Carbonic Anhydrase Inhibitors as Novel Drugs against Mycobacterial β-Carbonic Anhydrases: An Update on In Vitro and In Vivo Studies

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].