Bioorganic & Medicinal Chemistry 22 (2014) 4537–4543

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Bioorganic & Medicinal Chemistry

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Sulfonamide inhibition study of the carbonic anhydrases fromthe bacterial pathogen Porphyromonas gingivalis: Theb-class (PgiCAb) versus the c-class (PgiCA) enzymes

http://dx.doi.org/10.1016/j.bmc.2014.07.0480968-0896/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding authors. Tel.: +39 081 6132559; fax: +39 081 6132712 (C.C.);tel.: +39 055 4573005; fax: +39 055 4573385 (C.T.S.).

E-mail addresses: c.capasso@ibp.cnr.it (C. Capasso), claudiu.supuran@unifi.it(C.T. Supuran).

Sonia Del Prete a, Daniela Vullo b, Sameh M. Osman c, Andrea Scozzafava a, Zeid AlOthman c,Clemente Capasso b,⇑, Claudiu T. Supuran b,d,⇑a Istituto di Biochimica delle Proteine – CNR, Via P. Castellino 111, 80131 Napoli, Italyb Università degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino (Firenze), Italyc Department of Chemistry, College of Science, King Saud University, PO Box 2455, Riyadh 11451, Saudi Arabiad Università degli Studi di Firenze, Polo Scientifico, Dipartimento NEIROFABA;Sezione di Scienze Farmaceutiche e Nutraceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino (Firenze), Italy

a r t i c l e i n f o

Article history:Received 2 July 2014Revised 26 July 2014Accepted 29 July 2014Available online 7 August 2014

Keywords:Carbonic anhydraseBeta-class enzymeSulfonamidePorphyromonas gingivalis

a b s t r a c t

The oral pathogenic bacterium Porphyromonas gingivalis, encodes for two carbonic anhydrases (CAs, EC4.2.1.1) one belonging to the c-class (PgiCA) and another one to the b-class (PgiCAb). This last enzymehas been cloned and characterized here for its inhibition profile with the main class of CA inhibitors,the sulfonamides. Many of the clinically used sulfonamides as well as simple aromatic/heterocyclicsulfonamides were ineffective as PgiCAb inhibitors whereas better inhibition was observed with simplederivatives such as sulfanilamide, metanilamide, 4-aminoalkylbenzenesulfonamides (KIs of 364–475nM). The halogenosulfanilamides incorporating heavy halogens, 4-hydroxy- and 4-hydroxyalkyl-ben-zenesulfonamides, were also micromolar, ineffective PgiCAb inhibitors. The best inhibitors of the b-classenzyme were acetazolamide and ethoxzolamide, with KIs of 214–280 nM. Interestingly, the c-classenzyme was much more sensitive to sulfonamide inhibitors compared to the b-class one, PgiCAb. Identi-fication of potent and possibly selective inhibitors of PgiCAb/PgiCA may lead to pharmacological tools use-ful for understanding the physiological role(s) of these enzymes, since this bacterium is the main causativeagent of periodontitis and few treatment options are presently available.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Sulfa drugs were first used in the 1930s, and they revolution-ized medicine.1 After a few years, bacteria started to develop resis-tance to these drugs, and eventually penicillin replaced them as afirst-line treatment.2,3 While antibiotic resistance remains a prob-lem for most classes of antibiotics ever discovered afterwards, sulfadrugs are still commonly used to treat a variety of bacterial infec-tions, mainly in combination with other drug classes. Sulfa drugswork by binding to and inhibiting a specific bacterial enzyme,the dihydropteroate synthase (DHPS).2,4–6 This enzyme is criticalfor the synthesis of folate, an essential nutrient for bacteria. Itsbiosynthesis requires the chemical reaction between two mole-cules, 6-hydroxymethyl-7,8-dihydropterin-pyrophosphate (DHPP)

and p-aminobenzoic acid (PABA).7 Mammals get folate from theirdiet, but bacteria must synthesize this vitamin. Because of theirstructural similarity to PABA, the sulfonamides compete with thissubstrate for the bacterial enzyme, DHPS.8 Thus, they inhibit thesynthesis of bacterial dihydrofolic acid and, thereby, the formationof its essential cofactor forms. Bacteria that can obtain folates fromtheir environment are naturally resistant to these drugs. Acquiredbacterial resistance is generally irreversible and may be due to analtered dihydropteroate synthetase, a decreased cellular perme-ability of the sulfa drugs, or an enhanced production of the naturalsubstrate, PABA.8–10

Recently, a new class of enzymes, the carbonic anhydrases (CAs,EC 4.2.1.1) started to be investigated in detail in pathogenic bacte-ria, in the search of antibiotics with a novel mechanism ofaction.11–15 It has been demonstrated that in many bacteria, CAsare essential for the life cycle of the organism and that theirinhibition leads to growth impairment or growth defects of thepathogen.11,13 CAs are metalloenzymes that catalyze the reversible

4538 S. D. Prete et al. / Bioorg. Med. Chem. 22 (2014) 4537–4543

hydration of CO2 to bicarbonate and a proton, using Zn(II), Cd(II) orFe(II) at their active site. CAs have been found in virtually allmammalian tissues and cell types, where they function in CO2

transport, pH regulation, biosynthetic pathways and otherphysiological processes.11,12,14,16,17 CAs are also found in plants,cyanobacteria, and algae, where they appear to facilitate photosyn-thesis. In bacteria, CAs are involved in the transport of CO2 andbicarbonate and in related physiological processes.18–21 BacterialCAs exist in at least three genetically unrelated families ofenzymes, the a-, b-, and c-CAs.16,22–26 In the human CA family,all 15 isozymes that have been reported to date belong to thea class. Intriguingly, CA inhibitors (CAIs) include various anions,imidazole, phenols, hydroxamates, carboxylates, phosphates/phos-phonates, and various sulfonamide derivatives (R-SO2NH2) andtheir isosteres (sulfamides, sulfamates), which represent the mainclass of clinically used CAIs.18–21,27–30

Many common pathogenic bacteria encode such enzymes. Forexample Helicobacter pylori, is a pathogenic bacterium whichlives in the highly acidic environment of the stomach. Its gen-ome encodes three CAs: a-, b-and c-CA, respectively.31–36 Thecrucial role played by these CAs present in H. pylori is the acidacclimatization of the pathogen within the stomach. The inhibi-tion of these enzymes, in fact, led to the death of the bacteriaand a possible eradication of H. pylori from the stomach andhas been used clinically for the treatment of gastric ulcers. Thegenome of Vibrio cholerae, the causative agent of cholera,encodes for putative CAs belonging to each bacterial class: thea, b and c ones.37–39 Recently it was reported that sodium bicar-bonate induces cholera toxin (CT) expression is mediated bysome of these enzymes.36 In the presence of CA inhibitors (suchas the sulfonamide ethoxzolamide), a significant reduction in vir-ulence gene expression was observed, which inhibited virulencein vivo.36 Our group cloned, purified and characterized the a-CAfrom V. cholerae, named VchCA. This new enzyme showed signif-icant catalytic activity, being more active than the human iso-form hCA I or than the H. pylori a-class enzyme.37–39 Aninhibition study with a panel of sulfonamides and one sulfamateled to the detection of a large number of low nanomolar VchCAinhibitors, including methazolamide, acetazolamide, ethoxzola-mide, dorzolamide, brinzolamide, benzolamide, and indisulam(with KI in the range of 0.69�8.1 nM).

Porphyromonas gingivalis is one of the few major pathogensresponsible for the development of chronic periodontitis and asuccessful colonizer of the oral epithelium.11 The perturbationof epithelial cells by bacteria is the first stage in the initiationof inflammatory and immune processes causing eventuallydestruction of the tissues surrounding and supporting the teeth,which ultimately result in tooth loss.11,14,40 The genome of P.gingivalis encodes for a b- and a c-CA. Recently, our group puri-fied the recombinants c-CA (named PgiCA) and the b-CA (namedPgiCAb) identified in the genome of this pathogenic bacterium.PgiCA was shown to possess a significant catalytic activity forthe reaction that converts the CO2 to bicarbonate and protons,with a kcat of 4.1 � 105 s�1 and a kcat/K0 of 5.4 � 107 M�1 � s�1.11,14,40 Like most enzymes belonging to the CA superfamily,PgiCA was also inhibited by acetazolamide with an inhibitionconstant of 324 nM.11,14,40

In a previous work we explored the inhibition profile withsulfonamides of the c-CA from this pathogen.11 Here we reportthe sulfonamide inhibition study of the other enzyme, PgiCAb,comparing it with data obtained for the c-CA enzyme (PgiCA), assulfonamides are the classical, best investigated inhibitors of allCAs. Identification of potent and possibly selective inhibitors ofPgiCA and PgiCAb may lead to pharmacological tools useful forunderstanding the physiological role(s) of these enzymes.

2. Results and discussion

2.1. Cloning, sequence analysis and catalytic properties ofPgiCAb

IPTG induction of Escherichia coli BL21 (DE3) cells transformedwith the plasmid pET15-b/PgiCAb resulted in the production ofthe recombinant b-CA, named PgiCAb. The b-CA was isolated andpurified to homogeneity from the E. coli (DE3) cell extract. Mostof the CA activity was recovered in the soluble fraction of the cellextract after sonication and centrifugation. Using an affinity col-umn (His-select HF Nickel affinity gel), PgiCAb was purified tohomogeneity. Analysis by SDS-Page of PgiCAb showed two mainbands of about 25 kDA (monomeric form) and 50 kDA (dimericform) under reducing condition (data not shown). The full nucleo-tide sequence showed an open reading frame encoding a 242 res-idues polypeptide chain which contained all the typical features ofa b-CA, including the three residues that are involved in the cata-lytic mechanism of the enzyme (two cysteines and one histidine,more precisely Cys90, His143 and Cys146, see Fig. 1). Furthermore,the catalytic dyad (Asp92–Arg94) involved in the activation of thewater molecule coordinated to the zinc ion from the enzyme activesite, is also conserved in PgiCAb, as for the other investigated b-CAssuch as the two enzymes from Legionella pneumophila lpCA1 andlpCA2, the H. pylori enzyme HpyCA, the two Brucella suis enzymesBsuCA219 and 213, as well as the two b-CAs from Salmonellatyphimurium, stCA1 and stCA2 (Fig. 1). The predicted molecularmass of the enzyme from its amino acid sequence is of 26.1 kDa.Furrhemore, the phylogenetic analysis of these enzymes shownin Fig. 2 clearly shows that PgiCAb is evolutionarily more similarto one of the S. typhimurium enzymes, stCA2, and also with thetwo L. pneumophila enzymes lpCA 2 and lpCA1. These four b-CAsclustered together on the lower branch of the tree shown inFig. 2, whereas the remaining enzymes also clustered together,on a different branch, being thus less closely related to theP. gingivalis enzyme (Fig. 2).

PgiCAb showed a good catalytic activity, with a kcat of2.8 � 105 s�1 and a kcat/Km of 1.5 � 107 M�1 � s�1. PgiCAb was alsoinhibited by the clinically used sulfonamide acetazolamide, withan inhibition constant of 214 nM (Table 1). This is in fact to beexpected considering the fact that all these b-CAs of bacterialorigin investigated earlier showed catalytic properties in the samerange as PgiCAb, both considering the kinetic constant kcat or thecatalytic efficiency kcat/Km (Table 1). PgiCAb is however one ofthe least active catalysts for the CO2 hydration reaction amongthe bacterial b-CAs investigated so far.

2.2. Sulfonamide inhibition studies of PgiCAb and comparisonwith PgiCA

The library of 40 compounds, comprising 39 sulfonamides andone sulfamate investigated earlier as inhibitors of the c-classenzyme PgiCA, were also included in this study. Derivatives 1–24and AAZ-HCT are either simple aromatic/heterocyclic sulfona-mides widely used as building blocks for obtaining new familiesof such pharmacological agents,27 or they are clinically usedagents, among which acetazolamide AAZ, methazolamide MZA,ethoxzolamide EZA and dichlorophenamide DCP, are the classical,systemically acting antiglaucoma CA inhibitors (CAIs).27a Dorzola-mide DZA and brinzolamide BRZ are topically-acting antiglaucomaagents, benzolamide BZA is an orphan drug belonging to this classof pharmacological agents, whereas topiramate TPM, zonisamideZNS and sulthiame SLT are widely used antiepileptic drugs.27

Sulpiride SLP and indisulam IND were also shown by our groupto belong to this class of pharmacological agents,27 together with

PgiCAb --MKKIVLFSAAMAMLIACGNQTTQTKSDTPTAAVEGRIGEVLTQDIQQGLTPEAVLVGL lpCA1 -------MPKKLLIAAFLCNIFCNPSHLAYASSTEIPILGKTMTQAKQQQMTPRQALQRL lpCA2 ---------------------------------------MWTLTKEQQQAITPEKAIELL HpyCA ---------------------------------------------------------MKA Bsuis_CA_1 ----------------------------------------------------MADLPDSL Bsuis_CA_9 -------------------------------------------MPMKNDHSPDQRTLSEL stCA1 -----------------------------------------------------MKDIDTL stCA2 MEQNQPAQPSRRAILKQTLAVSALSVTGLAALSVPTISFAASLSKEERDGMTPDAVIEHF

90 92 94 PgiCAb QEGNARYVANKQLPRDLNAQAVAGLEGQFPEAIILSCIDSRVPVEYIFDKGIGDLFVGRV lpCA1 KDGNQRFLSNKPLARDYLKQAKQSAYGQYPFAVILNCMDSRSVPEFFFDQGLADLFTLRV lpCA2 KEGNKRFVSNLKLNRNLIQQVNETSQGQFPFAVILSCMDSRTPAELIFDQGLGDIFSIRV HpyCA FLGALEFQENE-YEELKELYESLKT-KQKPHTLFISCVDSRVVPNLITGTKPGELYVIRN Bsuis_CA_1 LAGYKTFMSEH-FAHETARYRDLAEKGQSPETLVVACCDSRAAPETIFNAAPGEIFVLRN Bsuis_CA_9 FEHNRQWAAEK-QEKDPEYFSRLSS-SQRTEFLWIGCSDSRVPANVVMGLQPGEVFVHRN stCA1 ISNNALWSKML-VEEDPGFFEKLAQ-AQKPRFLWIGCSDSRVPAERLTGLEPGELFVHRN stCA2 KQGNLRFRENRPAKHDYLAQKRNSIAGQYPAAVILSCIDSRAPAEIVLDAGIGETFNSRV : * . : : * *** : . . .: : *

143 146 PgiCAb AGNVV--------DDHMLGSLEYACEVSGSKVLLVLGHEDCGA------IKSAIKGVEMG lpCA1 AGNVL--------NDDILGSMEFATKVVGARLVVVLAHTSCGA------VAGACKDVKLG lpCA2 AGNIL--------NDDILGSIEFACQVVGVKLIAVVGHTQCGA------IKGACDGVKLG HpyCA MGNVIPPKTSHKESLSTMASIEYAIVHVGVQNLIICGHSDCGACGSTHLINDGXTKAKTP Bsuis_CA_1 VANLIPPYEPDGEYHAASAALEFAVQSLKVKHIVVMGHGRCGG------IKAALDTESAP Bsuis_CA_9 VANLV-----HRADLNLLSVLEFAVGVLEIKHIIVCGHYGCGG------VRAAMDGYGHG stCA1 VANLV-----IHTDLNCLSVVQYAVDVLEVEHIIICGHSGCGG------IKAAVENPELG stCA2 AGNIS--------NRDMLGSMEFACAVAGAKVVLVIGHTRCGA------VRCAIDNAELG .*: . :::* : : .* **. : .

PgiCAb N-----ITSLMEEIKPSV-EATQYTGERTYANKEFADAVVKENVIQTMDEIRRDSPILKK lpCA1 H-----LTDVINKIHPVVKPSMESTGIDNCSDPKLIDDMAKANALHVVKNILEQSPILNE lpCA2 N-----LTNLLNKINPVIQEAKKLDAKHDVHSPEFLNCVTSLNVKHTMNEITQRSDIVHQ HpyCA Y-----IADWIQFLEPIK-EELKNHPQFSNHFAKRSWLTERLNVRLQLNNLLS-YDFIQE Bsuis_CA_1 LSPSDFIGKWMSLISP---AAEAISGNALMTQSERHTALERISIRYSLANLRT-FPCVDI Bsuis_CA_9 I-----IDNWLQPIRDIA-QANQAELDTIENTQDRLDRLCELGVSSQVESLSR-TPVLQS stCA1 L-----INNWLLHIRDIWLKHSSLLGK--MPEEQRLDALYELNVMEQVYNLGH-STIMQS stCA2 N-----LTGLLDEIKPAI-AKTEYSGERKGSNYDFVDAVARKNVELTIENIRKNSPVLKQ : : : . . : .: :

PgiCAb LEEEGK-IKICGAIYEMSTGKVHFL----------------------------- lpCA1 LVKNKQ-IGIVAGIHDIKTGKVTFFEEKRSVPE--------------------- lpCA2 LLNEKR-IAIAGGLYQLETGEVQFFDE--------------------------- HpyCA RVVNNE-LKIFGWHYIIETGRIYNYNFESHFFEPIXETXKQRKSHENF------ Bsuis_CA_1 LEKKGK-LTLHGAWFDISTGELWVMDHRTGD----------------FK-RPEL Bsuis_CA_9 AWKDGKDIIVHGWMYNLKDGLLRDIGCDCTR------------NALQFACQPAE stCA1 AWKRGQNVTIHGWAYSINDGLLRDLDVTATNRETLENGYHKGISALSLKYIPHQ stCA2 LEDEKK-IKIVGSMYHLTGGKVEFFEV---------------------------

: : . . : * :

Figure 1. Amino acid sequences alignment of selected b-CAs from five bacterial species. The LpCA1 numbering system was used. Amino acid residues participating in thecoordination of the metal ion are indicated in red and bold (Cys90, His143 and Cys146, respectively), whereas the catalytic dyad involved in the activation of the watermolecule coordinated to zinc (Asp92–Arg94) is shown in blue and bold. The asterisk (⁄) indicates identity at a position; the symbol (:) designates conserved substitutions,whereas (.) indicates semi-conserved substitutions. The multiple alignment was performed with the program MUSCLE and refined using the program Gblocks. Legend:PgiCAb, Porphyromonas gingivalis (YP_001929649.1); lpCA1, Legionella pneumophila, isoform 1 (WP_014844650.1); lpCA2, Legionella pneumophila, isoform 2(WP_014842179.1); HypaCA, Helicobacter pylory (YP_005769368.1); Bsuis_CA_1, Brucella suis, isoform 1 (WP_012243428.1); Bsuis_CA_2, Brucella suis, isoform 2(YP_005616633.1); stCA1, Salmonella typhimurium, isoform 1 (NP_459176.1); stCA2, Salmonella typhimurium, isoform 2 (YP_007905873.1)

Figure 2. Phylogenetic analysis carried out on the b-CA amino acid sequences of thefive bacterial species reported in Figure 1. The tree was constructed using theprogram PhyML 3.0, phylogeny software based on the maximum-likelihoodprinciple. Branch support values are reported at branch points.

Table 1Kinetic parameters for the CO2 hydration reaction catalyzed by the human cytosolicisozymes hCA I and II (a-class CAs); bacterial b-CAs: PgiCAb, HpyCA, BsuCA219,BsuCA213, LpCA1 and LpCA2; bacterial c-CA: PgiCA. All the measurements were doneat 20 �C, pH 7.5 (a-class enzymes) and pH 8.3 (b- and c- CAs) by a stopped flow CO2

hydrase assay method40

Enzyme Class kcat

(s � 1)kcat/Km

(M�1 � s�1)KI (acetazolamide)(nM)

hCA I a 2.0 � 105 5.0 � 107 250hCA II a 1.4 � 106 1.5 � 108 12PgiCAb b 2.8 � 105 1.5 � 107 214HpyCA b 7.1 � 105 4.8 � 107 40BsuCA219 b 6.4 � 105 3.9 � 107 63BsuCA213 b 1.1 � 106 8.9 � 107 303LpCA1 b 3.4 � 105 4.7 � 107 76.8LpCA2 b 8.3 � 105 8.5 � 107 72.1PgiCA c 4.1 � 105 5.4 � 107 324

S. D. Prete et al. / Bioorg. Med. Chem. 22 (2014) 4537–4543 4539

4540 S. D. Prete et al. / Bioorg. Med. Chem. 22 (2014) 4537–4543

the COX2 ‘‘selective’’ inhibitors celecoxib CLX and valdecoxib VLX.Saccharin and the diuretic hydrochlorothiazide HCT are alsoknown to act as CAIs.27

SO2NH2

NH2

SO2NH2

NH2

SO2NH2

SO2NH2

SO2NH2

CH2NH2

SO2NH2

CH2CH2NH2

SO2NH2

NH2

F

SO2NH2

NH2

Cl

SO2NH2

NH2

Br

SO2NH2

OH

ClCl

SO2NH2

NH2

SO2NH2

CF3

SO2NH2

NH2

SO2NH2

Cl

S

NN

NH2 SO2NH2S

N

NH SO2NH2

N

CH3

SO2NH2

SO2NH2

(CH2)nOH

SO2NH2

COOH

12 3

5 6 7 8

9 10 11 12

1314

4

15: n = 016: n = 117: n = 2

18

S

NN

NH

SO2NH2S

O

O

NH2

SO2NH2NH

S

O

O

NH2

SO2NH2NH

N

N

NH2

NH

S

O

O SO2NH2

OH

O2N

20

22: n = 023: n = 124: n = 1

21

19

( )n

Data of Table 2 show the inhibition data of PgiCAb with the 40derivatives mentioned above, as obtained by a stopped-flow CO2

hydrase assay monitoring the physiologic reaction catalyzed byCAs.40 Inhibition data of the human (h), possibly off target isoformshCA I and II, of a b-class CA, HpyCA (from the bacterialathogen H.pylori) and of the other c-CA from P. gingivalis, PgiCA, are also pre-sented in Table 2, for comparison reasons. The following structure-activity relationship (SAR) data can be observed for the inhibitionof PgiCAb with this panel of sulfonamides/sulfamates:

(i) Several sulfonamides, such as 14, 15, 17, 18, and DCP, didnot significantly inhibit PgiCAb up to concentrations of20 lM, although many of them were rather effective PgiCAinhibitors. For example 17 showed an inhibition constantof 178 nM against PgiCA, although it does not inhibit the

b-class enzyme, as mentioned above. Apart 14 which is aheterocyclic derivative, the other compounds in thiscategory are benznesulfonamides incorporating hydroxyalkyl,

carboxyl or in the case of DCP, chlorine and sulfamoylmoieties.

(ii) A rather large number of the investigated sulfonamides,among which 7–13, 16, 19–24, DZA, BZA, TPM, and SLP-HCT, showed modest inhibitory properties against PgiCAb,with inhibition constants in the micromolar range, of1353–9240 nM. Again the first striking feature is the differ-ence in inhibitory constants of the b- and c-class enzymesfrom P. gingivalis, with the last one being more susceptiblein most cases (except TPM) than PgiCAb. Indeed, only topira-mate is not at all inhibitory against PgiCA, being a weak,micromolar inhibitor of PgiCAb (Table 2). It can also beobserved that these ineffective PgiCAb inhibitors belong toa variety of scaffolds, such as the halogenated sulfanilamides(7–10), the 1,3-disulfamoyl-benzene derivatives (11 and 12),

S SSO2NH2

O OMe

NHEt

S

NN

CH3CONH SO2NH2S

N

CH3CON SO2NH2

N

CH3

NS S

SO2NH2

O OMeO(CH2)3

NHEtSO2NH2

Cl

SO2NH2Cl

S

N

SO2NH2EtO

S

NN

NH

SO2NH2S

O

O O

O

OO

O

O

SNH2

O

O

SO2NH2

S

OO

NH

NH

ClN

SO2NH2

OMe

NH

O

ON

SO2NH2

AAZ MZA

ZRBAZD

EZA

DCP

BZA TPM

INDSLP

ZNS

NN

SO2NH2

F

FF

CH3

NO

SO2NH2

CH3

SNH

O

O O

SO2NH2N

S

O O

NH

NHS

Cl

SO2NH2O O

VLXCLX

SAC

SLT

HCT

S. D. Prete et al. / Bioorg. Med. Chem. 22 (2014) 4537–4543 4541

the 5-substituted-1,3,4-thiadiazole-2-sulfonamides (13, 20,BZA), the sulfonylated sulfanilamides (21–24) as well as tothe various heterocyclic systems to which the clinically usedsulfonamides/sulfamates DZA, TPM, and SLP-HCT belong.

(iii) More effective PgiCAb inhibitors were compounds 1–7, MZA,BRZ and ZNS, which showed inhibition constants rangingbetween 345 and 818 nM (Table 1). Most of them (exceptZNS) were rather ineffective as PgiCA inhibitors, which makethe two CAs from P. gingivalis rather antithetic: the effectiveinhibitors for the b-class enzyme are ineffective as c-CAinhibitors, and vice versa. Indeed, only ZNS has an inyterest-ing inhibition profile against both enzymes, with a KI of

345 nM against PgiCAb and 157 nM against PgiCA, respec-tively. It is interesting to note that these compounds withmedium potency as PgiCAb inhibitors incorporate quite sim-ple scaffolds of 3- or 4-substituted benzenesulfonamideswith compact moieties such as amino, methyl, sulfamoyl,aminoalkyl, etc. (compounds 1–6). Among the halogenatedsulfanilamides investigated here, only the fluoro-derivative7 showed such properties, whereas the compounds withheavier halogens were less effective as PgiCAb inhibitors(e.g., compounds 8–10).

(iv) Only two compounds, AAZ and EZA, showed KIs > 300 nMagainst PgiCAb. Indeed, with inhiibtion constants of 214

Table 2Inhibition of human isoforms hCA I and hCA II, and of the b-class bacterial enzymes from H. pylori (HypCA) and P. gingivalis (PgiCAb) with sulfonamides 1–24 and the clinicallyused drugs AAZ-HCT. Inhibition data of the c-class enzyme from this pathogen (PgiCA)11 are also given

Inhibitor/ enzyme class hCA Ia a hCA IIa a KI* (nM)

HpyCAb b PgiCAbc b PgiCA c

1 28,000 300 nt 477 42202 25,000 240 1845 715 8933 79c 8 nt 364 >100,0004 78,500 320 2470 710 9455 25,000 170 2360 783 36006 21,000 160 3500 475 38407 8300 60 1359 818 6808 9800 110 1463 4525 6629 6500 40 1235 6620 20110 7300 54 nt 5040 21811 5800 63 973 4765 71112 8400 75 640 3898 104013 8600 60 2590 7100 51014 9300 19 768 >20,000 59515 5500 80 nt >20,000 32616 9500 94 236 8955 22317 21,000 125 218 >20,000 17818 164 46 450 >20,000 56019 109 33 38 9150 68520 6 2 64 7645 145021 69 11 nt 6450 354022 164 46 nt 3405 410023 109 33 87 9240 465024 95 30 71 7960 3400AAZ 250 12 40 214 324MZA 50 14 176 393 343EZA 25 8 33 280 613DCP 1200 38 nt >20,000 1035DZA 50,000 9 73 2415 685BRZ 45,000 3 128 408 722BZA 15 9 54 2675 741TPM 250 10 32 4250 >100,000ZNS 56 35 254 345 157SLP 1200 40 35 1470 418IND 31 15 143 1353 131VLX 54,000 43 nt 2395 755CLX 50,000 21 nt 4150 169SLT 374 9 nt 3140 424SAC 18,540 5959 nt 2244 273HCT 328 290 nt 1572 380

nt = not tested.* Errors in the range of 5–10% of the shown data, from 3 different assays.40

a Human recombinant isozymes, stopped flow CO2 hydrase assay method, from Ref.27.b Recombinant bacterial enzyme, stopped flow CO2 hydrase assay method, from Ref.33.c Recombinant bacterial enzyme, this work.

4542 S. D. Prete et al. / Bioorg. Med. Chem. 22 (2014) 4537–4543

and 280 nM, respectively, these were the most effective Pgi-CAb inhibitors detected so far. AAZ is also one of the fewcompounds which inhibited better the b- than the c-classenzyme from this pathogen, whereas EZA was a muchweaker PgiCA inhibitor (Table 1).

The analyzed CAIs might be useful in the eradication of Por-phyromonas gingivalis infection, but one may expect that inhibitionof only one of the two CAs present in the pathogen may not beenough to eradicate the bacteria, as one enzyme might compensatethe role of the other one. Thus, the best possible drugs would bethe ones which most efficiently inhibit both PgiCAb as well asPgiCA, the c-class enzyme from Porphyromonas gingivalis. Amongthe compounds investigated so far, the best candidates would beAAZ, MZA and ZNS, which have KIs < 400 nM against bothenzymes. However, the detection of inhibitors with higher efficacyin inhibiting both these CAs may be feasible, considering that onlyone class of CAIs has been so far investigated for this purpose, i.e.,the sulfonamides. In future communications from our laboratories,the other classes of CAIs will be investigated for their inhibitoryaction against these enzymes.

3. Conclusions

Porphyromonas gingivalis encodes for two CAs, one belonging tothe c-class (PgiCA) and another one to the b-class (PgiCAb). Thislast enzyme has been cloned and characterized here for its inhibi-tion profile with the main class of CA inhibitors, the sulfonamides.PgiCAb shows a significant although smaller catalytic activity com-pared to other bacterial b-CAs for the hydration of CO2 to bicarbon-ate and protons. Many of the clinically used sulfonamides as wellas simple aromatic/heterocyclic sulfonamides were ineffective asPgiCAb inhibitors whereas better inhibition was observed withsimple derivatives such as sulfanilamide, metanilamide, 4-amin-oalkylbenzenesulfonamides (KIs of 364–475 nM). The haloge-nosulfanilamides incorporating heavy halogens, 4-hydroxy- and4-hydroxyalkyl-benzenesulfonamides, were also micromolar,ineffective as PgiCAb inhibitors. The best inhibitors of the b-classenzyme were acetazolamide and ethoxzolamide, with KIs of214–280 nM. Interestingly, the c-class enzyme was much moresensitive to sulfonamide inhibitors compared to the b-class onePgiCAb. Identification of potent and possibly selective inhibitorsof PgiCAb/PgiCA may lead to pharmacological tools useful for

S. D. Prete et al. / Bioorg. Med. Chem. 22 (2014) 4537–4543 4543

understanding the physiological role(s) of these enzymes, sincethis bacterium is the main causative agent of periodontitis andfew treatment options are presently available. Effective, dualinhibitors of both PgiCA and PgiCAb may lead to eradication of P.gingivalis infection.

4. Experimental protocols

4.1. Chemistry

Sulfonamides 1–24 and AAZ-HCT were commercially availableor reported earlier by us.27,41 All compounds were >95% purity,as assessed by HPLC.

4.2. Sequence and phylogenetic analysis

Multialignment of nucleotide sequences was performed usingthe programs PileUp (G.C.G.-Wiscon- sin)42 and ClustalW version1.7. A most parsimonious tree was constructed with the programPhyML.43

4.3. CA activity measurements and inhibition studies

A stopped-flow CO2 hydration assay with an applied photo-physics instrument has been used for measuring catalytic activityand inhibition of the new enzymes reported here. Phenol red (ata concentration of 0.2 mM) has been used as indicator, workingat the absorbance maximum of 557 nm, with 20 mM Hepes (pH7.4) or 20 mM Tris (pH 8.3) as buffers, and 20 mM Na2SO4 orNaClO4 (for maintaining constant the ionic strength). The initialrates of the CA-catalyzed CO2 hydration reaction were followedfor a period of 10–100 s.40 The concentrations of substrate (CO2)ranged from 1.7 to 17 mM for the determination of the kineticparameters and inhibition constants, with at least six traces ofthe initial 5–10% of the reaction being used for determining theinitial velocity, for each inhibitor. The uncatalyzed rates weredetermined subtracted from the total observed rates. Stock solu-tions of inhibitors (10 mM) were prepared in distilled-deionizedwater and dilutions up to 0.01 nM were done with the assay buffer.Enzyme and inhibitor solutions were preincubated prior to assayfor 15 min (at room temperature), in order to allow for the forma-tion of the E-I complex. The inhibition constants were obtained bynon-linear least-squares methods using PRISM 3 and the Cheng–Prusoff equation as reported earlier by our groups. The kineticparameters for the uninhibited enzymes were derived fromLineweaver–Burk plots, as reported earlier,11,14 and represent themean from at least three different determinations.

Acknowledgments

This work was supported in part by an FP7 EU Project (Gums &Joints, Grant agreement Number HEALTH-F2-2010-261460).

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