molecules
Re iew
Ca bonic Anhyd ase Inhibi o s as No el D ugs
agains Mycobac e ial β-Ca bonic Anhyd ases:
An Upda e on In Vi o and In Vi o S udies
Ashok Aspa wa 1,* , Jean-Y es Winum 2, Fab izio Ca a 3, Claudiu T. Supu an 3,
Milka Hamma en 1, Ma aleena Pa ikka 1,4 and Seppo Pa kkila 1,5
1Facul y o Medicine and Heal h Technology, Uni e si y o Tampe e, 33014 Tampe e, Finland;
milka.hamma en@s a .u a. i (M.H.); ma aleena.pa ikka@s a .u a. i (M.P.); seppo.pa kkila@s a .u a. i (S.P.)
2Ins i u des Biomolécules Max Mousse on (IBMM) UMR 5247 CNRS, ENSCM, Uni e si éde Mon pellie ,
34296 Mon pellie CEDEX 05, F ance; Jean-y es.winum@umon pellie .
3
Neu o a ba Depa men , Sezione di Chimica Fa maceu ica e Nu aceu ica, Uni e si àdegli S udi di Fi enze,
50019 Ses o Fio en ino (Fi enze), I aly; [email p o ec ed] (F.C.); [email p o ec ed] (C.T.S.)
4O al and Maxillo acial Uni , Tampe e Uni e si y Hospi al, 33521 Tampe e, Finland
5Fimlab L d. and Tampe e Uni e si y Hospi al, 33520 Tampe e, Finland
*Co espondence: ashok.aspa wa @s a .u a. i; Tel.: +35-846-596-2117
Recei ed: 23 Oc obe 2018; Accep ed: 6 No embe 2018; Published: 8 No embe 2018


Abs ac :
Mycobac e ia cause a a ie y o diseases, such as ube culosis, lep osy, and oppo unis ic
diseases in immunocomp omised people. The ea men o hese diseases is p oblema ic, necessi a ing
he de elopmen o no el ea men s a egies. Recen ly,
β
-ca bonic anhyd ases (
β
-CAs) ha e
eme ged as po en ial d ug a ge s in mycobac e ia. The genomes o mycobac e ia encode o
h ee
β
-CAs ha ha e been cloned and cha ac e ized om Mycobac e ium ube culosis (M b) and
he c ys al s uc u es o wo o he enzymes ha e been de e mined. Di e en classes o inhibi o
molecules agains M b
β
-CAs ha e subsequen ly been designed and ha e been shown o inhibi hese
mycobac e ial enzymes
in i o
. The inhibi ion o hese cen ally impo an mycobac e ial enzymes
leads o educed g ow h o mycobac e ia, lowe i ulence, and impai ed bio ilm o ma ion. Thus,
he inhibi ion o
β
-CAs could be a no el app oach o de eloping d ugs agains he se e e diseases
caused by pa hogenic mycobac e ia. In he p esen a icle, we e iew he da a ela ed o
in i o
and
in i o inhibi ion s udies in he ield.
Keywo ds: mycobac e ial diseases; β-ca bonic anhyd ases; Mycobac e ium ube culosis; d ug a ge s;
ca bonic anhyd ase inhibi o s; in i o inhibi ion; in i o inhibi ion
1. In oduc ion
Mycobac e ia a e od-shaped, non-mo ile, and acid- as bac e ia ha con ain a high amoun o G
+ C in hei genome [
1
,
2
]. The genus mycobac e ium includes a a ie y o clinically ele an human
pa hogens, including Mycobac e ium ube culosis (M b), he main causa i e agen o human ube culosis
(TB). A g oup o closely ela ed bac e ia e e ed o as he Mycobac e ium ube culosis complex (MTC)
composes o a a ie y o pa hogens causing TB in humans and o he mammals. These include M.
ube culosis,M. bo is,M. cap ae,M. a icanum,M. cane ii, M. mic o i,M. o ygis, and M. pinnipedii [
3
].
M b is ypically ansmi ed h ough ai by a d ople con ac . M b can a ec many o gans in humans,
bu he main a ge o gan is he lung, causing pulmona y TB in 80% o he pa ien s. In 2017, 10 million
new cases we e diagnosed [
4
]. In addi ion o he diagnosed ac i e M b in ec ions, he Wo ld Heal h
O ganiza ion (WHO) es ima es ha 23% o he wo ld’s popula ion has de eloped a la en TB [
4
], which
is asymp oma ic bu can become eac i a ed and cause a di icul - o- ea and po en ially le hal disease.
Molecules 2018,23, 2911; doi:10.3390/molecules23112911 www.mdpi.com/jou nal/molecules
Molecules 2018,23, 2911 2 o 14
M b is cu en ly one o he deadlies bac e ia killing 1.3 million people e e y yea . Mul i-d ug- esis an
s ains o M b a e on he ise making TB inc easingly di icul o ea . This de elopmen poses an
eno mous global h ea necessi a ing immedia e ac ion o ind new ways o ea his de as a ing
disease [4].
Lep osy is ano he example o a clinically ele an mycobac e ial disease. Lep osy is caused by
M. lep ae, which is ansmi ed h ough d ople s due o close and equen con ac wi h un ea ed
pa ien s. As he mul iplica ion a e o M. lep ae is e y slow, he incuba ion pe iod o disease anges
be ween 1 and 20 yea s. The disease mainly a ec s he skin, pe iphe al ne es, mucosa o he uppe
espi a o y ac , and eye. I le un ea ed, he disease usually causes pe manen issue damage.
In many de eloping coun ies, lep osy is s ill a se ious heal h p oblem and he people su e ing om
he disease o en ace social p oblems ha go hand in hand wi h he disease p og ession. The la es
WHO epo shows ha he e we e 216,108 new lep osy cases in 145 coun ies om he 6 WHO
egions [5].
The non ube culous mycobac e ia (NTM) g oup includes all mycobac e ia o he han MTC
and M. lep ae, and abou 40 species o hem a e pa hogenic [
6
]. NTM a e ubiqui ously ound in a
wide a ie y o en i onmen al ese oi s [
7
,
8
]. Al hough hey a e mos ly nonpa hogenic, hey a e
impo an oppo unis ic pa hogens o humans [
9
]. The species o NTM associa ed wi h human disease
a e: M. a ium, M. in acellula e,M. kansasii,M. o ui um,M. chelonae,M. szulgai,M. pa a ube culosis,
M. sc o ulaceum [
10
] as well as bac e ia belonging o he M. abscessus complex [
11
]. NTM can
cause pulmona y disease esembling ube culosis, lymphadeni is, and skin disease. The pulmona y
disease ep esen s abou 80% o in ec ions caused by NTM [
12
]. Recen epo s sugges ha he
NTM pulmona y disease is inc easing in se e al pa s o he wo ld [
13
,
14
]. Howe e , s anda dized
diagnos ics and e ec i e ea men p o ocols o NTM in ec ions a e lacking [15].
Genomes o many mycobac e ial species om bo h MTC and NTM ca ego ies ha e been
sequenced [
16
–
20
]. Bioin o ma ic and molecula analysis o mycobac e ial genomes e ealed ha hey
code o se e al no el p o eins ha a e essen ial o he al e na i e pa hways and c i ical o he li e
cycle o hese pa hogens [
21
–
23
]. Recen p og ess in he s uc u al and unc ional analyses o genomes
and p o eomes has opened new a enues o he design o mechanism-based d ugs a ge ing p o eins
c ucial o pa hogenesis o mycobac e ia [
24
]. Among many such p o eins,
β
-ca bonic anhyd ases
(
β
-CAs) o mycobac e ia could be possible a ge s o de eloping no el an imycobac e ial agen s wi h
he po en ial o ea e en in ec ions caused by d ug- esis an mycobac e ia.
M. ube culosis genome codes o h ee
β
-CA genes R 1284 (
β
-CA1), R 3588c (
β
-CA2) and R 3273
(
β
-CA3) as shown in Table 1[
25
]. Da abase sea ches and ou bioin o ma ic analyses showed he
p esence o all he h ee
β
-CAs in bo h NTM and MTC bac e ia [
26
–
30
].
β
-CAs ca alyze he e e sible
hyd a ion o CO
2
o HCO
3−
and H
+
, hus gene a ing a bu e ing weak base (bica bona e) and a s ong
acid (H
+
) [
31
,
32
]. Mycobac e ial
β
-CAs a e zinc-con aining me alloenzymes wi h cha ac e is ics simila
o many o he bac e ial
β
-CAs. All conse ed amino acid esidues ypical o
β
-CAs and in ol ed in
he ca aly ic cycle, i.e., he ou zinc-binding esidues, Cys42, Asp44, His97 and Cys101 a e shown in
Figu e 1.
Table 1. Ac i i y and inhibi ion p ope ies o M b β-CAs compa ed o human CA II.
CAs Gene ID P o ein Kca (s−1)Kca /Km (M−1s−1)Ac i i y KI(nM) aRe e ence
M b β-CA1 R 1284 163 aa 3.9 ×1053.7 ×107Mode a e 480 [27]
M b β-CA2 R 3588c 207 aa 9.8 ×1059.3 ×107High 9.8 [29]
M b β-CA3 R 3273 b215 aa 4.3 ×1054.0 ×107Mode a e 104 [28]
hCAII CA2 260 aa 1.4 ×1061.5 ×108Ve y high 12 [29]
aInhibi ion using ace azolamide. bCA domain o β-CA3.
Molecules 2018,23, 2911 3 o 14
Molecules 2018, 23, x FOR PEER REVIEW 2 o 14
po en ially le hal disease. M b is cu en ly one o he deadlies bac e ia killing 1.3 million people
e e y yea . Mul i-d ug- esis an s ains o M b a e on he ise making TB inc easingly di icul o
ea . This de elopmen poses an eno mous global h ea necessi a ing immedia e ac ion o ind new
ways o ea his de as a ing disease [4].
Lep osy is ano he example o a clinically ele an mycobac e ial disease. Lep osy is caused by
M. lep ae, which is ansmi ed h ough d ople s due o close and equen con ac wi h un ea ed
pa ien s. As he mul iplica ion a e o M. lep ae is e y slow, he incuba ion pe iod o disease anges
be ween 1 and 20 yea s. The disease mainly a ec s he skin, pe iphe al ne es, mucosa o he uppe
espi a o y ac , and eye. I le un ea ed, he disease usually causes pe manen issue damage. In
many de eloping coun ies, lep osy is s ill a se ious heal h p oblem and he people su e ing om
he disease o en ace social p oblems ha go hand in hand wi h he disease p og ession. The la es
WHO epo shows ha he e we e 216,108 new lep osy cases in 145 coun ies om he 6 WHO
egions [5].
The non ube culous mycobac e ia (NTM) g oup includes all mycobac e ia o he han MTC and
M. lep ae, and abou 40 species o hem a e pa hogenic [6]. NTM a e ubiqui ously ound in a wide
a ie y o en i onmen al ese oi s [7,8]. Al hough hey a e mos ly nonpa hogenic, hey a e
impo an oppo unis ic pa hogens o humans [9]. The species o NTM associa ed wi h human
disease a e: M. a ium, M. in acellula e, M. kansasii, M. o ui um, M. chelonae, M. szulgai, M.
pa a ube culosis, M. sc o ulaceum [10] as well as bac e ia belonging o he M. abscessus complex [11].
NTM can cause pulmona y disease esembling ube culosis, lymphadeni is, and skin disease. The
pulmona y disease ep esen s abou 80% o in ec ions caused by NTM [12]. Recen epo s sugges
ha he NTM pulmona y disease is inc easing in se e al pa s o he wo ld [13,14]. Howe e ,
s anda dized diagnos ics and e ec i e ea men p o ocols o NTM in ec ions a e lacking [15].
Genomes o many mycobac e ial species om bo h MTC and NTM ca ego ies ha e been
sequenced [16–20]. Bioin o ma ic and molecula analysis o mycobac e ial genomes e ealed ha
hey code o se e al no el p o eins ha a e essen ial o he al e na i e pa hways and c i ical o he
li e cycle o hese pa hogens [21–23]. Recen p og ess in he s uc u al and unc ional analyses o
genomes and p o eomes has opened new a enues o he design o mechanism-based d ugs a ge ing
p o eins c ucial o pa hogenesis o mycobac e ia [24]. Among many such p o eins, β-ca bonic
anhyd ases (β-CAs) o mycobac e ia could be possible a ge s o de eloping no el an imycobac e ial
agen s wi h he po en ial o ea e en in ec ions caused by d ug- esis an mycobac e ia.
M. ube culosis genome codes o h ee β-CA genes R 1284 (β-CA1), R 3588c (β-CA2) and
R 3273 (β-CA3) as shown in Table 1 [25]. Da abase sea ches and ou bioin o ma ic analyses showed
he p esence o all he h ee β-CAs in bo h NTM and MTC bac e ia [26–30]. β-CAs ca alyze he
e e sible hyd a ion o CO2 o HCO3− and H+, hus gene a ing a bu e ing weak base (bica bona e)
and a s ong acid (H+) [31,32]. Mycobac e ial β-CAs a e zinc-con aining me alloenzymes wi h
cha ac e is ics simila o many o he bac e ial β-CAs. All conse ed amino acid esidues ypical o β-
CAs and in ol ed in he ca aly ic cycle, i.e., he ou zinc-binding esidues, Cys42, Asp44, His97 and
Cys101 a e shown in Figu e 1.
Figu e 1.
C ys al S uc u e o R 1284 (
β
-CA1) om M. ube culosis. (
A
) S uc u e o
β
-CA1 (1YLK) [
22
].
Coo dina ion o he Zn(II) ion in he
β
-CA1 o M b. (
B
) Closed ac i e si e, wi h he Zn(II) ion ( iole
sphe e) coo dina ed by a his idine, wo cys eines and one aspa a e esidue. (
C
) Open ac i e si e, wi h
h ee p o ein ligands coo dina ed o Zn(II); he aspa a e makes a sal b idge wi h a conse ed a ginine
esidue in all
β
-CAs [
22
,
29
]. The images adap ed om Co a ubias e al. h ps://www. csb.o g/
s uc u e/1YLK [22]. Licensed unde CC BY 4.0.
The mycobac e ial
β
-CAs a e essen ial du ing s a a ion o he g ow h and su i al o he
bac e ia [
22
,
23
,
33
,
34
]. Recen s udies showed ha he bica bona e ion, which is a p oduc o e e sible
hyd a ion o CO
2
, is essen ial o he anspo o ex acellula DNA (eDNA) and he o ma ion o
bio ilm in NTM bac e ia
in i o
[
35
]. Inhibi ion o
β
-CAs using e hoxzolamide (EZA), a CA inhibi o ,
educed he anspo o eDNA and he o ma ion o bio ilm [
35
]. EZA also inhibi ed he PhoPR
egulon, a wo-componen egula o y sys em in M b, as well as Esx-1 p o ein sec e ion sys em cen ally
impo an o he i ulence o M b bac e ium, and showed e icacy in in ec ed mac ophages and
mice [
36
], sugges ing ha
β
-CAs pe o m e y impo an oles in mycobac e ial in ec ions. Using
M. ma inum, an NTM model bac e ium, we we e he i s o show ha di hioca bama e Fc14-584b,
a
β
-CA inhibi o impai s mycobac e ial g ow h in zeb a ish la ae
in i o
[
26
]. These essen ial enzymes
a e hus po en ial d ug a ge s and a e cu en ly unde in es iga ion by se e al g oups, including
ou s [
26
–
29
,
35
–
39
]. Simila ly, se e al
in i o
s udies ha e shown ha all he M b
β
-CAs could be
e icien ly (K
I
in nanomola anges) inhibi ed by sul onamides/sul ama es (Table 1). In he p esen
e iew, we upda e he da a on
in i o
and
in i o
s udies using CA inhibi o s on mycobac e ial
β
-CAs.
2. In Vi o Inhibi ion S udies o M. ube culosis β-CAs
2.1. Sul onamides as Inhibi o s o M. ube culosis β-CAs
The cloning and cha ac e iza ion o he M. ube culosis
β
-CAs we e done in he 2000s and hese
enzymes we e iden i ied as no el d ug a ge s o de eloping an i-TB agen s [
27
–
29
].
β
-CA1 was
he i s M b
β
-CA cloned and cha ac e ized and in he same s udy, he i s
in i o
inhibi ion
s udies we e pe o med using a panel o sul onamides, sul ama es and hei de i a i es [
27
].
Fo
in i o
inhibi ion s udies, he CO
2
hyd a ion ac i i y o
β
-CA1 was measu ed by applying
Applied Pho ophysics s opped- low ins umen using phenol ed as an indica o [
27
]. Among
he es ed sul onamides, mos o hem inhibi ed he ac i i y o
β
-CA1 in he ange o 1–10
µ
M.
Many o he de i a i es, including sul anilyl-sul onamides ace azolamide (ATZ) (1), me hazolamide,
dichlo ophenamide, do zolamide (DZA) (2), b inzolamide, benzolamide, and he sul ama e opi ama e,
exhibi ed sub-mic omola inhibi ion (K
I
alues o 0.481–0.905
µ
M) [
27
] (Table 2). Among he es ed
sul onamides 3-b omosul anilamide (3) and indisulam (4) inhibi ed he ac i i y o
β
-CAs mos
e icien ly (K
I
alues o 97–186 nM) (Table 2and Figu e 2). This was he i s s udy o show ha
M b
β
-CA1 is a po en ial a ge o de eloping an i-TB d ugs ha ha e a di e en mechanism o
ac ion [
27
]. Se e al s udies
in i o
inhibi ion s udies we e pe o med using hese inhibi o molecules
Molecules 2018,23, 2911 4 o 14
on M b
β
-CA1 and human CA II ha showed simila inhibi ion p o iles sugges ing eliabili y o he
me hod used o he s udies [29,39–45].
Molecules 2018, 23, x FOR PEER REVIEW 4 o 14
Figu e 2. Chemical s uc u es o ace azolamide (1) (ATZ), do zolamide (2) (DZA), 3-
b omosul anilamide (3) and indisulam (4).
Inhibi ion o M b β-CA2 was in es iga ed using a se ies o diazenylbenzenesul onamides (5)
ha we e de i ed om sul anilamide o me anilamide (Table 2) [40]. To inc ease he inhibi o y
p ope ies, new molecules we e syn hesized by diazo iza ion o aminosul onamide and by coupling
wi h phenols o amines [29]. The molecules we e subsequen ly inco po a ed wi h a ious R moie ies
in he molecule such as hyd oxy, amino, me hylamino and dime hylamino and sul ona e ha may
induce wa e solubili y o hese compounds as sodium sal s. The aminome hylene sodium sul ona e
de i a i es and hei co esponding N-me hyla ed analogue showed he bes inhibi ion cons an s (KIs
o 45–59 nM) [29]. In hese compounds, he pa a posi ion had bulky subs i uen wi h espec o he
sul onyl moie y, sugges ing ha his s a egy may be good in ob aining low nanomola ange
inhibi o s ha a e selec i e agains M b β-CA2 [29]. In addi ion o M b β-CA1 and β-CA2, he
diazenylbenzenesul onamides (5) we e also es ed o he inhibi ion o he M b β-CA3, and he
p on osil (6) (Table 2 and Figu e 3) was ound o be he bes inhibi o wi h inhibi ion cons an s in he
ange o (KIs) o 126–148 nM [39]. In ano he s udy, se e al compounds we e s udied o hei
inhibi o y p ope ies agains M b β-CA3 and among hem 2-amino-py imidin-4-yl-sul anilamide (7)
(KI 90 nM) and sul onyla ed sul onamide (KI o 170 nM) showed ha M b CA3 can be success ully
a ge ed using CAIs wi h a po en ial o de eloping agen s a ge ing mycobac e ia (Table 2) [28].
Figu e 3. Chemical s uc u es o diazenylbenzenesul onamides (5), p on osil (6) and 2-amino-
py imidin-4-yl-sul anilamide (7).
Inhibi ion s udies on M b β-CA1 and β-CA3 using sul onamides p epa ed by eac ion o
sul anilamide wi h a yl/alkyl isocyana es (U eido-sul onamides) (8) ha e been ca ied ou and he
KIs we e ound o be in he ange o 4.8–6500 nM and o 6.4–6850 nM, espec i ely (Table 2) [41].
Simila ly, inhibi ion s udies on all he h ee β-CAs o M b we e pe o med using a numbe o
halogena ed sul anilamides and halogena ed benzolamide (9) de i a i es ha showed he e icacies
o inhibi ion in he sub-mic omola o mic omola ange (Table 2 and Figu e 4). The inhibi ion ange
was dependen on he subs i u ion pa e n a he sul anilamide moie y/ agmen o he molecule. Bes
inhibi o s we e he halogena ed benzolamides (KIs in he ange o 0.12–0.45 µM), whe eas he
halogena ed sul anilamides we e sligh ly less inhibi o y (KIs in he ange o 0.41–4.74 µM) [42].
Figu e 2.
Chemical s uc u es o ace azolamide (
1
) (ATZ), do zolamide (
2
) (DZA),
3-b omosul anilamide (3) and indisulam (4).
Inhibi ion o M b
β
-CA2 was in es iga ed using a se ies o diazenylbenzenesul onamides (5) ha
we e de i ed om sul anilamide o me anilamide (Table 2) [
40
]. To inc ease he inhibi o y p ope ies,
new molecules we e syn hesized by diazo iza ion o aminosul onamide and by coupling wi h phenols
o amines [
29
]. The molecules we e subsequen ly inco po a ed wi h a ious R moie ies in he molecule
such as hyd oxy, amino, me hylamino and dime hylamino and sul ona e ha may induce wa e
solubili y o hese compounds as sodium sal s. The aminome hylene sodium sul ona e de i a i es and
hei co esponding N-me hyla ed analogue showed he bes inhibi ion cons an s (K
I
s o 45–59 nM) [
29
].
In hese compounds, he pa a posi ion had bulky subs i uen wi h espec o he sul onyl moie y,
sugges ing ha his s a egy may be good in ob aining low nanomola ange inhibi o s ha a e selec i e
agains M b
β
-CA2 [
29
]. In addi ion o M b
β
-CA1 and
β
-CA2, he diazenylbenzenesul onamides
(5) we e also es ed o he inhibi ion o he M b
β
-CA3, and he p on osil (6) (Table 2and Figu e 3)
was ound o be he bes inhibi o wi h inhibi ion cons an s in he ange o (K
I
s) o 126–148 nM [
39
].
In ano he s udy, se e al compounds we e s udied o hei inhibi o y p ope ies agains M b β-CA3
and among hem 2-amino-py imidin-4-yl-sul anilamide (7) (K
I
90 nM) and sul onyla ed sul onamide
(K
I
o 170 nM) showed ha M b CA3 can be success ully a ge ed using CAIs wi h a po en ial o
de eloping agen s a ge ing mycobac e ia (Table 2) [28].
Molecules 2018, 23, x FOR PEER REVIEW 4 o 14
Figu e 2. Chemical s uc u es o ace azolamide (1) (ATZ), do zolamide (2) (DZA), 3-
b omosul anilamide (3) and indisulam (4).
Inhibi ion o M b β-CA2 was in es iga ed using a se ies o diazenylbenzenesul onamides (5)
ha we e de i ed om sul anilamide o me anilamide (Table 2) [40]. To inc ease he inhibi o y
p ope ies, new molecules we e syn hesized by diazo iza ion o aminosul onamide and by coupling
wi h phenols o amines [29]. The molecules we e subsequen ly inco po a ed wi h a ious R moie ies
in he molecule such as hyd oxy, amino, me hylamino and dime hylamino and sul ona e ha may
induce wa e solubili y o hese compounds as sodium sal s. The aminome hylene sodium sul ona e
de i a i es and hei co esponding N-me hyla ed analogue showed he bes inhibi ion cons an s (KIs
o 45–59 nM) [29]. In hese compounds, he pa a posi ion had bulky subs i uen wi h espec o he
sul onyl moie y, sugges ing ha his s a egy may be good in ob aining low nanomola ange
inhibi o s ha a e selec i e agains M b β-CA2 [29]. In addi ion o M b β-CA1 and β-CA2, he
diazenylbenzenesul onamides (5) we e also es ed o he inhibi ion o he M b β-CA3, and he
p on osil (6) (Table 2 and Figu e 3) was ound o be he bes inhibi o wi h inhibi ion cons an s in he
ange o (KIs) o 126–148 nM [39]. In ano he s udy, se e al compounds we e s udied o hei
inhibi o y p ope ies agains M b β-CA3 and among hem 2-amino-py imidin-4-yl-sul anilamide (7)
(KI 90 nM) and sul onyla ed sul onamide (KI o 170 nM) showed ha M b CA3 can be success ully
a ge ed using CAIs wi h a po en ial o de eloping agen s a ge ing mycobac e ia (Table 2) [28].
Figu e 3. Chemical s uc u es o diazenylbenzenesul onamides (5), p on osil (6) and 2-amino-
py imidin-4-yl-sul anilamide (7).
Inhibi ion s udies on M b β-CA1 and β-CA3 using sul onamides p epa ed by eac ion o
sul anilamide wi h a yl/alkyl isocyana es (U eido-sul onamides) (8) ha e been ca ied ou and he
KIs we e ound o be in he ange o 4.8–6500 nM and o 6.4–6850 nM, espec i ely (Table 2) [41].
Simila ly, inhibi ion s udies on all he h ee β-CAs o M b we e pe o med using a numbe o
halogena ed sul anilamides and halogena ed benzolamide (9) de i a i es ha showed he e icacies
o inhibi ion in he sub-mic omola o mic omola ange (Table 2 and Figu e 4). The inhibi ion ange
was dependen on he subs i u ion pa e n a he sul anilamide moie y/ agmen o he molecule. Bes
inhibi o s we e he halogena ed benzolamides (KIs in he ange o 0.12–0.45 µM), whe eas he
halogena ed sul anilamides we e sligh ly less inhibi o y (KIs in he ange o 0.41–4.74 µM) [42].
Figu e 3. Chemical s uc u es o diazenylbenzenesul onamides (5), p on osil (6) and 2-amino-
py imidin-4-yl-sul anilamide (7).
Inhibi ion s udies on M b
β
-CA1 and
β
-CA3 using sul onamides p epa ed by eac ion o
sul anilamide wi h a yl/alkyl isocyana es (U eido-sul onamides) (8) ha e been ca ied ou and he K
I
s
we e ound o be in he ange o 4.8–6500 nM and o 6.4–6850 nM, espec i ely (Table 2) [
41
]. Simila ly,
inhibi ion s udies on all he h ee
β
-CAs o M b we e pe o med using a numbe o halogena ed
sul anilamides and halogena ed benzolamide (9) de i a i es ha showed he e icacies o inhibi ion in
he sub-mic omola o mic omola ange (Table 2and Figu e 4). The inhibi ion ange was dependen on
he subs i u ion pa e n a he sul anilamide moie y/ agmen o he molecule. Bes inhibi o s we e he
Molecules 2018,23, 2911 5 o 14
halogena ed benzolamides (K
I
s in he ange o 0.12–0.45
µ
M), whe eas he halogena ed sul anilamides
we e sligh ly less inhibi o y (KIs in he ange o 0.41–4.74 µM) [42].
Molecules 2018, 23, x FOR PEER REVIEW 5 o 14
Figu e 4. Gene al chemical s uc u es o u eido con aining sul onamides (8) and halogena ed benzene
sul onamides (9).
A new se ies o luo ine con aining sul onamides (T iazinyl sul onamides) (10) ha we e
inco po a ed wi h amino, amino alcohol and amino acid moie ies we e used o he inhibi ion o all
he h ee β-CAs o M b [43] (Figu e 5). Among he compounds es ed, some o hem inhibi ed β-CA2
e icien ly wi h KI alues in he nanomola ange and also wi h e y good po ency (KIs in sub-
mic omola ange) agains β-CA1 and β-CA3 [43] (Table 2). In a ecen s udy, no el sul onamides
we e ob ained om sul anilamide, which was N4-alkyla ed wi h e hyl b omoace a e, ollowed by
eac ion wi h hyd azine hyd a e and u he eac ed wi h a ious a oma ic aldehydes [44]. The
inhibi ion s udies using hese sul onamides showed KIs in he ange o 127 nM–2.12 µM o M b β-
CA3 [44].
Figu e 5. Gene al chemical s uc u e o iazinyl sul onamides (10).
2.2. Mono and Di hioca bama es
A se ies o N-mono- and N,N-disubs i u ed di hioca bama es (DTCs) (11,12) ha e been es ed
o inhibi ion o β-CA1 and β-CA3 om M b (Table 2 and Figu e 6) [45]. Bo h enzymes could be
inhibi ed wi h sub-nanomola o mic omola e icacies, 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. A yl, a ylalkyl-, he e ocyclic as well as
alipha ic and amino acyl moie ies led o po en M b β-CA1 and β-CA3 inhibi o s in bo h he N-mono-
and N,N-disubs i u ed di hioca bama e se ies [45].
Figu e 6. Chemical s uc u es o di hioca bama es (11) and (12).
2.3. Phenolic Na u al P oduc s and Phenolic Acids
Se e al s udies ha e shown ha sul onamides inhibi he M b β-CAs e icien ly as discussed
abo e. Simila ly, in an e o o disco e no el inhibi o s ha could selec i ely inhibi β-CAs h ough
no el mechanism o ac ion, Supu an’s g oup sc eened a se ies o phenolic-based na u al p oduc s
(NPs) agains he M b β-CAs [46]. Enzyme inhibi ion p ope ies o 21 NP compounds we e
Figu e 4.
Gene al chemical s uc u es o u eido con aining sul onamides (
8
) and halogena ed benzene
sul onamides (9).
A new se ies o luo ine con aining sul onamides (T iazinyl sul onamides) (10) ha we e
inco po a ed wi h amino, amino alcohol and amino acid moie ies we e used o he inhibi ion o
all he h ee
β
-CAs o M b [
43
] (Figu e 5). Among he compounds es ed, some o hem inhibi ed
β
-CA2 e icien ly wi h K
I
alues in he nanomola ange and also wi h e y good po ency (K
I
s in
sub-mic omola ange) agains
β
-CA1 and
β
-CA3 [
43
] (Table 2). In a ecen s udy, no el sul onamides
we e ob ained om sul anilamide, which was N4-alkyla ed wi h e hyl b omoace a e, ollowed
by eac ion wi h hyd azine hyd a e and u he eac ed wi h a ious a oma ic aldehydes [
44
].
The inhibi ion s udies using hese sul onamides showed K
I
s in he ange o 127 nM–2.12
µ
M o
M b β-CA3 [44].
Molecules 2018, 23, x FOR PEER REVIEW 5 o 14
Figu e 4. Gene al chemical s uc u es o u eido con aining sul onamides (8) and halogena ed benzene
sul onamides (9).
A new se ies o luo ine con aining sul onamides (T iazinyl sul onamides) (10) ha we e
inco po a ed wi h amino, amino alcohol and amino acid moie ies we e used o he inhibi ion o all
he h ee β-CAs o M b [43] (Figu e 5). Among he compounds es ed, some o hem inhibi ed β-CA2
e icien ly wi h KI alues in he nanomola ange and also wi h e y good po ency (KIs in sub-
mic omola ange) agains β-CA1 and β-CA3 [43] (Table 2). In a ecen s udy, no el sul onamides
we e ob ained om sul anilamide, which was N4-alkyla ed wi h e hyl b omoace a e, ollowed by
eac ion wi h hyd azine hyd a e and u he eac ed wi h a ious a oma ic aldehydes [44]. The
inhibi ion s udies using hese sul onamides showed KIs in he ange o 127 nM–2.12 µM o M b β-
CA3 [44].
Figu e 5. Gene al chemical s uc u e o iazinyl sul onamides (10).
2.2. Mono and Di hioca bama es
A se ies o N-mono- and N,N-disubs i u ed di hioca bama es (DTCs) (11,12) ha e been es ed
o inhibi ion o β-CA1 and β-CA3 om M b (Table 2 and Figu e 6) [45]. Bo h enzymes could be
inhibi ed wi h sub-nanomola o mic omola e icacies, 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. A yl, a ylalkyl-, he e ocyclic as well as
alipha ic and amino acyl moie ies led o po en M b β-CA1 and β-CA3 inhibi o s in bo h he N-mono-
and N,N-disubs i u ed di hioca bama e se ies [45].
Figu e 6. Chemical s uc u es o di hioca bama es (11) and (12).
2.3. Phenolic Na u al P oduc s and Phenolic Acids
Se e al s udies ha e shown ha sul onamides inhibi he M b β-CAs e icien ly as discussed
abo e. Simila ly, in an e o o disco e no el inhibi o s ha could selec i ely inhibi β-CAs h ough
no el mechanism o ac ion, Supu an’s g oup sc eened a se ies o phenolic-based na u al p oduc s
(NPs) agains he M b β-CAs [46]. Enzyme inhibi ion p ope ies o 21 NP compounds we e
Figu e 5. Gene al chemical s uc u e o iazinyl sul onamides (10).
2.2. Mono and Di hioca bama es
A se ies o N-mono- and N,N-disubs i u ed di hioca bama es (DTCs) (11,12) ha e been es ed o
inhibi ion o
β
-CA1 and
β
-CA3 om M b (Table 2and Figu e 6) [
45
]. Bo h enzymes could be inhibi ed
wi h sub-nanomola o mic omola e icacies, 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. A yl, a ylalkyl-, he e ocyclic as well as alipha ic
and amino acyl moie ies led o po en M b
β
-CA1 and
β
-CA3 inhibi o s in bo h he N-mono- and
N,N-disubs i u ed di hioca bama e se ies [45].
Molecules 2018, 23, x FOR PEER REVIEW 5 o 14
Figu e 4. Gene al chemical s uc u es o u eido con aining sul onamides (8) and halogena ed benzene
sul onamides (9).
A new se ies o luo ine con aining sul onamides (T iazinyl sul onamides) (10) ha we e
inco po a ed wi h amino, amino alcohol and amino acid moie ies we e used o he inhibi ion o all
he h ee β-CAs o M b [43] (Figu e 5). Among he compounds es ed, some o hem inhibi ed β-CA2
e icien ly wi h KI alues in he nanomola ange and also wi h e y good po ency (KIs in sub-
mic omola ange) agains β-CA1 and β-CA3 [43] (Table 2). In a ecen s udy, no el sul onamides
we e ob ained om sul anilamide, which was N4-alkyla ed wi h e hyl b omoace a e, ollowed by
eac ion wi h hyd azine hyd a e and u he eac ed wi h a ious a oma ic aldehydes [44]. The
inhibi ion s udies using hese sul onamides showed KIs in he ange o 127 nM–2.12 µM o M b β-
CA3 [44].
Figu e 5. Gene al chemical s uc u e o iazinyl sul onamides (10).
2.2. Mono and Di hioca bama es
A se ies o N-mono- and N,N-disubs i u ed di hioca bama es (DTCs) (11,12) ha e been es ed
o inhibi ion o β-CA1 and β-CA3 om M b (Table 2 and Figu e 6) [45]. Bo h enzymes could be
inhibi ed wi h sub-nanomola o mic omola e icacies, 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. A yl, a ylalkyl-, he e ocyclic as well as
alipha ic and amino acyl moie ies led o po en M b β-CA1 and β-CA3 inhibi o s in bo h he N-mono-
and N,N-disubs i u ed di hioca bama e se ies [45].
Figu e 6. Chemical s uc u es o di hioca bama es (11) and (12).
2.3. Phenolic Na u al P oduc s and Phenolic Acids
Se e al s udies ha e shown ha sul onamides inhibi he M b β-CAs e icien ly as discussed
abo e. Simila ly, in an e o o disco e no el inhibi o s ha could selec i ely inhibi β-CAs h ough
no el mechanism o ac ion, Supu an’s g oup sc eened a se ies o phenolic-based na u al p oduc s
(NPs) agains he M b β-CAs [46]. Enzyme inhibi ion p ope ies o 21 NP compounds we e
Figu e 6. Chemical s uc u es o di hioca bama es (11) and (12).

Molecules 2018,23, 2911 6 o 14
2.3. Phenolic Na u al P oduc s and Phenolic Acids
Se e al s udies ha e shown ha sul onamides inhibi he M b
β
-CAs e icien ly as discussed
abo e. Simila ly, in an e o o disco e no el inhibi o s ha could selec i ely inhibi
β
-CAs h ough
no el mechanism o ac ion, Supu an’s g oup sc eened a se ies o phenolic-based na u al p oduc s (NPs)
agains he M b
β
-CAs [
46
]. Enzyme inhibi ion p ope ies o 21 NP compounds we e in es iga ed
agains
β
-CAs o M b as well as agains human
α
-CAs I and II o compa ison. Sul onamides ha a e
used clinically inhibi ed human CAs e icien ly (a nM ange), whe eas
β
-CAs equi ed mic omola
concen a ions. In con as , 8 and 7 o he 21 phenolic compounds had sub-mic omola a ini y o
β
-CA1 and
β
-CA3, espec i ely. The selec i i y o some compounds was signi ican ly highe agains
β
-CAs han human
α
-CAs ( he inhibi ion ange being 8
µ
M o 430
µ
M) [
46
]. These NPs a e he i s
nonclassical CA inhibi o s ha a e mo e po en agains mycobac e ia
β
-CAs compa ed o hos CA
enzymes, sugges ing use ulness o NPs o a ge ing
β
-CA o M b [
46
]. In addi ion o na u al phenolic
p oduc s, a se ies o phenolic acids and hei es e s, de i a i es o ca eic, e ulic, and p-couma ic acids
we e es ed agains all he
β
-CAs o M b [
47
]. Among he sc eened compounds, es e s 6–9 showed
good inhibi o y ac i i y agains all he M b
β
-CAs (K
I
s 1.87 Mm–7.05
µ
M), whe eas hey showed no
inhibi o y ac i i y agains human CAI and CAII, sugges ing ha hey could be po en ially de eloped
as an i-mycobac e ial compounds [
47
]. Compu a ional analysis o binding mode o he compounds
sugges ed ha he inhibi o s ancho o he zinc-coo dina ed wa e molecule om he CA ac i e si e
in e e ing wi h he nucleophilic a ack o he zinc hyd oxide on he subs a e CO
2
[
47
]. These esul s
p o ided insigh s in o mechanism o inhibi ion o
β
-CAs, which may be aluable o de eloping new
mycobac e ial agen s wi h a no el mechanism o ac ion [47].
Table 2.
Compounds ha inhibi M. ube culosis
β
-CAs a nM concen a ions compa ed o inhibi ion o
human CAII enzyme in i o.
CAIs hCAII β-CA1 β-CA2 β-CA3 Re e ence
3-b omosul anilamide 40 186 NS NS [27]
Diazenylbenzenesul onamides 105 - 45–955 - [29]
2-amino-py imidin-4-yl-sul anilamide 33 - NA 91 [28]
Sul onyla ed sul onamide NS - - 170 [28]
p on osil NS 126 148 [39]
Halogena ed benzolamides NS 120–580 410–450 170–340 [42]
N-mono- and N,N-di hioca bama es 0.7–325 0.9–481 NA 0.91–431 [45]
U eido-sul onamides 2.1–226 5–560 NA 6.4–533 [41]
Cinnamoyl glycosides NS 140 130–640 - [48]
T iazinyl Sul onamides 4.9–5.5 42–580 8.1–10 2.1–210 [43]
aAce azolamide (ATZ) 12 481 9 104 [29]
aE hoxzolamide (EZA) 8 - 594 27 [29]
aDo zolamide (DZA) 9 744 99 137 [29]
aIndisulam 15 97 NS NS [27]
a
Clinically, he mos ele an o p omising compounds ha inhibi human CAs e icien ly. NA—no
assayed, NS–non-signi ican .
In ano he s udy, inhibi ion p o iles o se ies o C-cinnamoyl glycosides (13) con aining he phenol
moie y we e in es iga ed agains he h ee
β
-CAs o M b [
48
] (Table 2and Figu e 7). Among he
compounds in es iga ed, mos o hem (compounds
1
–
3
and
5
–
7
) inhibi ed M b
β
-CA2 a nanomola
concen a ions (K
I
130-640 nM), and o M b
β
-CA1 (compounds
5
and
6
) he K
I
ange was be ween
140–930 nM and showed p e e ence o
β
-CA1 o e human CAII. Only one compound inhibi ed M b
β-CA1 a nanomola quan i ies (KI140 nM) o e human CAII [48].
Molecules 2018,23, 2911 7 o 14
Molecules 2018, 23, x FOR PEER REVIEW 6 o 14
in es iga ed agains β-CAs o M b as well as agains human α-CAs I and II o compa ison.
Sul onamides ha a e used clinically inhibi ed human CAs e icien ly (a nM ange), whe eas β-CAs
equi ed mic omola concen a ions. In con as , 8 and 7 o he 21 phenolic compounds had sub-
mic omola a ini y o β-CA1 and β-CA3, espec i ely. The selec i i y o some compounds was
signi ican ly highe agains β-CAs han human α-CAs ( he inhibi ion ange being 8 µM o 430 µM)
[46]. These NPs a e he i s nonclassical CA inhibi o s ha a e mo e po en agains mycobac e ia β-
CAs compa ed o hos CA enzymes, sugges ing use ulness o NPs o a ge ing β-CA o M b [46]. In
addi ion o na u al phenolic p oduc s, a se ies o phenolic acids and hei es e s, de i a i es o ca eic,
e ulic, and p-couma ic acids we e es ed agains all he β-CAs o M b [47]. Among he sc eened
compounds, es e s 6–9 showed good inhibi o y ac i i y agains all he M b β-CAs (KIs 1.87 Μm–7.05
µM), whe eas hey showed no inhibi o y ac i i y agains human CAI and CAII, sugges ing ha hey
could be po en ially de eloped as an i-mycobac e ial compounds [47]. Compu a ional analysis o
binding mode o he compounds sugges ed ha he inhibi o s ancho o he zinc-coo dina ed wa e
molecule om he CA ac i e si e in e e ing wi h he nucleophilic a ack o he zinc hyd oxide on he
subs a e CO2 [47]. These esul s p o ided insigh s in o mechanism o inhibi ion o β-CAs, which
may be aluable o de eloping new mycobac e ial agen s wi h a no el mechanism o ac ion [47].
In ano he s udy, inhibi ion p o iles o se ies o C-cinnamoyl glycosides (13) con aining he
phenol moie y we e in es iga ed agains he h ee β-CAs o M b [48] (Table 2 and Figu e 7). Among
he compounds in es iga ed, mos o hem (compounds 1–3 and 5–7) inhibi ed M b β-CA2 a
nanomola concen a ions (KI 130-640 nM), and o M b β-CA1 (compounds 5 and 6) he KI ange was
be ween 140–930 nM and showed p e e ence o β-CA1 o e human CAII. Only one compound
inhibi ed M b β-CA1 a nanomola quan i ies (KI 140 nM) o e human CAII [48].
Figu e 7. Chemical s uc u e o C-cinnamoyl glycoside (13).
2.4. Ca boxylic Acids
Weak acids a e known o inhibi he g ow h o mycobac e ium bu he mechanism o ac ion o
hese compounds is no known. Ca boxylic acids (14) ha con ain sca olds such as benzoic acids,
nipeco ic acid, o ho and pa a couma ic acid and e ulic acid we e in es iga ed o he inhibi ion o
all he h ee β-CAs o M b (Figu e 8). These compounds inhibi ed all he h ee β-CA enzymes o M b
a sub-mic omola o mic omola concen a ion ange (KIs in he ange o 0.11–0.97 µM). The KIs o
he inhibi ion o β-CA2 was in he ange o 0.59–8.10 µM, whe eas agains β-CA1, he ca boxylic acids
showed inhibi ion cons an s in he ange o 2.25–7.13 µM [49]. This class o ela i ely unde explo ed
β-CA inhibi o s wa an u he in i o s udies, as hey may ha e he po en ial o de eloping
an imycobac e ial agen s.
Figu e 8. Gene al chemical s uc u es e ulic and couma ic acids (14).
Figu e 7. Chemical s uc u e o C-cinnamoyl glycoside (13).
2.4. Ca boxylic Acids
Weak acids a e known o inhibi he g ow h o mycobac e ium bu he mechanism o ac ion o
hese compounds is no known. Ca boxylic acids (14) ha con ain sca olds such as benzoic acids,
nipeco ic acid, o ho and pa a couma ic acid and e ulic acid we e in es iga ed o he inhibi ion o all
he h ee
β
-CAs o M b (Figu e 8). These compounds inhibi ed all he h ee
β
-CA enzymes o M b a
sub-mic omola o mic omola concen a ion ange (K
I
s in he ange o 0.11–0.97
µ
M). The K
I
s o he
inhibi ion o
β
-CA2 was in he ange o 0.59–8.10
µ
M, whe eas agains
β
-CA1, he ca boxylic acids
showed inhibi ion cons an s in he ange o 2.25–7.13
µ
M [
49
]. This class o ela i ely unde explo ed
β
-CA inhibi o s wa an u he
in i o
s udies, as hey may ha e he po en ial o de eloping
an imycobac e ial agen s.
Molecules 2018, 23, x FOR PEER REVIEW 6 o 14
in es iga ed agains β-CAs o M b as well as agains human α-CAs I and II o compa ison.
Sul onamides ha a e used clinically inhibi ed human CAs e icien ly (a nM ange), whe eas β-CAs
equi ed mic omola concen a ions. In con as , 8 and 7 o he 21 phenolic compounds had sub-
mic omola a ini y o β-CA1 and β-CA3, espec i ely. The selec i i y o some compounds was
signi ican ly highe agains β-CAs han human α-CAs ( he inhibi ion ange being 8 µM o 430 µM)
[46]. These NPs a e he i s nonclassical CA inhibi o s ha a e mo e po en agains mycobac e ia β-
CAs compa ed o hos CA enzymes, sugges ing use ulness o NPs o a ge ing β-CA o M b [46]. In
addi ion o na u al phenolic p oduc s, a se ies o phenolic acids and hei es e s, de i a i es o ca eic,
e ulic, and p-couma ic acids we e es ed agains all he β-CAs o M b [47]. Among he sc eened
compounds, es e s 6–9 showed good inhibi o y ac i i y agains all he M b β-CAs (KIs 1.87 Μm–7.05
µM), whe eas hey showed no inhibi o y ac i i y agains human CAI and CAII, sugges ing ha hey
could be po en ially de eloped as an i-mycobac e ial compounds [47]. Compu a ional analysis o
binding mode o he compounds sugges ed ha he inhibi o s ancho o he zinc-coo dina ed wa e
molecule om he CA ac i e si e in e e ing wi h he nucleophilic a ack o he zinc hyd oxide on he
subs a e CO2 [47]. These esul s p o ided insigh s in o mechanism o inhibi ion o β-CAs, which
may be aluable o de eloping new mycobac e ial agen s wi h a no el mechanism o ac ion [47].
In ano he s udy, inhibi ion p o iles o se ies o C-cinnamoyl glycosides (13) con aining he
phenol moie y we e in es iga ed agains he h ee β-CAs o M b [48] (Table 2 and Figu e 7). Among
he compounds in es iga ed, mos o hem (compounds 1–3 and 5–7) inhibi ed M b β-CA2 a
nanomola concen a ions (KI 130-640 nM), and o M b β-CA1 (compounds 5 and 6) he KI ange was
be ween 140–930 nM and showed p e e ence o β-CA1 o e human CAII. Only one compound
inhibi ed M b β-CA1 a nanomola quan i ies (KI 140 nM) o e human CAII [48].
Figu e 7. Chemical s uc u e o C-cinnamoyl glycoside (13).
2.4. Ca boxylic Acids
Weak acids a e known o inhibi he g ow h o mycobac e ium bu he mechanism o ac ion o
hese compounds is no known. Ca boxylic acids (14) ha con ain sca olds such as benzoic acids,
nipeco ic acid, o ho and pa a couma ic acid and e ulic acid we e in es iga ed o he inhibi ion o
all he h ee β-CAs o M b (Figu e 8). These compounds inhibi ed all he h ee β-CA enzymes o M b
a sub-mic omola o mic omola concen a ion ange (KIs in he ange o 0.11–0.97 µM). The KIs o
he inhibi ion o β-CA2 was in he ange o 0.59–8.10 µM, whe eas agains β-CA1, he ca boxylic acids
showed inhibi ion cons an s in he ange o 2.25–7.13 µM [49]. This class o ela i ely unde explo ed
β-CA inhibi o s wa an u he in i o s udies, as hey may ha e he po en ial o de eloping
an imycobac e ial agen s.
Figu e 8. Gene al chemical s uc u es e ulic and couma ic acids (14).
Figu e 8. Gene al chemical s uc u es e ulic and couma ic acids (14).
3. In Vi o Inhibi ion o Mycobac e ial s ains Using CA Inhibi o s
A new class o compounds p epa ed by eac ion o 6-me cap opu ine wi h sul ony/sul enyl
halides known as 9-sul onyla ed/sul enyla ed-6-me cap opu ines inhibi g ow h o M b H37R , a wild
ype bacilli in he ange o 0.39–3.39
µ
g/mL [
50
] (Table 3). In addi ion, one o he de i a i es showed an
app eciable (minimal inhibi o y concen a ion (MIC) unde 1
µ
g/mL) inhibi o y ac i i y agains se e al
d ug esis an s ains o M b [
50
]. The compounds ha exhibi MIC o less han 1
µ
g/mL a e conside ed
as excellen leads and we e he i s CAIs wi h an i- ube cula ac i i y. Thus, hese compounds may
indeed cons i u e in e es ing leads o disco e ing mo e e icien an imycobac e ial d ugs. Simila ly,
C-cinnamoyl glycosides con aining he phenol moie y ha inhibi M b
β
-CA1 and
β
-CA2 in nanomola
quan i ies we e es ed o inhibi ion o he M b H
37
R s ain, leading o he iden i ica ion o compounds
ha ing an i- ube cula ac i i y (Table 3) [
48
]. The MIC o he C-cinnamoyl glycosides was 100
µ
g/mL;
hough high, he compounds inhibi ed he g ow h o he bac e ium comple ely. In e es ingly, one o
he C-cinnamoyl glycosides, (E)-1-(2,3,4,6- e a-O-ace yl-
β
-D-glucopy anosyl)-4-(3-hyd oxyphenyl)
bu -3-en-2-one inhibi ed he g ow h o he bac e ium e icien ly (3.125–6.25
µ
g/mL) on a solid medium
(Table 3) [48].
Molecules 2018,23, 2911 8 o 14
Table 3.
S udies on minimal inhibi o y concen a ions (MICs) o he CA inhibi o s in
mycobac e ial cul u es.
Inhibi o Bacilli Concen a ion Re e ence
(E)-1-(2,3,4,6- e a-O-ace yl-
β
-D-glucopy anosyl)-
4-(3-hyd oxyphenyl) bu -3-en-2-one M b H37R b3.125–6.25 µg/mL [48]
100 µg/mL [48]
DTCs (Fc14-584b and Fc14-594a) M. ma inum 17–18 µg/mL [26]
9-sul onyla ed/sul enyla ed-6-me cap opu ines M b H37R 0.39–3.39 µg/mL [50]
9-sul onyla ed/sul enyla ed-6-me cap opu ines aM b 1 µg/mL [50]
aD ug esis an M b s ains. bAn imycobac e ial ac i i y on solid medium.
Inhibi ion s udies on M. ma inum, an NTM and a close ela i e o M b, we e ca ied ou in liquid
cul u es using DTCs Fc14-584b and Fc14-594a. These d ugs we e p epa ed by eac ion o co esponding
amine wi h ca bon disul ide in he p esence o a base and shown o be speci ic inhibi o s o M b
β
-CA1
and
β
-CA3 [
45
].
In i o
inhibi ion s udies showed ha he concen a ion equi ed o he inhibi ion o
he M. ma inum was 17–18
µ
g/mL o bo h compounds a e six days o exposu e o he inhibi o s.
Fu he s udies o ind i he compounds we e bac e ios a ic o bac e icidal showed ha he e was no
g ow h esump ion o M.ma inum wi h inhibi o concen a ion below MIC a e inhibi o dilu ion by
1:4, sugges ing ha hese compounds we e bac e icidal [26] (Table 3).
Simila o o he bac e ia ha con ain ex acellula DNA (eDNA) in he ma ix o he bac e ial
bio ilms, NTM bac e ia also con ain signi ican amoun s o eDNA in hei bio ilms and a e esponsible
o pheno ypic esis ance o he bac e ia o an ibio ics, in addi ion o o he biological unc ions [
51
].
A ecen s udy showed ha bica bona e ion posi i ely in luences eDNA expo in NTM and i is
well es ablished ha bica bona e is gene a ed by he hyd a ion o ca bon dioxide ia CA [
31
,
35
].
Sc eening o a mu an lib a y o eDNA expo in NTM bac e ia M. a ium iden i ied mu an s ha
we e inac i a ed o CA gene and hese mu an s when complemen ed wi h he CA gene es o ed
he anspo o eDNA, sugges ing ha CAs play impo an oles in he anspo o eDNA and
o ma ion o bio ilms in NTM [
35
]. The su ace exposed p o eome o M. a ium in eDNA con aining
bio ilms showed p esence o abundan CAs and inhibi ion s udies exposing hese bac e ia o
6-e hoxy-1,3-benza hiazole-2-sul onamide/e hoxzolamide (
15
) (EZA) showed educ ion in eDNA
anspo signi ican ly (Table 4and Figu e 9) [
35
]. Thus, in addi ion o ha ing an e ec on mycobac e ial
g ow h, CA-inhibi ion may also be a po en ial s a egy o inhibi bio ilm o ma ion o mycobac e ia.
Table 4. De ails o in i o inhibi ion s udies on di e en mycobac e ial species.
Inhibi o Bac e ium Inhibi o y E ec Re e ences
EZA M. ube culosis aA enua es i ulence, inhibi s PhoPR [36]
EZA M. a ium bT anspo o eDNA and bio ilm o ma ion [35]
Di hioca bama e 12 M. ma inum bcImpai s g ow o bac e ium in he la ae [26]
aMycobac e ium ube culosis complex. bNon ube culous mycobac e ia. cIn zeb a ish la al model.
Molecules 2018, 23, x FOR PEER REVIEW 8 o 14
o 6-e hoxy-1,3-benza hiazole-2-sul onamide/e hoxzolamide (15) (EZA) showed educ ion in eDNA
anspo signi ican ly (Table 4 and Figu e 9) [35]. Thus, in addi ion o ha ing an e ec on
mycobac e ial g ow h, CA-inhibi ion may also be a po en ial s a egy o inhibi bio ilm o ma ion o
mycobac e ia.
Figu e 9. Chemical s uc u e o e hoxzolamide (15).
Table 3. S udies on minimal inhibi o y concen a ions (MICs) o he CA inhibi o s in mycobac e ial
cul u es.
Inhibi o
Bacilli Concen a ion Re e
ence
(E)-1-(2,3,4,6- e a-O-ace yl-β-D-glucopy anosyl)-
4-(3-hyd oxyphenyl) bu -3-en-2-one M b H37R
b 3.125–6.25 µg/mL [48]
100 µg/mL [48]
DTCs (Fc14-584b and Fc14-594a) M. ma inum 17–18 µg/mL [26]
9-sul onyla ed/sul enyla ed-6-me cap opu ines M b H37R 0.39–3.39 µg/mL [50]
9-sul onyla ed/sul enyla ed-6-me cap opu ines a M b 1 µg/mL [50]
a D ug esis an M b s ains. b An imycobac e ial ac i i y on solid medium.
4. CA Inhibi o s and In Vi o Inhibi ion o Mycobac e ia
The i s in i o s udy o show he e ec o CA inhibi o on M b was published in 2015 by Johnson
e al. [36]. The au ho s showed ha EZA (Figu e 10) inhibi s he signaling o PhoPR in M b [36,52].
EZA is a sul onamide compound (Figu e 10) ha is a gene al inhibi o o CA enzyme ac i i y and is
an FDA app o ed d ug used in he ea men o glaucoma, epilepsy and duodenal ulce s and is a
diu e ic.
Figu e 9. Chemical s uc u e o e hoxzolamide (15).
4. CA Inhibi o s and In Vi o Inhibi ion o Mycobac e ia
The i s
in i o
s udy o show he e ec o CA inhibi o on M b was published in 2015 by Johnson
e al. [
36
]. The au ho s showed ha EZA (Figu e 10) inhibi s he signaling o PhoPR in M b [
36
,
52
].
Molecules 2018,23, 2911 9 o 14
EZA is a sul onamide compound (Figu e 10) ha is a gene al inhibi o o CA enzyme ac i i y and is an
FDA app o ed d ug used in he ea men o glaucoma, epilepsy and duodenal ulce s and is a diu e ic.
Molecules 2018, 23, x FOR PEER REVIEW 9 o 14
(A)
(B)
Figu e 10. No el compounds ha inhibi M. ube culosis β-CAs a nanomola quan i ies. (Uppe
panel; A): s uc u es o he compounds wi h a po en ial o be de eloped as an i-mycobac e ial agen s
o ea ing he mycobac e ial diseases caused by MTC and NTM bac e ia ha a e esis an o
clinically used d ugs. (Lowe panel; B): compounds ha e icien ly inhibi he M. ube culosis β-CAs
in addi ion o human CAII and a e in clinical use o es ing o ea human diseases.
The s udy showed ha M b ea ed wi h EZA induces pheno ypes simila o he mu an s o he
PhoPR, down egula ing PhoPR egulon, educing he p oduc ion o i ulence-associa ed lipids, and
inhibi ing Esx-1 p o ein sec e ion (Table 4) [36]. In addi ion, quan i a i e single cell imaging o a
PhoPR dependen luo escen epo e s ain showed ha EZA inhibi s PhoPR egula ed genes in
in ec ed mac ophages and mouse lungs [36]. Simila ly, he e icacy assessmen in M b-in ec ed mice,
o ally ea ed wi h EZA, showed a signi ican educ ion in bac e ial g ow h in he lungs compa ed o
he mock- ea ed con ol g oup [36].
Figu e 10.
No el compounds ha inhibi M. ube culosis
β
-CAs a nanomola quan i ies. (
Uppe panel
;
A
): s uc u es o he compounds wi h a po en ial o be de eloped as an i-mycobac e ial agen s o
ea ing he mycobac e ial diseases caused by MTC and NTM bac e ia ha a e esis an o clinically
used d ugs. (
Lowe panel
;
B
): compounds ha e icien ly inhibi he M. ube culosis
β
-CAs in addi ion
o human CAII and a e in clinical use o es ing o ea human diseases.
The s udy showed ha M b ea ed wi h EZA induces pheno ypes simila o he mu an s o he
PhoPR, down egula ing PhoPR egulon, educing he p oduc ion o i ulence-associa ed lipids, and
inhibi ing Esx-1 p o ein sec e ion (Table 4) [
36
]. In addi ion, quan i a i e single cell imaging o a PhoPR
dependen luo escen epo e s ain showed ha EZA inhibi s PhoPR egula ed genes in in ec ed
mac ophages and mouse lungs [
36
]. Simila ly, he e icacy assessmen in M b-in ec ed mice, o ally
ea ed wi h EZA, showed a signi ican educ ion in bac e ial g ow h in he lungs compa ed o he
mock- ea ed con ol g oup [36].