.5 Cin
an incuba o , as desc ibed p e iously.
17
The 1–2 h pos e iliza ion
(hp ) emb yos we e collec ed om b eede anks using a sie e
and insed wi h emb yonic medium [5.0 mM NaCl, 0.17 mM KCl,
0.33 mM CaCl
2
, 0.33 mM MgSO
4
, and 0.1% w/ Me hylene Blue
(Sigma-Ald ich, Ge many)]. All zeb a ish expe imen s we e done a
he zeb a ish co e acili y, Uni e si y o Tampe e, using zeb a ish
la ae younge han 7 days pos e iliza ion (dp ) (p ojec license
no needed). The zeb a ish co e acili y a Uni e si y o Tampe e
has an es ablishmen au ho iza ion g an ed by he Na ional
Animal Expe imen Boa d (ESAVI/7975/04.10.05/2016). To a oid
unnecessa y s ess o he ish, T icaine (Sigma-Ald ich, S . Louis,
MO) was used as an anes he ic p io o in ec ing and o eu haniz-
ing p io o end-poin analysis.
E alua ion o sa e y and oxici y o he DTCs
De e mina ion o LC
50
To de e mine an LC
50
alue o compound Fc14–594 A, 8 g oups
o 30 wild ype zeb a ish emb yos we e exposed o a ying
concen a ions o he d ug (0, 1, 10, 20, 25, and 30 mM). A e
emo ing coun s o any emb yos which died wi hin 24 hp , su -
i al a ios we e hen used o calcula e a dose esponse cu e
(DRC) using he DRM module o he DRC R(15) package.
16
Simila ly, a DRC was p oduced o compound Fc14–584B a e 13
g oups o 30 ish we e exposed o a ying concen a ions o he
d ug (0, 300, 500, 600 mM). As he con ol g oup, we included an
equal numbe o wild ype la ae (un ea ed). The expe imen s
we e ca ied ou in 24-well pla es (Co ning
V
R
Cos a
V
R
cell cul u e
pla es). In each well, we placed h ee 1–2 hp emb yos in 1 ml o
emb yonic medium con aining ei he dilu ed inhibi o o wi hou
any d ug. In o al, i e se s o expe imen s we e ca ied ou o
each inhibi o . Emb yo su i al was checked e e y 24 h un il 5 days
a e i s exposu e.
Pheno ypic analysis o inhibi o - ea ed and con ol emb yos
and la ae
The ish we e moni o ed e e y 24 h, and dead ish and deb is
we e emo ed. Six pheno ypic pa ame e s (mo emen , yolk sack,
ha ching, hea bea , body shape and edema) o he 0–5 dp ish
we e eco ded. The images we e aken using a Luma V1.12 luo -
escence s e eomic oscope a ached o a came a wi h a 1.5lens
(Ca l Zeiss Mic oImaging GmbH, G€
o ingen, Ge many). The images
we e analyzed wi h AxioVision so wa e e sions 4.7 and 4.8. We
used 5 dp ish o mo phological examina ion using his ochemical
s aining.
His ochemical analysis
Rep esen a i e la ae we e collec ed o his ological examina ion
om each g oup, a he end o 5 dp . The his ological s udies
we e done o analyze he mo phology o 5 dp zeb a ish la ae
exposed o di e en concen a ions o DTCs, and con ol g oup
zeb a ish la ae. A he end o 5 dp , he la ae we e washed wi h
PBS and ixed in 4% pa a o maldehyde (PFA) in PBS o 3 h a
oom empe a u e. A e he ixa ion, he la ae we e ans e ed
o 70% e hanol and s o ed a 4 C be o e being embedded in pa -
a in. The pa a in embedded samples we e sec ioned in o 5 mM
slices o he his ochemical s aining. The ixed sec ions con aining
samples we e depa a inized in xylene, ehyd a ed in an alcohol
se ies, and his ologically s ained wi h Maye 's Hema oxylin and
Eosin Y (bo h om Sigma-Ald ich). A e dehyd a ion, he slides
we e moun ed wi h En ellanNeu
TM
(Me ck; Da ms ad , Ge many).
The slides con aining he issues we e examined o he p esence
o pa hological changes acco ding o OECD guidelines
18
and pho-
og aphed using a Nikon Mic opho mic oscope (Nikon Mic opho -
FXA, Japan). All he p ocedu es we e ca ied ou a oom
empe a u e.
Isola ion o o al RNA and e e se ansc ip ion
Th ee s ains o M. ma inum (ATCC 927, ATCC BAA-535/M, and
E11) we e cul u ed, as desc ibed in he Ma e ials and Me hods sec-
ion “M. ma inum in ec ions o zeb a ish la ae”, bu wi hou
Hyg omycin B. The RNA ex ac ion was pe o med om bac e ial
pelle s o 30 mg using RNeasy
V
R
Mini ki (Qiagen, Hilden, Ge many),
ollowing he manu ac u e 's ins uc ions. Pu i y and concen a ion
o o al RNAs om bac e ial samples we e de e mined using a
NanoD op Spec opho ome e (The moScien i ic, Wal ham, MA) a
260 and 280 nm. A e e se ansc ip ase- eac ion was pe o med
o 50 ng o o al RNA in a olume o 50 ml using a Fi s S and
cDNA Syn hesis ki (High-Capaci y cDNA Re e se T ansc ip ion Ki s,
Figu e 1. Chemical s uc u es o he compounds used in he s udy: The DTCs
Fc14–584B and Fc14–494 A a e a new class o po en b-CA inhibi o s ha bind
he zinc ion om he enzyme ac i e si e in monoden a e manne . Bo h enzymes
we e inhibi ed wi h e icacies be ween he subnanomola o he mic omola
ange (Ki ¼0.94–893 nM), depending on he subs i u ion pa e n a he ni ogen
a om om he di hioca bama e zinc-binding g oup.
13
JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY 833
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Applied Biosys ems, Fos e Ci y, CA), andom p ime s and M-MuLV
e e se ansc ip ase, acco ding o he p o ocol ecommended by
he manu ac u e .
Phylogene ic and sequence analyzes
A selec ion o insec , pa asi e and mycobac e ium b-CA amino
acid sequences we e e ie ed om UniP o . An analysis o he M.
ma inum genome was made using he exone a e p og am
22
o
iden i y any b-CA sequences he ein. A simila analysis was pe -
o med using he genome o T. spi alis. The wo M. ma inum
sequences and one T. spi alis sequence p oduced om hese p e-
dic ions we e included wi h he o he UniP o sequences o
phylogene ic analysis. A maximum likelihood phylogene ic analysis
o he inal 9 b-CAs was pe o med using PhyML.
23
Fo his ana-
lysis, he LG amino acid subs i u ion model was used du ing a un
o 1000 boo s aps. The alpha, ansi ion/ ans e sion, and p opo -
ion o in a iable si es pa ame e s we e all se o empi ical, wi h
all o he pa ame e s as de aul . The esul s we e isualized using
he FigT ee p og am (h p:// ee.bio.ed.ac.uk/so wa e/ ig ee/).
Exp ession analysis o b-CAs om M. ma inum
P ime s o polyme ase chain eac ion (PCR) o h ee b-CAs o M.
ma inum (b-CA1 F 50-a gcccaacaccaa ccga a-30,R5
0-gccga a cacc-
gaca gg c-30;b-CA2, F1 50-g gacgg accgacgac acc-30,R15
0-
cg gacc cg gag gc-30; and b-CA3, F2 50-a cc cga ggcg gacga-30,
R2 50-cccg g ga cgacc cg -30) we e manually designed o ull
leng h o ansc ip . The PCR eac ions we e pe o med wi h an
ini ial dena u a ion s ep a 95 C o 3 min ollowed by 35 cycles,
55 C annealing empe a u e and 72 C o 10 s elonga ion s ep.
Following he PCR, he samples we e analyzed on a 0.7% aga ose
gel using a s anda d DNA ma ke s (100 bp and 1 kb) (P omega).
The e hidium b omide gels we e obse ed unde UV-ligh
(GelDoc) and pho og aphed.
Quan i a i e eal- ime PCR
P ime s o Quan i a i e Real-Time PCR (qRT-PCR) we e designed
based on cDNA sequences aken om NCBI/Unip o using he p o-
g am P ime Exp ess
V
R
So wa e 2.0 (Applied Biosys ems) (b-CA1,F
50gcggca gc cac cac30,R5
0cgg c cg cc gga cc30;b-CA2 F
50cccaacaccaa ccga aacc30,R5
0gcgacgaa cgc cg ac30;b-CA3
50cgaagaaca gccgacga 30,5
0g c ggc cccgcga ag30). The qRT-PCR
was pe o med using he SYBR G een PCR Mas e Mix Ki in an ABI
PRISM 7000 De ec ion Sys em
TM
acco ding o he manu ac u e 's
ins uc ions (Applied Biosys ems). The PCR condi ions consis ed o
an ini ial dena u a ion s ep a 95 C o 10 min ollowed by 40
cycles a 95 C o 15 s (dena u a ion) and 60 C o 1 min (elong-
a ion). The da a we e analyzed using he ABI PRISM 7000 SDS
TM
so wa e (Applied Biosys ems). E e y PCR was pe o med in a o al
eac ion olume o 15 ml con aining 2 ml o i s s and cDNA (20 ng
cDNA), 1 Powe SYBR g een PCR Mas e Mix
TM
(Applied
Biosys ems, Fos e Ci y, CA, USA), and 0.5 mM o each p ime . The
inal esul s a e gi en as ela i e exp ession alues, calcula ed
acco ding o he P a l's Equa ion.
24
De e mina ion o minimal inhibi o y concen a ion in in i o
cul u es o M. ma inum
Fo he de e mina ion o minimal inhibi o y concen a ion (MIC),
he p o ocol used he e was modi ied om Hall e al.
25
B ie ly, wild
ype M. ma inum (ATCC 927) was g own on Middleb ook 7H10
aga pla es (BD) o 6 days a þ29 C. Bac e ial mass was sc aped
om he pla e and ans e ed in o PBS pH 7.4 con aining 0.2%
Tween 80 (SIGMA) o ob ain an OD600 o 0.08-0.100. 200 ml o his
bac e ial suspension was mixed wi h 11 ml o Middleb ook 7H9
B o h OADC (BD) (no ween, no glyce ol) by o exing. The bac e -
ial concen a ion was de e mined by pla ing on 7H10 aga (BD)
and pu i y by pla ing on LB aga (SIGMA). Pla es we e incuba ed
o 6 days a þ29 C. The bac e ial concen a ion was be ween
1.4 10
5
and 4.7 10
5
c u/ml. 50 ml o his bac e ial suspension
was pipe ed pe well on o s e ile, clea 96-well issue cul u e
ea ed pla es (Co ning Cos a om SIGMA). The il e s e ilized
inhibi o s dissol ed in Middleb ook 7H9 B o h OADC (no ween,
no glyce ol) we e added on op o bac e ia in a olume o 50 ml. A
concen a ion ange o 0.3 pM–300 nM using a 10- old dilu ion se -
ies was es ed in wo sepa a e expe imen s on 2–6 eplica e wells.
A concen a ion ange o 18.75-300 nM using a 2- old dilu ion se -
ies was in wo sepa a e expe imen s on six eplica e wells. The lids
we e sealed on o he pla es wi h pa a ilm and he cul u es we e
incuba ed a þ28.5 C o 5 days. The esul was de e mined by
assessing he u bidi y o he cul u es bo h by isual inspec ion
and by an OD600 measu emen using Pe kin Elme En ision mul i-
eade scan measu emen . Fi e ho izon al and i e e ical poin s
0.72 mm apa we e measu ed om each well. The sum o he
eadings was calcula ed o each sample. The backg ound signal
om wells con aining medium only was sub ac ed om all
alues.
The na u e o inhibi ion o he es ed agen s was also exam-
ined. Bac e ia we e g own as o MIC de e mina ion by making
se ial inhibi o dilu ions. A e ini ial incuba ion, esh 7H9 medium
was added o dilu e he inhibi o s a a a io o 1:2 and 1:4.
Cul u es we e analyzed a e six days o incuba ion as in MIC
de e mina ion by compa ing o undilu ed duplica e cul u e wells.
M. ma inum ans o ma ion
A luo escen M. ma inum (wasabi) s ain was gene a ed using a
modi ied p o ocol o M. ube culosis elec opo a ion.
26
M. ma inum
ATCC 927 was g own in Middleb ook 7H9 B o h OADC (BD) wi h
0.2% Tween 80 (SIGMA) s a ing om an OD600 o 0.07–0.100 o
an OD600 o 0.700–0.800. 20 ml o cul u e was ha es ed o bac-
e ia and washed h ee imes in 10% glyce ol. 0.1–1mg o pu i ied
pTEC15 plasmid DNA was mixed wi h 500 mlM. ma inum in 10%
glyce ol and incuba ed o 5 min. Cells we e ans o med in 2 mm
cu e es wi h a single pulse o 2.5 kV, 25 mF (1000 X esis ance)
using Genepulse II Elec opo a ion Sys em (Bio-Rad). T ans o med
cells we e esuspended in 4 ml 7H9 medium. A e o e nigh incu-
ba ion a 29 C wi h gen le shaking, ans o man s we e selec ed
on 7H10 aga pla es wi h 75 mg/ml hyg omycin B. Co ec ans-
o man s we e e i ied by luo escence mic oscopy. pTEC15 was a
gi om Lali a Ramak ishnan (Addgene plasmid #30174).
M. ma inum in ec ions o zeb a ish emb yos
M. ma inum ATCC 927 con aining pTEC15 plasmid o cons i u i e
g een luo escence
21
was g own o 4 days in Middleb ook 7H9
B o h OADC (BD) wi h 0.2% Tween 80 (SIGMA) and 75 mg/ml o
Hyg omycin B (VWR) s a ing om an OD600 o 0.07-0.100 o an
OD600 o 0.760–0.890. The bac e ia we e pelle ed and dissol ed in
he app op ia e olume o PBS con aining 0.6 mg/ml o phenol
ed as a ace (SIGMA). Wild- ype (AB) zeb a ish emb yos we e
manually decho iona ed a 22-24 hp and pu in o E3-wa e
27
con-
aining 0.00045% phenyl hiou ea (PTU)(SIGMA) o inhibi
834 A. ASPATWAR ET AL.
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pigmen a ion. The emb yos we e anes he ized wi h 0.02% T icaine
(SIGMA). Using aluminosilica e capilla y needles and a Pneuma ic
PicoPump PV820 (Wo ld P ecision Ins umen s) 1 nl o bac e ial
suspension was injec ed in o he caudal ein o he zeb a ish 27-
31 hp and he bac e ial concen a ion was e i ied by pla ing
28
.
The in ec ed ish we e kep a 28.5 C in 1 ml o PTU-E3 medium
wi h o wi hou he inhibi o on 24-well pla es.
De e mina ion o bac e ial load om in ec ed zeb a ish la ae
On day 5 pos in ec ion he ish we e eu hanized wi h an o e dose
o T icaine (SIGMA), ans e ed on o a black 96-well P oxypla e
(Pe kinElme ), and embedded on hei side in he middle o he
well in 50 ml o 1% low mel aga ose (SIGMA). Eigh non-in ec ed
ish we e embedded o measu ing backg ound alues. P io o
g een luo escence scan measu emen wi h Pe kin Elme En ision
mul i eade , 50 ml o PBS was added on op o each sample. The
scan measu emen was ca ied ou on 5 ho izon al and 5 e ical
do s 0.5 mm apa om 6.5 mm heigh wi h 100% exci a ion a
493 nm, 509 nm emission and 500 lashes pe poin . The a e age
signal ( ela i e luo escence uni s, RFU) om each well was calcu-
la ed. The a e age backg ound signal was sub ac ed om he
measu ed samples.
S a is ical analysis
The G aphPad P ism so wa e (5.02) was used o pe o m s a is ical
analysis. Due o small sample numbe s, we used a non-pa ame ic
wo- ailed Mann–Whi ney es he de e mina ion o s a is ical sig-
ni icance o di e ences be ween he d ug ea ed and non- ea ed
g oup. Fo s a is ical analysis o he oxici y pa ame e s, a wo-
ailed Fishe ’s es was used. p alues below .05 we e conside ed
signi ican .
Resul s
Exp ession analysis shows ansc ip ion o all h ee b-CA genes
in M. ma inum
Bioin o ma ic analysis o he M. ma inum genome showed he
p esence o h ee b-CA genes. We expe imen ally alida ed he
exp ession o he b-CA genes in log-phase cul u es o M. ma inum
s ain ATCC 927 using RT-PCR. The PCR bands we e 500 bp o
b-CA 1, 490 bp o b-CA 2, and 600 pb o b-CA3 (Figu e 2(A))as
expec ed. In qRT-PCR compa ing b-CAs exp ession in h ee
di e en s ains o M. ma inum (M, ATCC 927 and E11), we ound
he exp ession o be highes in ATCC 927 (Figu e 2(B–D)). (Ou
molecula analysis hus con i med he p esence o he b-CA genes
in M. ma inum.)
M. ma inum b-CA sequences a e simila o he b-CA sequences
o M. ube culosis
M b con ains h ee b-CA sequences; wo o he enzymes (b-CA 1
and b-CA 2) can be speci ically inhibi ed using DTCs in i o.
13
To
in es iga e i he b-CA sequences o M. ma inum a e closely
ela ed o M b b-CAs, we i s pe o med mul iple sequence align-
men (MSA) s udies. The MSA showed e y high conse a ion o
nucleo ides be ween he sequences (M b ca1 s. M. ma inum
ca2 ¼83.95, M b ca2 s. M. ma inum ca1 ¼83.17 and M b ca3 s.
M. ma inum ca3 ¼68.86) (The names and numbe ing o he b-CAs
we e based on he UniP o en y) (Supplemen a y Figu e 1 online).
The subcellula localiza ion in o ma ion accessed om ube culis
(h p:// ube culis .ep l.ch) da abase sugges ed ha b-CA 1 and 2
a e cy oplasmic, and b-CA 3 is a memb ane-associa ed p o ein and
hese p edic ed localiza ions ma ch hose o he M b b-CAs.
4
The
phylogene ic analysis showed ha each o he h ee M b b-CA
sequences a e mos closely ela ed o a co esponding M. ma inum
b-CA sequence (Figu e 3).
DTC Fc14–584B is sa e compa ed o Fc14–594 A in zeb a ish
emb yos/la ae
The oxice ec so DTCsFc14–584B and Fc14–594 A on
de eloping zeb a ish emb yos we e dose-dependen (Figu es 4 and
5). Fc14–594 A had an LC
50
alue o 18.5 mM(Figu e 5(A)). The DTC
Fc14–584B was less oxic, and had an LC
50
alue o 498.1 mM(Figu e
5(B)).
To assess he oxici y o he wo inhibi o s, Fc14–594 A and
Fc14–584B, in a p elimina y expe imen , we examined 1–5 dp
zeb a ish exposed o di e en concen a ions o he d ugs, and
compa ed he obse able de elopmen al pa ame e s wi h hose o
non- ea ed ish. Figu e 4 shows ep esen a i e pic u es o la ae
subjec ed o di e en concen a ions o inhibi o s. As Fc14–584B
was clea ly be e ole a ed a highe concen a ions, we ca ied
ou a mo e de ailed analysis wi h his d ug a 300 and 500 mM
concen a ions (Figu e 6). A 300 mM concen a ion did no a ec
su i al, ha ching, mo emen , edema, o yolk sack u iliza ion du -
ing he i s 5 dp . Some la ae subjec ed o 300 mM o Fc14–584B
showed mild abno mali ies in body shape (cu ing o he back)
Figu e 2. Exp ession analysis o b-CAs om M. ma inum: (A) The quali a i e exp ession analysis o h ee b-CA genes using PCR showed p esence o all he h ee b-CAs
in M. ma inum s ain ATCC 927 which was used o in i o in ec ion and d ug ea men s udies. The mo Scien i ic GeneRule 1 kb DNA Ladde was used as ma ke .
Genomic DNA was used as posi i e con ol. The es samples used we e cDNAs o he analysis o exp ession o b-CAs om M. ma inum. (B–D) Rela i e exp ession ana-
lysis o h ee b-CA genes om h ee M. ma inum s ains using RT-qPCR acco ding o he P a l me hod.
24
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and hea bea (di e ence o con ols no s a is ically signi ican ).
Pheno ypic s udies sugges ed ha Fc14–584B, is sa e han
Fc14–594 A and has limi ed ad e se e ec s on he emb yos a
300 mM concen a ion. Thus, Fc14–584B was selec ed o u he in
i o es ing.
The DTCs Fc14–584B and Fc14–594 A did no induce any
his ological de ec s in he zeb a ish la ae
To see any damage o he issues o he zeb a ish emb yos, we
s udied he his ological s uc u es o 5 dp la ae ea ed wi h di -
e en concen a ions o Fc14–594 A and Fc14–584B and compa ed
he indings wi h he con ol g oup o 5 dp zeb a ish la ae. The
Semi hin (5 mm) sagi al sec ions o he la ae s ained wi h hema-
oxylin and eosin did no show any appa en mo phological
changes compa ed o he con ol g oup la ae. The his ochemical
s udies o he d ug ea ed la ae sugges ed ha hese d ugs do
no cause any his ological damage o he in e nal issue a he
LC
50
dose o lowe .
DTCs Fc14–584B and Fc14–594 A inhibi he g ow h o
M. ma inum in i o
We hen sough o de e mine, whe he he selec ed DTCs inhibi
he g ow h o M. ma inum in i o. We ca ied ou s anda d MIC-
es s using liquid cul u es o M. ma inum on a 96-well pla e. In
addi ion o a isual inspec ion, he op ical densi y o he cul u es
was measu ed a e 6 days. In a p elimina y expe imen , we
i a ed a concen a ion ange om 3 nM o 300 mM wi h a 10- old
dilu ion se ies and ound no g ow h a 300 mM concen a ion and
educed g ow h a 30 mM (da a no shown). Based on hese esul s
we ca ied ou wo ounds o MIC es s using concen a ions
be ween 18.75 mM and 300 mM wi h a 2- old dilu ion se ies. A dose
esponse in g ow h inhibi ion was seen o bo h compounds
(Figu e 7(A–D)). The MIC o bo h compounds was 75 mM(Figu e
7(A–D)). In e ms o g ow h esump ion, none o he inhibi ed
mycobac e ial cul u es showed any signs o e i al wi h inhibi o
concen a ions below MIC a e inhibi o dilu ion by 1:4. This sug-
ges s ha he es ed agen s ac ed as bac e icidal a he han bac-
e ios a ic inhibi o s (da a no shown).
DTC Fc14–584B inhibi s he g ow h o M. ma inum in i o in
zeb a ish la ae
Based on ou esul s om oxici y and MIC es ing, we con inued
wi h compound Fc14–584B o in i o es ing. We in ec ed ish
1-dp wi h g een luo escen M. ma inum (a e age in ec ion dose
471 ± 143 bac e ia) (Figu e 8(A)). Fc14–584B was added o he
emb yonic medium a a concen a ion o 300 mM. As zeb a ish la -
ae can be kep anspa en , he bac e ial load a 6 days pos
in ec ion (dpi) could be measu ed by luo escence. The luo escen
Figu e 3. E olu iona y ela ionship o he b-CAs om M. ma inum and M. ube culosis. The phylogene ic analysis o all h ee b-CAs om bo h bac e ial species showed
ha he b-CA sequences a e e olu iona ily closely ela ed.
836 A. ASPATWAR ET AL.
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Figu e 5. LC
50
de e mina ion o he wo compounds: The LC
50
dose o he d ugs, Fc14–584B and Fc14–594 A was de e mined based on cumula i e mo ali y o 5 days
a e he exposu e o emb yos o he di e en concen a ion o he d ugs. The LC
50
we e de e mined a e h ee independen expe imen s wi h simila expe imen al
condi ions (n¼30).
Figu e 4. E ec s o di hioca bama es Fc14–594 A and Fc14–584B on de eloping emb yos. De elopmen al images o 1–5 dp emb yos exposed o di e en con-
cen a ions o Fc14–594 A and Fc14–584B b-CA inhibi o compounds. (A) Row shows he images o con ol g oup emb yos (no ea ed wi h inhibi o s) wi h no -
mal emb yonic de elopmen . (B) Row shows he images o zeb a ish emb yos exposed o Fc14–594 A. The emb yos exposed o 20 lM concen a ion o
Fc14–594 A showed sho and cu ed body s uc u e wi h mild edema (a ows), cu ed ail (bulle ), and unu ilized yolk sac (a ow head) and he emb yos
exposed o 30 lM Fc14–594 A did no su i e beyond 3 dp . (C) Row shows he images o emb yos exposed o Fc14–584B. The emb yos exposed o concen a-
ions up o 300 lMo Fc14–584B gene ally had a no mal emb yonic de elopmen wi h no signi ican pheno ypic de ec s. The emb yos exposed o 600 lMo
Fc14–584B did no su i e beyond 3 dp .
JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY 837
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Figu e 6. E ec o di hioca bama e Fc14–584B on pheno ypic pa ame e s o he de eloping zeb a ish emb yos. The e ec o 300 and 500 lM concen a ions o
Fc14–584B on su i al, mo emen , yolk sack, ha ching, hea bea , body shape, and edema o he zeb a ish emb yos was eco ded 1–5 dp . Fo each concen a ion,
n¼30. p<.05 by wo- ailed Fishe ’s es .
Figu e 7. Di hioca bama es Fc14–584B and Fc14–594 A inhibi he g ow h o M. ma inum. MIC was de e mined in liquid cul u es by isual inspec ion and u bidi y
measu emen in wo sepa a e expe imen s (AB and CD). In bo h expe imen s n¼6. p alues in he pic u es a e wo- ailed - es alues o compa isons o 0 lM.
838 A. ASPATWAR ET AL.
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signal co ela es wi h he bac e ial load measu ed om he same
samples wi h an M. ma inum speci ic qPCR me hod.
17
In he h ee
sepa a e expe imen s, he ea ed g oups had signi ican ly lowe
bac e ial numbe s compa ed o he con ols ( ange om 2.9 o
8.9- old di e ence (p<.05) in median bac e ial loads, a ep esen a-
i e expe imen shown in Figu e 8(C). These esul s p o ide e i-
dence o he in i o e icacy o DTC Fc14–584B as an
an imycobac e ial d ug.
Discussion
Tube culosis is cu en ly one o he deadlies in ec ions, causing
almos 2 million yea ly dea hs wo ldwide.
2
The TB epidemic is a
global p oblem ha is agg a a ed by he ecen eme gence o
mul id ug- esis an s ains o M. ube culosis ha a e un ea able
wi h common an ibio ic egimens. This ala ming si ua ion empha-
sizes he need o de elop no el an ibio ics agains p e iously
unexploi ed a ge s.
Recen s udies ha e shown ha inhibi o s o CAs, such as sul o-
namides, couma ines, and sul amides, a e e y e ec i e in inhibi -
ing he ac i i y o CA enzymes o M b a subnanomola
concen a ions in i o, hus alida ing he CA enzymes as po en-
ial an i ube culosis a ge s.
12,14,29
DTCs, Fc14–594 A and
Fc14–584B, belonging o a s uc u ally dis inc class o CA inhibi-
o s ha e been de eloped and iden i ied in i o as po en ial d ug
candida es agains b-CA enzymes.
13,16
Despi e he p omise o
b-CA enzymes as a ge s o de eloping an i-TB agen s, so a , no
in i o s udies ha e explo ed he po en ial o a ge ing he b-CA
enzyme. The aim o ou s udy was o e alua e he sa e y and ox-
ici y o he wo no el DTCs (Fc14–594 A and Fc14–584B), and sub-
sequen ly use he less oxic inhibi o o in i o s udies using
M. ma inum and zeb a ish la ae as model o ganisms. Zeb a ish
ha e been widely used o acu e and ch onic oxici y es ing
20,30
as well as o s udying de elopmen al oxici y.
19
Zeb a ish
emb yos de elop ex u e o and he chemicals can be easily added
o ish ank wa e , making i a highly easible model o s udying
many aspec s o oxici y. As de eloping emb yos a e mo e easily
a ec ed by chemicals han adul ish, assays wi h emb yos a e
mo e sensi i e and able o de ec e en low le els o oxici y.
Fo he oxici y e alua ion o he DTCs in 1–5 dp ish, we
s udied he e ec s o hese d ugs on he pheno ype o he ish,
and quan i a i e pa ame e s, such as mo ali y, ha ching a e,
hea bea , and mo emen pa e n. We also s udied his opa hology
o he issues o check he e ec o he d ugs on he issues
du ing emb yonic de elopmen . The sa e y sc eening showed ha
Fc14–584B was less oxic wi h an LC
50
dose o 498.1 mM compa ed
o Fc14–594 A (LC
50
dose o 18.5 mM). A 300 mM concen a ion,
Fc14–584B showed no signi ican pheno ypic changes in he as
majo i y o indi iduals, ye in 25% o he ish, hea bea and body-
pa e n we e mildly a ec ed. His ochemical analysis showed no
damage o he issues o 5 dp la ae. Based on he 75 mM MIC o
Fc14–584B o M. ma inum and he esul s o sa e y s udies, we
op ed o 300 mM concen a ion o e alua e he in i o e icacy o
Fc14–584B agains M. ma inum. The M. ma inum in ec ed emb yos
ea ed wi h 300 mM Fc14–584B showed signi ican ly lowe numbe
o bac e ia 6 dpi. Ou esul s on he b-CA inhibi o Fc14–584B
highligh i s po en ial as a lead compound upon which o de elop
an imycobac e ial d ugs.
Fu he mo e, we ha e succeeded in using a g een luo escen
s ain o M. ma inum o demons a e he an i-mycobac e ial ac i -
i y o Fc14–584B. Wi h he inco po a ion o a g een luo escen
wasabi epo e unde a s ong cons i u i e p omo e , he bac e ia
in in ec ed zeb a ish can be apidly quan i ied using a luo ome e .
This sys em can be adop ed o high h oughpu sc eening o any
an ibac e ial agen s o oxici y and sa e y using zeb a ish as a e -
eb a e animal model, as well as o in i o s udies o inhibi ion o
mic obial g ow h. In addi ion, he echniques and expe imen s
ou lined he e may be used o p obe o he iden i ied inhibi o s o
a- and b-CA enzymes ha can be de eloped in o no el d ugs a -
ge ing bac e ial, pa asi ic, o ungal diseases. This me hod is he
i s o i s kind o sc een o oxici y in zeb a ish emb yos ollowed
by in i o inhibi ion o mycobac e ial species in a ube culosis
zeb a ish la al model. This app oach allows he iden i ica ion o
compounds ha a e no oxic o e eb a es and a e ac i e agains
he pa hogenic bac e ia.
In conclusion, we ha e iden i ied DTC Fc14–584B as a po en
an i-mycobac e ial d ug candida e ha a ge s mycobac e ial b-CA
enzymes. Fc14–584B speci ically inhibi s pu i ied b-CAs o M b a
nanomola and subnanomola concen a ions. In a bac e ial cul-
u e, he compound Fc14–584B e ec i ely inhibi s he g ow h o
M. ma inum. We ha e also demons a ed ha he compound is
sa e o use in zeb a ish and causes no signi ican pheno ypic o
his ological abno mali ies in 5 dp zeb a ish la ae. Impo an ly,
we ha e demons a ed ha Fc14–584B signi ican ly impai s he
g ow h o M. ma inum in i o in he zeb a ish la al model. To ou
knowledge, his is he i s epo on in asi e M. ma inum and i s
suscep ibili y o inhibi o s o b-CA in i o in a e eb a e model.
Cu en ly, he DTC Fc14–584B is in p ocess owa ds p eclinical
Figu e 8. Di hioca bama e Fc14–584B inhibi s he g ow h o M. ma inum in i o in zeb a ish la ae. Zeb a ish la ae we e in ec ed wi h g een luo escen M. ma inum
wasabi s ain wi h an a e age in ec ion dose o 471 ± 143 bac e ia and we e analyzed a 6 dpi. P io o luo escence measu emen wi h a pla e eade , he ish we e
checked unde a luo escence mic oscope o ensu e success ul in ec ion based on gene al bac e ial luo escence in he con ol g oup. Rep esen a i e images o ypical
ou comes o in ec ion 6 dpi (A) wi hou ea men (B) wi h 300 lMo Fc14–584B. (C) Fo quan i ica ion, he end-poin bac e ial load was assessed in a luo ome ic
measu emen o bac e ia inside anspa en ish la ae. The median ela i e luo escence uni s (RFU) alue o he g oup is shown as a ho izon al line (n
ea ed
¼15,
n
con ol
¼16). The esul is a ep esen a i e o h ee sepa a e expe imen s.
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