International Journal of Molecular Sciences

Review Mycobacterium tuberculosis β-Carbonic Anhydrases: Novel Targets for Developing Antituberculosis Drugs

Ashok Aspatwar 1,* , Visvaldas Kairys 2 , Sangeetha Rala 3, Mataleena Parikka 1, Murat Bozdag 4 , Fabrizio Carta 4 , Claudiu T. Supuran 4 and Seppo Parkkila 1,5

1 Faculty of Medicine and Health Technology, Tampere University, FI-33014 Tampere, Finland; mataleena.parikka@tuni.fi (M.P.); seppo.parkkila@tuni.fi (S.P.) 2 Department of Bioinformatics, Institute of Biotechnology, Life Sciences Centre, Vilnius University, Sauletekio˙ al. 7, LT-10257 Vilnius, Lithuania; [email protected] 3 Tampere University of Applied Sciences, Kuntokatu 3, FI-33520 Tampere, Finland; sangeetha.rala@tuni.fi 4 Neurofarba Department, Sezione di Chimica Farmaceutica e Nutraceutica, Università degli Studi di Firenze, Via U. Schiff 6, I-50019 Sesto Fiorentino (Firenze), Italy; bozdag.murat@unifi.it (M.B.); fabrizio.carta@unifi.it (F.C.); claudiu.supuran@unifi.it (C.T.S.) 5 Fimlab Ltd. and Tampere University Hospital, FI-33520 Tampere, Finland * Correspondence: ashok.aspatwar@tuni.fi; Tel.: +358-46-596-2117

 Received: 16 September 2019; Accepted: 15 October 2019; Published: 17 October 2019 

Abstract: The genome of Mycobacterium tuberculosis (Mtb) encodes three β-carbonic anhydrases (CAs, EC 4.2.1.1) that are crucial for the life cycle of the bacterium. The Mtb β-CAs have been cloned and characterized, and the catalytic activities of the have been studied. The crystal structures of two of the enzymes have been resolved. In vitro inhibition studies have been conducted using different classes of inhibitors (CAIs). In vivo inhibition studies of pathogenic bacteria containing β-CAs showed that β-CA inhibitors effectively inhibited the growth of pathogenic bacteria. The in vitro and in vivo studies clearly demonstrated that β-CAs of not only mycobacterial species, but also other pathogenic bacteria, can be targeted for developing novel antimycobacterial agents for treating tuberculosis and other microbial infections that are resistant to existing drugs. In this review, we present the molecular and structural data on three β-CAs of Mtb that will give us better insights into the roles of these enzymes in pathogenic bacterial species. We also present data from both in vitro inhibition studies using different classes of chemical compounds and in vivo inhibition studies focusing on M. marinum, a model organism and close relative of Mtb.

Keywords: Mycobacterium tuberculosis; β-carbonic anhydrases; drug targets; tuberculosis; carbonic anhydrase inhibitors; in vitro inhibition; antituberculosis drugs

1. Introduction Diseases caused by Mycobacterium tuberculosis (Mtb) and other mycobacterial species affect a large number of people in the world [1]. According to recent World Health Organization (WHO) estimates approximately 10 million people developed TB disease in 2017 [2]. Worldwide, tuberculosis (TB) is one of the top 10 causes of death and the leading cause of a single infectious agent. In 2017, TB disease caused approximately 1.6 million deaths [2]. In addition, drug-resistant TB continues to be a public health problem. The best estimate is that in 2017 558,000 people (range, 483,000–639,000) developed TB that was resistant to rifampicin (RR-TB), the most effective first-line drug, and 82% of these cases had multidrug-resistant TB (MDR-TB) [2]. The genome-sequencing project of Mtb [3] and other pathogenic bacteria including M. marinum (Mmar), a model bacterium, have greatly increased our knowledge about the evolution

Int. J. Mol. Sci. 2019, 20, 5153; doi:10.3390/ijms20205153 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 5153 2 of 16 of mycobacteria [4,5]. Sequencing of the genomes of pathogenic mycobacteria also helped us to identify new drug targets, which might lead to the development of anti-TB drugs possessing a novel mechanism of action and thus resolve the multidrug resistance (MDR) challenge of mycobacteria that has emerged recently [3–7]. Among these targets, three novel proteins have been identified as carbonic anhydrases (CAs, EC 4.2.1.1). The CA enzymes are ubiquitous in nature and belong to eight different unrelated families (α-, β-, γ-, δ-, ζ-, η-, θ-, and ι-CAs) [8,9] and are known to perform important physiologic roles in living organisms [8–11]. The CAs of vertebrates belong to the α-CA gene family, which contains multiple isoforms of the . CAs are involved in a fundamental reaction in which is reversibly hydrated to generate both and hydrogen ions for the regulation of pH homeostasis. In addition, CAs participate in biosynthetic reactions, such as gluconeogenesis and ureagenesis in mammals. Due to their association with various diseases, α-CA enzymes are considered important targets for the design of new drugs, such as antidiuretics, antiglaucoma, antiepileptic, and anticancer agents [11]. In the recent past, CAs belonging to the β-CA family have emerged as potential targets for developing drugs against diseases caused by bacterial and parasitic pathogens. Indeed, several in vitro inhibition studies have shown that the β-CAs from pathogenic bacteria and parasitic pathogens can be inhibited by certain compounds [12–24]. Among these inhibitors are sulfonamides and their bioisosteres, which are the main classes of CA inhibitors that have been in clinical use for more than 50 years [25–28]. In addition, many studies have shown that pathogenic organisms are susceptible to CA inhibitors in vivo as well [9,25,29–31]. Analysis of Mtb CA sequences has revealed that Mtb CAs belong to the β-CA gene family, are encoded by Rv1284, Rv3588c, and Rv3273, and are denoted as β-CA1, β-CA2, and β-CA3, respectively [3,9,32]. The Mtb β-CAs are zinc-containing metalloenzymes with characteristics similar to those of many other bacterial β-CAs, including all the conserved amino acid residues involved in the catalytic cycle, i.e., the four zinc-binding residues Cys42, Asp44, His97, and Cys101 [9]. The three Mtb β-CAs have been cloned and purified, and kinetic studies of these CAs have been carried out [33–37]. The X-ray crystal structures of two of the Mtb β-CAs (Rv1284 and Rv3588c) have been resolved [36,37]. Studies conducted on mycobacterial β-CAs have shown that they are involved in the invasion and survival of pathogens in the host environment and are thus considered potential drug targets for developing novel anti-TB agents [9]. Several in vitro inhibition studies have shown that Mtb β-CAs can be efficiently inhibited by different classes of inhibitors [9]. In this review, we present the molecular data on the gene and protein sequences as well as the information on 3D structures of Mtb β-CAs that is available in the literature. We also review the available data on the roles of β-CAs in Mtb and nontuberculous mycobacteria (NTM), which include information on Mmar, a model organism for studying the physiological roles of β-CAs in mycobacteria and a close relative of Mtb. Similarly, we also discuss the data from in vitro inhibition studies using different classes of chemical compounds.

2. Molecular Biology The β-CA genes of Mtb (Rv1284, Rv3588c, and Rv3273) have been cloned, recombinant proteins of these CAs have been produced, and activity and inhibition studies have been carried out [34–37]. The X-ray crystal structures of two of the enzymes (Mtb β-CA1 and Mtb β-CA2) have been resolved [36,37]. The details of the molecular characterization of the Mtb β-CA genes are presented in the following sections.

2.1. M. tuberculosis Rv1284 The gene Rv1284 encodes Mtb β-CA1, and the length of the corresponding peptide is 163 amino acid residues (Figure1). The protein product of Rv1284 was identified in the membrane fraction of Mtb strain H37Rv by SDS-PAGE [38] and uLC-MS/MS [39]. The mRNA transcript is upregulated after 4 and 24 h of starvation, and the upregulation is highest at 96 h [40]. The gene product is required for the growth of bacteria in the C57BL/6J mouse spleen detected by transposon site hybridization (TraSH) Int. J. Mol. Sci. 2019, 20, 5153 3 of 16 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 3 of 16 in H37Rvthe growth [41]. The of bacteriaMtb Rv3588c in the C57BL/6J gene was mouse cloned, spleen and detected the crystal by transposon structure ofsite the hybridization corresponding recombinant(TraSH) in protein H37Rv was [41] studied. The Mtb in Rv3588c 2005 by gene Suarez was Covarrubias cloned, and et the al. crystal [36]. structure In a later of report the in 2009,corresp kineticonding studies recombinant of the recombinant protein was enzyme studied werein 2005 carried by Suarez out Covarrubias by Minakuchi et al. et [36] al.. (TableIn a later1)[ 34]. report in 2009, kinetic studies of the recombinant enzyme were carried out by Minakuchi et al. (Table The analysis of the cloned sequence showed no differences to the original sequence available in the 1) [34]. The analysis of the cloned sequence showed no differences to the original sequence available UniProtin the database UniProt [database3,34]. [3,34].

FigureFigure 1. The 1. Theβ-CA β-CA sequence sequence of ofMtb. Mtb.The The proteinprotein (Rv1284) sequence sequence shows shows the theamino amino acid acidresidues residues (boxed(boxed and and with with blue blue arrows) arrows) that that are are involved involved inin the coordination coordination of ofZn(II) Zn(II) ions ions in the in active the of site of the enzyme.the enzyme.

TableTable 1. Activity 1. Activity and and inhibition inhibition kinetics kinetics of Mtb ββ-CAs-CAs compared compared to tohuman human CA CAII. II.

CAs Gene ID Protein Kcat (S−1) 1 Kcat/Km (M−1S−1) 1KI (nM1 ) a Ref. a CAs Gene ID Protein Kcat (S− ) Kcat/Km (M− S− ) KI (nM) Ref. Mtb β- Mtb β-CA1 Rv1284Rv1284 163163 aa aa 3.93.9 × 10510 5 3.7 × 103.77 107 480 480[34] [34] CA1 × × Mtb β-CA2Mtb β Rv3588c- 207 aa 9.8 105 9.3 107 9.8 [35] Rv3588c 207 aa 9.8 × 10× 5 9.3 × 107 × 9.8 [35] Mtb β-CA3CA2 Rv3273 764 aa b 4.3 105 4.0 107 104 [33] Mtb β- × × hCA II CA2Rv3273 260764 aa aa b 4.31.4 × 10510 6 4.0 × 101.57 108 104 12[33] [35] CA3 × × a b hCAInhibition II usingCA2 acetazolamide; 260 aa Sulfate1.4 × 10 transporter6 1.5 domain × 108 (amino acids12 121–414). [35] a Inhibition using acetazolamide; b Sulfate transporter domain (amino acids 121–414). Interestingly, Mtb β-CA1 is a smaller protein compared to other β-CA enzymes and more specifically Interestingly, Mtb β-CA1 is a smaller protein compared to other β-CA enzymes and more compared to the β-CA2 enzyme of mycobacteria [34]. In β-CA1 the Zn(II) ion is coordinated by an specifically compared to the β-CA2 enzyme of mycobacteria [34]. In β-CA1 the Zn(II) ion is aspartatecoordinated residue, by possessing an aspartate a residue, so-called possessing ‘closed’ a active so-called site ‘closed conformation,’ active site which conformation, is also observed which in someis algal also observeβ-CA enzymesd in some [ 34algal]. Itβ- hasCA enzymes also been [34] shown. It has that also manybeen shown amino that acids many are amino different acids in areβ-CA1 compareddifferent to βin-CA2, β-CA1 suggesting compared to a structuralβ-CA2, suggesting basis for a structural the difference basis infor catalyticthe difference activity in catalytic and affi nity to CAactivity inhibitors, and affinity as shown to CA in in Tablehibitors,1[ 34as ].shown Interestingly, in Table 1 a[34] study. Interestingly, carried out a study in 2015 carried by Hofmann’s out in group2015 showed by Hofmann’s that Rv1284 group is susceptible showed that to redox Rv1284 conditions is susceptible in the to host redox environment, conditions linking in the oxidative host stimulienvironment, to changes linking in the oxidative pH homeostasis stimuli to changes of the in pathogen the pH homeostasis [42]. The of inactivation the pathogen of [42] Rv1284. The due to oxidationinactivation stops of Rv1284 the enzymatic due to oxidation hydration stops of the carbon enzymatic dioxide hydration and thus of carbon the production dioxide and of thus protons; the production of protons; therefore, the decrease in the pH within the vicinity of action of Rv1284 therefore, the decrease in the pH within the vicinity of action of Rv1284 could be a strategy employed could be a strategy employed by mycobacteria for survival in the host [42]. by mycobacteria for survival in the host [42]. 2.2. M. tuberculosis Rv3588c 2.2. M. tuberculosis Rv3588c The protein encoded by gene Rv3588c, also known in the databases as CanB, is a β-CA and Thecontains protein 207 aminoencoded acid by residues gene Rv3588c, (Figure 2) also [36]. known Biochemical in the experiments databases have as CanB, shown is that a β-CA the and containsprotein 207 product amino acidof Rv3588c residues is similar (Figure to2 )[the36 putative]. Biochemical 213 aa CA experiments Q9CBJ1 of Mycobacterium have shown thatleprae the [36] protein. productThe of protein Rv3588c was is identified similar to by the mass putative spectrometry 213 aa CA in Q9CBJ1the membrane of Mycobacterium protein fraction leprae of[ 36Mtb]. H37Rv The protein was identified[43] and the by proteomic mass spectrometry data supported in the the membrane translation protein start site fraction [44]. Transposon of Mtb H37Rv hybridization [43] and the proteomic(TraSH) data assay supported in H37Rv theshowed translation that thestart gene siteis required [44]. Transposon for bacterial hybridization growth in C57BL/6J (TraSH) mouse assay in H37Rvspleen showed [41]. that the gene is required for bacterial growth in C57BL/6J mouse spleen [41].

Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 4 of 16

Int. J. Mol. Sci. 2019, 20, 5153 4 of 16 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 4 of 16

Figure 2. The β-CA2 sequence of Mtb. The β-CA (Rv3588c) protein sequence shows the amino acid residues (boxed and with blue arrows) that are involved in the coordination of the Zn(II) ion at the active site of the enzyme.

The available data from sequence alignment and from crystallographic studies have shown that Mtb β-CA2 has zinc ligands that are present in other bacterial β-CAs [35]. Kinetic studies have shown Figurethat theFigure 2. productThe 2. Theβ-CA2 of β -theCA2 sequence Rv3588c sequence ofgene ofMtb. Mtb. of The MtbThe βpossessesβ-CA-CA (Rv3588c) the highest protein protein catalytic sequence sequence activity shows shows the for amino theCO amino2 hydration acid acid residuesamongresidues the (boxed three (boxed andMtb and withβ- CAswith blue (Tableblue arrows) arrows) 1) [35] that that. areare involved in in the the coordination coordination of the of theZn(II) Zn(II) ion at ion the at the activeactive site ofsite the of enzyme.the enzyme. 2.3. M. tuberculosis Rv3273 The available data from sequence alignment and from crystallographic studies have shown that The availableThe Rv3273 data gene from encoding sequence β-CA3 alignment was identified and fromin 2002 crystallographic from the genome studies of Mtb strain have shownH37Rv that Mtb β-CA2 has zinc ligands that are present in other bacterial β-CAs [35]. Kinetic studies have shown Mtb β[45]-CA2. It hasis believed zinc ligands to be a that transmembrane are present inprotein other based bacterial on itsβ -CAshydrophobic [35]. Kinetic N-terminus studies [38,46,47] have shown. that the product of the Rv3588c gene of Mtb possesses the highest catalytic activity for CO2 hydration that theBased product on the of information the Rv3588c from gene UniProt of Mtb andpossesses NCBI gene the highestdatabases, catalytic the Rv3273 activity protein for COproducthydration is among the three Mtb β-CAs (Table 1) [35]. 2 amongpredicted the three to Mtb containβ-CAs 764 (Table amino 1 acids)[35 ].[3,45]. It is a bifunctional protein with a sulfate transporter domain at the N-terminal end (amino acids 121–414) and a β-CA domain (amino acids 571–741) [33]. 2.3. M. tuberculosis Rv3273 2.3. M.The tuberculosis schematic structure Rv3273 of the protein product is shown in Figure 3. The domain organization of the proteinThe is Rv3273 shown gene to be encoding similar to β -thatCA3 of was the identified other bacterial in 2002 proteins from the found genome in Arthrobacter of Mtb strain aurescens H37Rv The[45](YP_94911. Rv3273It is believed6), geneLeptospira encodingto be borgpetersenii a transmembraneβ-CA3 was(YP_798800), identified protein Legionellabased in 2002 on frompneumophilaits hydrophobic the genome (YP_126096), N of-terminusMtb andstrain [38,46,47] Leptospira H37Rv. [45]. It is believedBasedinterrogans on to the be(NP_710760), information a transmembrane from which UniProt protein are putative and based NCBI bifunctionalon gene its hydrophobic databases, proteins the N-terminus andRv3273 contain protein [38 , both46 product,47 sulfate]. Based is on the informationpredictedtransporter to and from con βtain-CA UniProt 764domains amino and [45] acids NCBI. [3,45] gene. It databases, is a bifunctional the Rv3273 protein protein with a product sulfate transporter is predicted to containdomain 764The aminoat β the-CA N domainacids-terminal [3 (a,45 endmino]. It(amino acids is a bifunctional a 550cids–764) 121 –of414) the protein and Rv3273 a β- withCA gene domain a sulfate was (amino cloned, transporter acids characterized, 571 domain–741) [33] and at. the N-terminalThedenominated schematic end (amino as structure β-CA3 acids [33] of 121–414)the. The protein amino and product acid a β sequence-CA is shown domain of inthe Figure (amino cloned 3. DNA acidsThe domain was 571–741) shown organization [ 33to ].be The similar of schematic the to structureproteinthe amino of theis shown acidprotein sequenceto productbe similar of is Rv3273 to shown that thatof in the Figure is other available3 .bacterial The in domain databases proteins organization found[3,45] within Arthrobacter of the the exception protein aurescens is of shown a (YP_949116), Leptospira borgpetersenii (YP_798800), Legionella pneumophila (YP_126096), and Leptospira to besubstitution similar to that of of amino the other acid Arg606 bacterial to proteins Cys. Mtb found β-CA3 in hasArthrobacter three conserved aurescens zinc(YP_949116), binding residuesLeptospira interroganspresent in the(NP_710760), β-CA1 and β which-CA2 of are Mtb putative [34,36,37] bifunctional, which are Cys584, proteins His642, and containand probably both Asp586 sulfate borgpetersenii (YP_798800), Legionella pneumophila (YP_126096), and Leptospira interrogans (NP_710760), transporter[33]. Activity and and β -inhibitionCA domains studies [45]. of β-CA3 have also been carried out, and the details of the kinetics which are putative bifunctional proteins and contain both sulfate transporter and β-CA domains [45]. are shownThe β -inCA Table domain 1. (amino acids 550–764) of the Rv3273 gene was cloned, characterized, and denominated as β-CA3 [33]. The amino acid sequence of the cloned DNA was shown to be similar to the amino acid sequence of Rv3273 that is available in databases [3,45] with the exception of a substitution of amino acid Arg606 to Cys. Mtb β-CA3 has three conserved zinc binding residues present in the β-CA1 and β-CA2 of Mtb [34,36,37], which are Cys584, His642, and probably Asp586 [33]. Activity and inhibition studies of β-CA3 have also been carried out, and the details of the kinetics are shown in Table 1.

FigureFigure 3. Schematic 3. Schematic presentation presentation of ofMtb Mtbβ -CA3β-CA3 (Rv3273).(Rv3273). (A (A) )The The complete complete β-CA3β-CA3 protein protein showing showing bothboth the sulfatethe sulfate transporter transporter and and CA CA domains domains atat thethe N-N- and C C-terminal-terminal ends, ends, respectively. respectively. (B) The (B) The recombinant Mtb β-CA3 protein produced for kinetic studies [33]. recombinant Mtb β-CA3 protein produced for kinetic studies [33].

The β-CA domain (amino acids 550–764) of the Rv3273 gene was cloned, characterized, and denominated as β-CA3 [33]. The amino acid sequence of the cloned DNA was shown to be similar Figure 3. Schematic presentation of Mtb β-CA3 (Rv3273). (A) The complete β-CA3 protein showing to the aminoboth the acid sulfate sequence transporter of Rv3273 and CA domains that is availableat the N- and in C databases-terminal ends, [3,45 respectively.] with the (B exception) The of a substitutionrecombinant of amino Mtb acid β-CA3 Arg606 protein produced to Cys. forMtb kineticβ-CA3 studies has [33] three. conserved zinc binding residues present in the β-CA1 and β-CA2 of Mtb [34,36,37], which are Cys584, His642, and probably Asp586 [33]. Activity and inhibition studies of β -CA3 have also been carried out, and the details of the kinetics are shown in Table1. Int. J. Mol. Sci. 2019, 20, 5153 5 of 16

3. M. marinum Is a Suitable Model for Studying the Roles of β-CAs M. marinum, an NTM, is found in water and is a natural TB pathogen of zebrafish [4]. The Mmar bacillus hasInt. J. several Mol. Sci. 2019 characteristics,, 20, x FOR PEER REVIEW such as slow growth, immotility, no spores, and Gram-positive5 of 16 staining. 3.Mmar M. marinumis of Is great a Suitable scientific Model interestfor Studying because the Roles it isof β genetically-CAs related to Mtb, a pathogen that causes TB in humans, and interestingly, the experimental infection of Mmar in fish mimics M. marinum, an NTM, is found in water and is a natural TB pathogen of zebrafish [4]. The Mmar the pathogenesisbacillus has of several TB [48 characteristics,]. The genome such ofas slow the Mgrowth, strain immotility, of Mmar no containsspores, and a G 6,636,827-bpram-positive circular .staining.Mmar Mmaris is widely of great used scientific as a interest model because organism it is genetically to study related TB, and to Mtb, genome a pathogen comparisons that have confirmedcauses the close TB in genetic humans, relationship and interestingly, between the experimental these two infection species of Mmar showing in fish that mimics they the share 3000 pathogenesis of TB [48]. The genome of the M strain of Mmar contains a 6,636,827-bp circular orthologs with an average amino acid identity of 85% [4]. chromosome. Mmar is widely used as a model organism to study TB, and genome comparisons have confirmed the close genetic relationship between these two species showing that they share 3000 Sequence andorthologs Phylogenetic with an average Analysis amino of β acid-CAs identity of 85% [4].

BioinformaticSequence and studies Phylogenetic of Analysis different of β-MmarCAs strains showed the presence of three β-CAs in the bacterium [29]. Analysis of Mmar β-CA sequences showed that they are closely related to the Mtb β-CA Bioinformatic studies of different Mmar strains showed the presence of three β-CAs in the sequencesbacterium [29]. Sequence [29]. Analysis alignment of Mmar β of-CAβ-CA sequences protein showed sequences that they are from closely both related organisms to the Mtb showed β- very high conservationCA sequences of amino[29]. Sequence acids alignment between of the β-CA sequences protein sequences (identities from both for organismsβ-CA1: 85.2%, showed βvery-CA2: 86.8%, and β-CA3:high 72.8%). conservation The names,of amino acids numbering between the and sequences sequences (identities of the forβ β-CAs-CA1: were85.2%, basedβ-CA2: 86.8%, on the UniProt entries (Figureand β4-CA3:). 72.8%). The names, numbering and sequences of the β-CAs were based on the UniProt entries (Figure 4).

Figure 4. Alignment of the Mtb and Mmar β-CA protein sequences. Conserved amino acid residues Figure 4. Alignment of the Mtb and Mmar β-CA protein sequences. Conserved amino acid residues between the three Mtb CAs (UniProt IDs: Rv1284/CA1, Rv3588c/CA2, and Rv3273/CA3) and the between thecorresponding three Mtb MmarCAs β-CA (UniProt sequences IDs: are indicated Rv1284 /byCA1, asterisks. Rv3588c (A) Shows/CA2, the and alignment Rv3273 of β/-CA3)CA1 and the correspondingsequencesMmar of Mtbβ-CA and sequencesMmar; (B) Shows are the indicated alignment by of β asterisks.-CA2 sequences (A) Showsof Mtb and the Mmar alignment; and (C) of β-CA1 sequences of Mtb and Mmar;(B) Shows the alignment of β-CA2 sequences of Mtb and Mmar; and (C) shows the alignment of β-CA3 sequences of Mtb and Mmar. The boxed yellow and blue highlighted parts show the N-terminal sulfate transporter and C-terminal CA domains, respectively. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 6 of 16 Int. J. Mol. Sci. 2019, 20, 5153 6 of 16 shows the alignment of β-CA3 sequences of Mtb and Mmar. The boxed yellow and blue highlighted parts show the N-terminal sulfate transporter and C-terminal CA domains, respectively. Phylogenetic analysis showed that each of the three Mmar β-CAs are more closely related to the correspondingPhylogeneticMtb β-CA analysis compared showed to that the βeach-CA of of the other three mycobacterial Mmar β-CAs are species more (Figure closely5 relatedA). A subcellular to the localizationcorresponding prediction Mtbusing β-CA a compared web-based to tool the [β49-CA] (and of other the information mycobacterial accessed species from (Figure the TubercuList5A). A (http:subcellular//tuberculist.epfl.ch localization) databaseprediction suggestedusing a web that-basedMtb toolβ-CA1 [49] (and and theβ-CA2 information are cytoplasmic accessed from (data not shown),the Mtb TubercuListβ-CA3 is (htt ap://tuberculist.epfl.ch membrane-associated) database protein suggested (Figure5B), that and Mtb these β-CA1 predicted and β- localizationsCA2 are matchcytoplasmic those of the (dataMmar not βshown),-CAs (Figure Mtb β-CA35C) [is49 a]. membrane-associated protein (Figure 5B), and these predicted localizations match those of the Mmar β-CAs (Figure 5C) [49].

FigureFigure 5. Phylogeny 5. Phylogeny of of mycobacterial mycobacterial β-βCA-CA sequences sequences and andtransmembrane transmembrane domain domain prediction prediction of Mtb of and Mmar β-CA3 protein sequences. The phylogenetic tree in panel A shows the evolutionary Mtb and Mmar β-CA3 protein sequences. The phylogenetic tree in panel (A) shows the evolutionary relationship of the Mtb and Mmar β-CA protein sequences in comparison with β-CAs from other relationship of the Mtb and Mmar β-CA protein sequences in comparison with β-CAs from other organisms (Phatc = Phaeodactylum tricornutum; Babo = Brucella abortus; Tspi = Trichinella spiralis; Dmel organisms (Phatc = Phaeodactylum tricornutum; Babo = Brucella abortus; Tspi = Trichinella spiralis; = Drosophila melanogaster; Mpor = Mycolicibacterium porcinum; and Ehis = Entamoeba histolytica). Panel Dmel = Drosophila melanogaster; Mpor = Mycolicibacterium porcinum; and Ehis = Entamoeba histolytica). Panel (B) shows the predicted transmembrane plot of Mtb β-CA3, and panel (C) shows the predicted

transmembrane plot of Mmar β-CA3 [49]. Int. J. Mol. Sci. 2019, 20, 5153 7 of 16

Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 7 of 16 4. Structures of Mtb β-CAs B shows the predicted transmembrane plot of Mtb β-CA3, and panel C shows the predicted The X-ray crystal structures of the two Mtb β-CAs have been resolved [36,37]. The Protein Data transmembrane plot of Mmar β-CA3 [49]. Bank (PDB) contains the X-ray structures of two out of the three Mtb β-CAs: Rv1284 (PDB IDs 1YLK, 4YF4,4. 4YF5,Structures and 4YF6)of Mtb andβ-CAs Rv3588c (PDB IDs 1YM3 and 2A5V). The structure of the catalytic domain of Rv3273The is availableX-ray crystal as a structures homology ofmodel the two in Mtb SWISS-MODEL β-CAs have been Repository resolved [[36,37]50]. The. The latter Protein structure Data was generatedBank (PDB) by the contains automated the X SWISS-MODEL-ray structures of homologytwo out of the modeling three Mtb pipeline β-CAs: Rv1284 based on (PDBβ-CA IDs Can2 1YLK, from a Cryptococcus4YF4, 4YF5 neoformans, and 4YF6)template and Rv3588c (2W3N, (PDB 26.3%IDs 1YM3 identity). and 2A5V). The The structures structure of ofMtb the catalyticβ-CAs aredomain shown in Figureof 6Rv3273. The catalyticis available domains as a homology of the model three inMtb SWISSβ-CAs-MODEL possess Repository the same [50]β/.α Thefold, latter which structure is unique to thewasβ-CAs generated [51]; therefore,by the automated their secondary SWISS-MODEL structures homology share modeling many similarities, pipeline based as on shown β-CA inCan2 Figure 6. The mostfrom a important Cryptococcus catalytic neoformans property template of the(2W3N, protein 26.3% is theidentity). difference The structures in the environment of Mtb β-CAs around are the zincshown in the in active Figure site. 6. The The catalytic active sitesdomains of β of-CAs the three are classified Mtb β-CAs into possess ‘active’ the same or ‘open’ β/α fold, and which ‘inactive’ is or ‘closed’unique conformations to the β-CAs depending [51]; therefore, on thetheir coordination secondary structures of the zinc share ion many [51]. similarities,Mtb Rv3588c as shown was shown in to Figure 6. The most important catalytic property of the protein is the difference in the environment switch between active and inactive conformations depending on the pH [37]; presumably the same around the zinc in the active site. The active sites of β-CAs are classified into ‘active’ or ‘open’ and could‘inactive also hold’ or ‘ trueclosed for’ conformations the other two dependingMtb β-CAs. on the The coordination environment of the of zinc the zincion [51] center. Mtb ofRv3588c each of the Mtb βwas-CAs shown is shown to switch in Figure between7. The active active and and inactive inactive conformations forms of β-CAs depending are characterized on the pH by [37] diff; erent conformationspresumably of the the same Asp could side also chain hold in thetrue activefor the site. other In two the Mtb inactive β-CAs. form, The environment Asp is directly of the coordinated zinc to thecenter Zn ionof each (Figure of the7B). Mtb In β the-CAs active is shown form, in AspFigure forms 7. The a saltactive bridge and inactive with a nearbyforms of Argβ-CAs side are chain (Figurecharacterized7A,C,D). In by addition, different conformations this conformation of the is Asp further side chain stabilized in the byactive a hydrogen site. In the bond inactive from form, the Asp carboxyAsp is group directly to coor thedinated Arg backbone to the Zn amide ion (Figure (also 7B). shown In the in active Figure form,7A, Asp C, forms D). As a salt shown bridge in with Figure 7, the switcha nearby between Arg side the chain two (Figureβ-CA forms6A,C,D). is accompaniedIn addition, this by conformation both an Asp is sidefurther chain stabilized rotation by anda by hydrogen bond from the Asp carboxy group to the Arg backbone amide (also shown in Figure 7A, a movement of the backbone on which the Asp is located. By coordinating to Zn, this residue acts C, D). As shown in Figure 7, the switch between the two β-CA forms is accompanied by both an Asp as an autoinhibiting agent by not allowing water molecules to bind to zinc (cf. Figure7B,C) thus side chain rotation and by a movement of the backbone on which the Asp is located. By coordinating preventingto Zn, this the residue catalytic acts activity.as an autoinhibiting It should alsoagent be by noted not allowing that the water enzyme molecules activity to bind can to be zinc aff ected(cf. by environmentalFigure 7B,C) redox thus preventing conditions the leading catalytic to activity. a chemical It should modification also be noted of thethat activethe enzyme site, asactivity shown by applyingcan be redox affected agents by environmental to Rv1284 [42 redox]. Oxidative conditions conditions leading to resulta chemical in the modification removal of of cysteinethe active Cys35 (Figuresite,7 A)as shown from the by applying Zn coordination redox agents sphere to Rv1284 by forming [42]. Oxidative a disulfide conditions bond between result in the Cys35 remo andval aof more remotelycysteine positioned Cys35 (Figure Cys61 7A) (not from shown), the Zn whichcoordination could sphere even leadby forming to the a loss disulfide of zinc bond ions. between It was also shownCys35 that and the a Tyr120more remotely residue positio may bened critical Cys61 in(not the shown), oxidative which inactivation could even oflead Rv1284 to the [loss42]. of Notably, zinc in Rv3588cions. andIt was Rv3273, also shown there that is no the Cys Tyr120 in the residue homologous may be critical position in the of Cys61,oxidative implying inactivation a higher of Rv1284 resistance [42]. Notably, in Rv3588c and Rv3273, there is no Cys in the homologous position of Cys61, implying to oxidative stress in these two Mtb β-CAs [42]. a higher resistance to oxidative stress in these two Mtb β-CAs [42].

FigureFigure 6. Structures 6. Structures of ofMtb Mtbβ-CA β-CA catalytic catalytic domains.domains. (A (A) )X X-ray-ray structure structure of Rv1284 of Rv1284 (1YLK); (1YLK); (B) X- (rayB) X-ray structurestructure of Rv3588c of Rv3588c (1YM3); (1YM3); (C )(C homology) homology model model of of Rv3273 Rv3273 from from SWISS-MODEL SWISS-MODEL Repository. The The two chainstwo of chains the homodimers of the homodimers are colored are colored blue blue and and red. red. Zinc Zinc ions ions are are colored colored green green[ 36[36,37],37]..

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Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 8 of 16

FigureFigure 7. The 7. The active active sitesite of of MtbMtb β-CAs.β-CAs. (A) Rv1284, (A) Rv1284, PDB ID: PDB 1YLK, ID: (B 1YLK,) open conformation (B) open conformation of Rv3588c, of PDB ID: 1YM3, (C) closed conformation of Rv3588c, PDB ID: 2A5V, (D) homology model of Rv3273 Rv3588c, PDB ID: 1YM3, (C) closed conformation of Rv3588c, PDB ID: 2A5V, (D) homology model of Figure[36,37] .7. Zinc The is active shown site as of a grayMtb βsphere.-CAs. ( AIn) theRv1284, inactive PDB conforma ID: 1YLK,tion, (B )Asp open in conformation the binding site of ligatesRv3588c, to Rv3273 [36,37]. Zinc is shown as a gray sphere. In the inactive conformation, Asp in the binding site PDBZn, and ID: in1YM3, the active (C) closed conformation, conformation Asp formsof Rv3588c, a salt bridgePDB ID: with 2A5V, Arg ( Dand) homology a hydrogen model bond of with Rv3273 the ligates[36,37]Arg to backbone Zn,. Zinc and is inshownN. theThe active aszinc a gray coordination conformation, sphere. In bonds theAsp inactive are forms shown conforma a saltas dashedtion, bridge Asp lines, with in theand Arg binding the and hydrogen asite hydrogen ligates bonds to bond withZn, thebetween and Arg in backboneAsp the and active Arg N. conformation, are The shown zinc as coordination Aspgreen forms lines. a In bondssalt the bridge active are with shownform, Arg Zn as andis dashed ligated a hydrogen to lines, either andbond water the with ( hydrogenA )the or bondsArgother between backbone ligands Asp ( BN.– and DThe). Argzinc arecoordination shown as bonds green are lines. shown In the as dashed active form,lines, and Zn isthe ligated hydrogen to either bonds water (A) orbetween other ligands Asp and ( BArg–D are). shown as green lines. In the active form, Zn is ligated to either water (A) or 5. Expressionother ligands Analysis (B–D). of β-CAs from Mmar 5. Expression Analysis of β-CAs from Mmar Expression analysis of the three β-CA genes was carried out in the Mmar ATCC 927 strain using 5. Expression Analysis of β-CAs from Mmar ExpressionPCR, and the analysis presence of of the transcripts three β-CA was genesseen as was bands carried of 500 out bp infor theβ-CA1,Mmar 490ATCC bp for 927β-CA2 strain, and using PCR,600 and bpExpression the for presence β-CA3 analysis (Figure of transcripts of 8 A)the [29]three. Similarly, wasβ-CA seen genes quantitative as was bands carried of expression 500 out bpin the for analysis Mmarβ-CA1, ATCC of 490 the 927 bpβ-CA strain for genesβ -CA2,using in and 600 bpPCR,different for andβ-CA3 strains the presence (Figure of Mmar8 ofA) (M, transcripts [ ATCC29]. Similarly, 927 was and seen E11) quantitative as was bands also of performed 500 expression bp for using β-CA1, analysis RT -490qPCR, bp of andfor the β theβ-CA2-CA highest, genesand in different600expression bp strains for βwas of-CA3Mmar found (Figure(M, in the8 ATCCA) ATCC[29] 927. Similarly, 927 and strain E11) quantitative compared was also performedtoexpression the M and analysis using E11 RT-qPCR,strains. of the Theβ-CA and molecular genes the highestin different strains of Mmar (M, ATCC 927 and E11) was also performed using RT-qPCR, and the highest expressionanalysis was thus found confirmed in the the ATCC presence 927 of strainall three compared β-CA genes to in the Mmar M and(Figure E11 8). strains. The molecular expression was found in the ATCC 927 strain compared to the M and E11 strains. The molecular analysis thus confirmed the presence of all three -CA genes in Mmar (Figure8). analysis thus confirmed the presence of all three ββ-CA genes in Mmar (Figure 8).

Figure 8. Expression analysis of β-CAs from Mmar: (A) The qualitative expression analysis of Mmar β-CA genes showed the presence of all three β-CAs in the ATCC 927 strain. Genomic DNA was used FigureFigureas 8.a positiveExpression 8. Expression control. analysis (analysisB–D) Relative of ofβ-CAs β-CAs expression from fromMmar Mmar analysis:(: (AA)) Theof The three qualitative qualitative β-CA genes expression expression from the analysis three analysis straof Mmarins of ofMmar βMmar-CA genes using showedRT-qPCR the [29] presence. of all three β-CAs in the ATCC 927 strain. Genomic DNA was used β-CA genes showed the presence of all three β-CAs in the ATCC 927 strain. Genomic DNA was used as as a positive control. (B–D) Relative expression analysis of three β-CA genes from the three strains of a positive control. (B–D) Relative expression analysis of three β-CA genes from the three strains of Mmar using RT-qPCR [29]. Mmar using RT-qPCR [29].

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6. In Vitro Inhibition of Mycobacterial β-CAs

6.1. Inhibition of Mtb β-CAs Using Sulfonamides and Their Derivatives The inhibition studies on Mtb β-CA1 were performed using a panel of sulfonamides, sulfamates, and their derivatives, some of which are in clinical use [34]. Most of the tested compounds showed high affinity for β-CA1 (inhibition range of 1–10 µM). The compounds with inhibition in the submicromolar concentration range (KI values of 0.481-0.905 µM) included sulfanilyl-sulfonamides, such as acetazolamide, methazolamide, dichlorophenamide, dorzolamide, brinzolamide, benzolamide, and the sulfamate topiramate [34]. The most efficient inhibitors (KI values of 97–186 nM) of this enzyme were 3-bromosulfanilamide and indisulam. The promising inhibition profiles of sulfonamides and their derivatives used in this study suggested that the Mtb β-CA1 enzyme is a potential target for developing anti-TB agents with a different mechanism of action [34]. Similarly, the inhibition profiles of a series of diazenylbenzenesulfonamides derived from sulfanilamide or metanilamide were shown to inhibit Mtb β-CA2 in the range of 45–955 nM [52]. Subsequently, to obtain inhibitory molecules with better inhibition properties, new compounds were synthesized by diazotization of aminosulfonamide and by coupling with phenols or amines [35]. These molecules were subsequently incorporated with various R moieties in the molecule—such as hydroxy, amino, methylamino, dimethylamino, and sulfonate—which could induce water solubility of these compounds as sodium salts. Among the molecules thus prepared, the aminomethylene sodium sulfonate derivatives and their corresponding N-methylated analogues showed the most efficient inhibition properties (KIs of 45–59 nM) [35]. Later, the diazenylbenzenesulfonamides were also tested for the inhibition of Mtb β-CA3, and among them, prontosil was found to be the best inhibitor (KI 126–148 nM) [53]. In another study, several compounds were tested for their inhibition efficiency against Mtb β-CA3. Compounds that inhibited the activity of this enzyme in the nanomolar range included 2-amino-pyrimidin-4-yl-sulfanilamide (KI 90 nM) and sulfonylated sulfonamide (KI of 170 nM). These studies suggested that Mtb β-CA3 could be targeted using CAIs with the potential to develop agents that target Mtb [33]. The sulfonamides prepared by reaction of sulfanilamide with aryl/alkyl isocyanates showed inhibition of Mtb β-CA1 and β-CA3 in the range of 4.8–6500 nM and 6.4–6850 nM, respectively. The structural activity relationship related to inhibition has been shown to be associated with the nature of the moiety substituting the second ureido nitrogen, and it is the determining factor in controlling the inhibitory power. This may be due to flexibility of the ureido linker and the ability of this moiety to orientate in different subpockets of the active site cavities of these enzymes [54]. The inhibition of all three Mtb β-CAs using a number of halogenated sulfanilamides and halogenated benzolamide derivatives showed inhibition efficiency in the submicromolar to micromolar range. The substitution pattern at the sulfanilamide moiety/fragment of the molecule is crucial for the inhibition efficiency of the molecule. The best inhibitors were the halogenated benzolamides (KIs in the range of 0.12–0.45 µM), whereas the halogenated sulfanilamides were slightly less inhibitory (KIs in the range of 0.41–4.74 µM) [55]. Similarly, the inhibition of the three Mtb β-CAs was carried out using a series of fluorine-containing sulfonamides that were modified with amino, amino alcohol, and amino acid moieties [56]. Among these, some showed efficient inhibition of β-CA2 (KI values in the nanomolar range) and moderate inhibition efficiency against β-CA1 and β-CA3 (KI in the low micromolar range) [56]. Recently, a novel sulfonamide molecule obtained from sulfanilamide, which was N4-alkylated with ethyl bromoacetate followed by a reaction with hydrazine hydrate and further reacted with various aromatic aldehydes, showed KIs in the range of 127 nM–2.12 µM for Mtb β-CA3 [57]. Int. J. Mol. Sci. 2019, 20, 5153 10 of 16

6.2. Mono and Dithiocarbamates as Inhibitors of Mycobacterial β-CAs Studies have shown that both mono-(MTCs) and dithiocarbamates (DTCs) selectively inhibit Mtb β-CAs. The inhibition of Mtb β-CA1 and β-CA3 using a series of N-MTCs and N,N-disubstituted DTCs showed inhibition efficiencies in the subnanomolar to the micromolar range [58]. The inhibition properties of these compounds were dependent on the substitution pattern at the nitrogen atom of the DTC zinc-binding group. Replacement with aryl, arylalkyl-, heterocyclic as well as aliphatic and aminoacyl moieties led to potent β-CA1 and β-CA2 inhibitors in both the N-mono-and N,N-di-substituted DTC series [58].

6.3. Phenolic Natural Products and Phenolic Acids as Mycobacterial β-CA Inhibitors In search of novel inhibitors that could selectively inhibit Mtb β-CAs, a series of phenolic natural products (NPs) were screened against Mtb β-CA1 and Mtb β-CA3. It was shown that NPs were able to selectively inhibit Mtb β-CA1 and β-CA3 with KIs in the submicromolar range [59]. Similarly, screening of a series of phenolic acids and their derivatives against all three Mtb β-CAs showed good inhibition efficiencies (KIs 1.87 µM–6.09 µM). In addition, these compounds showed no inhibitory activity against human CA I or CA II, suggesting that the phenolic compounds could potentially be developed as anti-TB agents [60]. Computational analysis of the binding mode of the compounds suggested that the inhibitors anchored to the zinc-coordinated water molecule in the CA active site and interfered with the nucleophilic attack of the zinc hydroxide on the substrate CO2 [60]. Screening of a series of C-cinnamoyl glycosides containing the phenol moiety against the three Mtb β-CAs also showed that most of the compounds were highly efficient inhibitors of Mtb β-CA2 (KI 130–940 nM), showing a preference for β-CA2 over human CA II [61].

6.4. Carboxylic Acids as Inhibitors of Mycobacterial β-CA Carboxylic acids are known to inhibit Mtb β-CAs, but the inhibitory mechanism of these acids is not known. Screening of scaffolds containing carboxylic acids, such as benzoic acid, nipecotic acid, ortho and para coumaric acid and ferulic acid, against all three Mtb β-CAs showed inhibition efficacy in the micromolar range (KI 0.11–0.97 µM). The KIs for the inhibition of β-CA2 were in the range of 0.59–8.10 µM, whereas against β-CA1, the carboxylic acids showed inhibition constants in the range of 2.25–7.13 µM[62]. This class of inhibitors has not been well explored, and further in vitro inhibition studies are needed to document the real potential of these inhibitors as anti-TB agents.

7. Inhibition of Mycobacterial Strains in Culture In addition to studies on the in vitro inhibition of Mtb β-CAs, CA inhibitors have been used for the inhibition of Mtb growth in culture [9]. Studies using 6-mercaptopurine with sulfony/sulfenyl halides known as 9-sulfonylated/sulfenylated-6-mercaptopurines showed that the compounds inhibited growth of the wild-type Mtb bacilli H37Rv in the range of 0.39–3.39 µg/mL [63]. Among these compounds, one of the derivatives showed a minimal inhibitory concentration (MIC) of approximately 1 mg/mL against several drug-resistant Mtb strains [63]. The compounds with MICs below 1 µg/mL were considered excellent leads [63]. The inhibition of Mmar, a model mycobacterium, was recently investigated in liquid cultures using DTCs Fc14-584a and Fc14-584a. The DTCs were prepared by the reaction of a corresponding amine with carbon disulfide in the presence of a base. The obtained compounds were shown to be specific inhibitors of Mtb β-CA1 and β-CA3 [58]. This study showed that the minimum concentration (MIC) required for complete inhibition of Mmar was 75 µM. Further studies using the concentration below MIC (dilution 1:4) have shown no growth of Mmar, suggesting that the DTCs are bactericidal [29]. These studies demonstrate that CAIs, in addition to inhibiting the activity of mycobacterial β-CAs, impair the growth of both Mtb and NTM in culture and have significant potential as antimycobacterial compounds. Int. J. Mol. Sci. 2019, 20, 5153 11 of 16

8. In Vivo Inhibition of Mycobacteria by CA Inhibitors In vivo studies have shown that 6-ethoxy-1,3-benzathiazole-2-sulfonamide (ETZ) impairs the signaling of PhoPR in Mtb [64,65]. ETZ is a sulfonamide compound that is a general inhibitor of CA activity and is an FDA-approved drug used in the treatment of glaucoma, epilepsy, and duodenal ulcers and is also a diuretic [9]. ETZ treatment of Mtb induces phenotypes similar to the PhoPR mutants downregulating the PhoPR regulon, a reduction in virulence-associated lipids, and inhibition of Esx-1 protein secretion [64] (Table2). Further studies on single-cell imaging of a PhoPR-dependent fluorescent reporter strain showed that ETZ inhibited PhoPR-regulated genes in infected macrophages and in mouse lungs [64]. A mouse model infected with bacteria containing a fluorescent reporter showed a reduction in bacteria in the lungs and significantly reduced GFP fluorescence compared to the control group [64]. The extracellular DNA (eDNA) in NTM is responsible for the phenotypic resistance of the bacteria to antibiotics and plays an important biological role in the bacteria [66]. Studies have shown that bicarbonate ion helps in the export of eDNA in NTM, and it is well established that the enzymatic activity of CAs is involved in the production of bicarbonate ions via the hydration of carbon dioxide [11,67]. Furthermore, it has been demonstrated that a mutant M. avium in which CA genes were inactivated was unable to export eDNA [67]. However, when the CA genes were restored, the transport of eDNA was also restored, suggesting that CAs play important roles in the transport of eDNA and the formation of biofilms by the bacteria [67] (Table2). The surface-exposed proteome of M. avium in eDNA-containing biofilms showed that CAs are abundantly present, and the inhibition studies showed a significant reduction in eDNA transport in the presence of ETZ [67].

Table 2. In vivo inhibition studies showing the effects of CA inhibitors.

CA Inhibitor Mycobacterium Group Effect on the Bacterium References Inhibits PhoPR regulon, Ethoxzolamide Mtb MTC [64] attenuates virulence Reduces the transport of eDNA Ethoxzolamide M. avium NTM [67] and biofilm formation Impairs growth of Mmar in Dithiocarbamate Mmar NTM [29] zebrafish larvae

DTCs, another class of compounds that strongly inhibit Mtb β-CAs in vitro, have been used in vivo for the inhibition of Mmar [29,58]. The in vivo studies have shown that a DTC, FC14-384b (Figure9B and Table2), significantly impaired the growth of Mmar in zebrafish larvae [29]. The zebrafish larvae infected with the Mmar wasabi strain with an infection dose of 471 143 (CFUs) bacteria and treated ± with 300 µM FC14-384b showed a significant reduction (p > 0.0096) in bacterial load (Figure9B) compared to the zebrafish larvae not treated with the inhibitor (Figure9A). These studies suggest that the compounds that selectively inhibit the activity of β-CAs could be useful as a new class of antimycobacterial agents that can potentially treat MDR-TB. Int. J. Mol. Sci. 2019, 20, 5153 12 of 16 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 12 of 16

FigureFigure 9. Inhibition 9. Inhibition of ofMmar Mmarin in aa zebrafishzebrafish model. model. (A () AGreen) Green fluorescence fluorescence showing showing the infection the infection in in zebrafishzebrafish larvae larvae at at six six days days post post infection not not treated treated with with the the inhibitor inhibitor;; and and (B) (zebrafishB) zebrafish larvae larvae infected with Mmar and treated with 300 μM Fc14-584b showing the absence of bacteria. The figure infected with Mmar and treated with 300 µM Fc14-584b showing the absence of bacteria. The figure is is modified from Aspatwar et al. [29]. modified from Aspatwar et al. [29].

9. Conclusions9. Conclusions The experimental data on the biochemical and molecular characterization of Mtb β-CAs have The experimental data on the biochemical and molecular characterization of Mtb β-CAs have provided us with better insights into the diverse roles of these enzymes in the Mtb pathogen. In provided us with better insights into the diverse roles of these enzymes in the Mtb pathogen. In addition addition to their role in the hydration of CO2 required for pH homeostasis in the pathogen, these to theirenzymes role inhave the been hydration implicated of COin several2 required nonenzymatic for pH homeostasis functions that in are the required pathogen, for the these survival enzymes haveof been Mtb implicated and pathogenesis in several of TBnonenzymatic disease in the functions host. However, that are precise required physiological for the survival roles of Mtb the and pathogenesisindividual of enzymes TB disease have in yet the to host. be discovered. However, In precise vitro physiologicalinhibition studies roles using ofthe different individual classes enzymes of haveinhibitors yet to be have discovered. shown thatIn vitroMtb βinhibition-CAs are druggable studies usingtargets di withfferent potential classes as ofanti inhibitors-TB agents have with showna that Mtbdiverseβ-CAs mechanism are druggable of action targets compared with to potentialthe drugs asthat anti-TB are in agentsclinical withuse and a diverse to which mechanism the Mtb of actionstrains compared have developed to the drugs various that degrees are in of clinical resistance. use and to which the Mtb strains have developed various degreesInterestingly, of resistance. recent studies have shown that CA inhibitors that selectively inhibit Mtb β-CAs in vitro also inhibit the growth of Mmar in vivo in zebrafish. Similarly, ETZ, a general CA inhibitor and Interestingly, recent studies have shown that CA inhibitors that selectively inhibit Mtb β-CAs a clinically used drug, inhibits the secretion of virulence factors and attenuates Mtb. These findings in vitrorepresentalso inhibit an in thevivo growth proof of of conceptMmar inthat vivo thein Mtb zebrafish. β-CA enzymes Similarly, are indeed ETZ, a promising general CA targets inhibitor for and a clinicallydeveloping used antimycobacterial drug, inhibits the agents. secretion However, of virulence we need to factors design and novel attenuates CA inhibitorsMtb .that These are findingsnot representonly selective an in vivo againstproof the ofmycobacterial concept that β-CAs the Mtbbut areβ-CA also enzymesmembrane arepermeable indeed and promising safe for clinical targets for developinguse in humans antimycobacterial when tested in agents. strict preclinical However, and we clinical need settings. to design novel CA inhibitors that are not only selective against the mycobacterial β-CAs but are also membrane permeable and safe for clinical use inAuthor humans Contributions: when tested A.A. in is strict responsible preclinical for conceiving and clinical the idea settings. and for preparing the manuscript; V.K. contributed to part of the manuscript that involved structural and bioinformatic analysis-related data; S.R. contributed to the molecular biology part of the review; A.A., V.K., S.R., M.B., F.C., C.T.S., M.P., and S.P. Author Contributions: A.A. is responsible for conceiving the idea and for preparing the manuscript; V.K. contributedcontributed to part to the of writing the manuscript of the article that and involvedhave read and structural approved and the bioinformaticfinal version of analysis-relatedthe manuscript. data; S.R. contributedFunding: to theThe molecularwork was supported biology part by ofgrants the review;from the A.A., Sigrid V.K., Jusélius S.R., Foundation M.B., F.C., (S.P., C.T.S., M.P.), M.P., Finnish and S.P. Cultural contributed to theFoundation writing of (A.A.), the article Academy and haveof Finland read and(M.P. approved and S.P.), theJane finaland Aatos version Erkko of the Foundation manuscript. (M.P. and S.P.), and Funding:TampereThe Tuberculosis work was Foundation supported (M.P.). by grants from the Sigrid Jusélius Foundation (S.P. and M.P.), Finnish CulturalConflicts Foundation of Interest: (A.A.), The Academy authors declare of Finland no conflict (M.P. of and interest. S.P.), Jane and Aatos Erkko Foundation (M.P. and S.P.), and Tampere Tuberculosis Foundation (M.P.).

Conflicts of Interest: The authors declare no conflict of interest.

Int. J. Mol. Sci. 2019, 20, 5153 13 of 16

Abbreviations The following abbreviations are used in this manuscript:

CA Carbonic anhydrase Mtb Mycobacterium tuberculosis Mmar Mycobacterium marinum CAI Carbonic anhydrase inhibitor MDR-TB Multidrug-resistant tuberculosis WHO World Health Organization TB Tuberculosis MTBC Mycobacterium tuberculosis complex NTM Nontuberculous mycobacteria ETZ Ethoxzolamide β-CA1 Beta-carbonic anhydrase 1 β-CA2 Beta-carbonic anhydrase 2 β-CA3 Beta-carbonic anhydrase 3 RR-TB Resistant to rifampicin Mycobacterium tuberculosis eDNA Extracellular DNA PDB Protein Data Bank MTC Monothiocarbamates DTC Dithiocarbamates NP Natural products MIC Minimal inhibitory concentration

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