Bioorganic & Medicinal Chemistry 22 (2014) 4537–4543

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Sulfonamide inhibition study of the carbonic anhydrases from the bacterial pathogen Porphyromonas gingivalis: The b-class (PgiCAb) versus the c-class (PgiCA) enzymes 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, Italy Università degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino (Firenze), Italy c Department of Chemistry, College of Science, King Saud University, PO Box 2455, Riyadh 11451, Saudi Arabia d Università degli Studi di Firenze, Polo Scientifico, Dipartimento NEIROFABA;Sezione di Scienze Farmaceutiche e Nutraceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino (Firenze), Italy b

a r t i c l e

i n f o

Article history: Received 2 July 2014 Revised 26 July 2014 Accepted 29 July 2014 Available online 7 August 2014 Keywords: Carbonic anhydrase Beta-class enzyme Sulfonamide Porphyromonas gingivalis

a b s t r a c t The oral pathogenic bacterium Porphyromonas gingivalis, encodes for two carbonic anhydrases (CAs, EC 4.2.1.1) one belonging to the c-class (PgiCA) and another one to the b-class (PgiCAb). This last enzyme has 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/heterocyclic sulfonamides were ineffective as PgiCAb inhibitors whereas better inhibition was observed with simple derivatives such as sulfanilamide, metanilamide, 4-aminoalkylbenzenesulfonamides (KIs of 364–475 nM). The halogenosulfanilamides incorporating heavy halogens, 4-hydroxy- and 4-hydroxyalkyl-benzenesulfonamides, were also micromolar, ineffective PgiCAb inhibitors. The best inhibitors of the b-class enzyme were acetazolamide and ethoxzolamide, with KIs of 214–280 nM. Interestingly, the c-class enzyme was much more sensitive to sulfonamide inhibitors compared to the b-class one, PgiCAb. Identification of potent and possibly selective inhibitors of PgiCAb/PgiCA may lead to pharmacological tools useful for understanding the physiological role(s) of these enzymes, since this bacterium is the main causative agent of periodontitis and few treatment options are presently available. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Sulfa drugs were first used in the 1930s, and they revolutionized medicine.1 After a few years, bacteria started to develop resistance to these drugs, and eventually penicillin replaced them as a first-line treatment.2,3 While antibiotic resistance remains a problem for most classes of antibiotics ever discovered afterwards, sulfa drugs are still commonly used to treat a variety of bacterial infections, mainly in combination with other drug classes. Sulfa drugs work by binding to and inhibiting a specific bacterial enzyme, the dihydropteroate synthase (DHPS).2,4–6 This enzyme is critical for the synthesis of folate, an essential nutrient for bacteria. Its biosynthesis requires the chemical reaction between two molecules, 6-hydroxymethyl-7,8-dihydropterin-pyrophosphate (DHPP) ⇑ 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: [email protected] (C. Capasso), claudiu.supuran@unifi.it (C.T. Supuran). http://dx.doi.org/10.1016/j.bmc.2014.07.048 0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

and p-aminobenzoic acid (PABA).7 Mammals get folate from their diet, but bacteria must synthesize this vitamin. Because of their structural similarity to PABA, the sulfonamides compete with this substrate for the bacterial enzyme, DHPS.8 Thus, they inhibit the synthesis of bacterial dihydrofolic acid and, thereby, the formation of its essential cofactor forms. Bacteria that can obtain folates from their environment are naturally resistant to these drugs. Acquired bacterial resistance is generally irreversible and may be due to an altered dihydropteroate synthetase, a decreased cellular permeability of the sulfa drugs, or an enhanced production of the natural substrate, 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 bacteria, in the search of antibiotics with a novel mechanism of action.11–15 It has been demonstrated that in many bacteria, CAs are essential for the life cycle of the organism and that their inhibition leads to growth impairment or growth defects of the pathogen.11,13 CAs are metalloenzymes that catalyze the reversible

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hydration of CO2 to bicarbonate and a proton, using Zn(II), Cd(II) or Fe(II) at their active site. CAs have been found in virtually all mammalian tissues and cell types, where they function in CO2 transport, pH regulation, biosynthetic pathways and other physiological processes.11,12,14,16,17 CAs are also found in plants, cyanobacteria, and algae, where they appear to facilitate photosynthesis. In bacteria, CAs are involved in the transport of CO2 and bicarbonate and in related physiological processes.18–21 Bacterial CAs exist in at least three genetically unrelated families of enzymes, 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 the a class. Intriguingly, CA inhibitors (CAIs) include various anions, imidazole, phenols, hydroxamates, carboxylates, phosphates/phosphonates, and various sulfonamide derivatives (R-SO2NH2) and their isosteres (sulfamides, sulfamates), which represent the main class of clinically used CAIs.18–21,27–30 Many common pathogenic bacteria encode such enzymes. For example Helicobacter pylori, is a pathogenic bacterium which lives in the highly acidic environment of the stomach. Its genome encodes three CAs: a-, b-and c-CA, respectively.31–36 The crucial role played by these CAs present in H. pylori is the acid acclimatization of the pathogen within the stomach. The inhibition of these enzymes, in fact, led to the death of the bacteria and a possible eradication of H. pylori from the stomach and has been used clinically for the treatment of gastric ulcers. The genome of Vibrio cholerae, the causative agent of cholera, encodes for putative CAs belonging to each bacterial class: the a, b and c ones.37–39 Recently it was reported that sodium bicarbonate induces cholera toxin (CT) expression is mediated by some of these enzymes.36 In the presence of CA inhibitors (such as the sulfonamide ethoxzolamide), a significant reduction in virulence gene expression was observed, which inhibited virulence in vivo.36 Our group cloned, purified and characterized the a-CA from V. cholerae, named VchCA. This new enzyme showed significant catalytic activity, being more active than the human isoform hCA I or than the H. pylori a-class enzyme.37–39 An inhibition study with a panel of sulfonamides and one sulfamate led to the detection of a large number of low nanomolar VchCA inhibitors, including methazolamide, acetazolamide, ethoxzolamide, dorzolamide, brinzolamide, benzolamide, and indisulam (with KI in the range of 0.69 8.1 nM). Porphyromonas gingivalis is one of the few major pathogens responsible for the development of chronic periodontitis and a successful colonizer of the oral epithelium.11 The perturbation of epithelial cells by bacteria is the first stage in the initiation of inflammatory and immune processes causing eventually destruction 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 purified the recombinants c-CA (named PgiCA) and the b-CA (named PgiCAb) identified in the genome of this pathogenic bacterium. PgiCA was shown to possess a significant catalytic activity for the 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 inhibition constant of 324 nM.11,14,40 In a previous work we explored the inhibition profile with sulfonamides of the c-CA from this pathogen.11 Here we report the sulfonamide inhibition study of the other enzyme, PgiCAb, comparing it with data obtained for the c-CA enzyme (PgiCA), as sulfonamides are the classical, best investigated inhibitors of all CAs. Identification of potent and possibly selective inhibitors of PgiCA and PgiCAb may lead to pharmacological tools useful for understanding the physiological role(s) of these enzymes.

2. Results and discussion 2.1. Cloning, sequence analysis and catalytic properties of PgiCAb IPTG induction of Escherichia coli BL21 (DE3) cells transformed with the plasmid pET15-b/PgiCAb resulted in the production of the recombinant b-CA, named PgiCAb. The b-CA was isolated and purified to homogeneity from the E. coli (DE3) cell extract. Most of the CA activity was recovered in the soluble fraction of the cell extract after sonication and centrifugation. Using an affinity column (His-select HF Nickel affinity gel), PgiCAb was purified to homogeneity. Analysis by SDS-Page of PgiCAb showed two main bands of about 25 kDA (monomeric form) and 50 kDA (dimeric form) under reducing condition (data not shown). The full nucleotide sequence showed an open reading frame encoding a 242 residues polypeptide chain which contained all the typical features of a b-CA, including the three residues that are involved in the catalytic 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 the water molecule coordinated to the zinc ion from the enzyme active site, is also conserved in PgiCAb, as for the other investigated b-CAs such as the two enzymes from Legionella pneumophila lpCA1 and lpCA2, the H. pylori enzyme HpyCA, the two Brucella suis enzymes BsuCA219 and 213, as well as the two b-CAs from Salmonella typhimurium, stCA1 and stCA2 (Fig. 1). The predicted molecular mass of the enzyme from its amino acid sequence is of 26.1 kDa. Furrhemore, the phylogenetic analysis of these enzymes shown in Fig. 2 clearly shows that PgiCAb is evolutionarily more similar to one of the S. typhimurium enzymes, stCA2, and also with the two L. pneumophila enzymes lpCA 2 and lpCA1. These four b-CAs clustered together on the lower branch of the tree shown in Fig. 2, whereas the remaining enzymes also clustered together, on a different branch, being thus less closely related to the P. gingivalis enzyme (Fig. 2). PgiCAb showed a good catalytic activity, with a kcat of 2.8  105 s 1 and a kcat/Km of 1.5  107 M 1  s 1. PgiCAb was also inhibited by the clinically used sulfonamide acetazolamide, with an inhibition constant of 214 nM (Table 1). This is in fact to be expected considering the fact that all these b-CAs of bacterial origin investigated earlier showed catalytic properties in the same range as PgiCAb, both considering the kinetic constant kcat or the catalytic efficiency kcat/Km (Table 1). PgiCAb is however one of the least active catalysts for the CO2 hydration reaction among the bacterial b-CAs investigated so far. 2.2. Sulfonamide inhibition studies of PgiCAb and comparison with PgiCA The library of 40 compounds, comprising 39 sulfonamides and one sulfamate investigated earlier as inhibitors of the c-class enzyme PgiCA, were also included in this study. Derivatives 1–24 and AAZ-HCT are either simple aromatic/heterocyclic sulfonamides widely used as building blocks for obtaining new families of such pharmacological agents,27 or they are clinically used agents, among which acetazolamide AAZ, methazolamide MZA, ethoxzolamide EZA and dichlorophenamide DCP, are the classical, systemically acting antiglaucoma CA inhibitors (CAIs).27a Dorzolamide DZA and brinzolamide BRZ are topically-acting antiglaucoma agents, benzolamide BZA is an orphan drug belonging to this class of pharmacological agents, whereas topiramate TPM, zonisamide ZNS and sulthiame SLT are widely used antiepileptic drugs.27 Sulpiride SLP and indisulam IND were also shown by our group to belong to this class of pharmacological agents,27 together with

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PgiCAb lpCA1 lpCA2 HpyCA Bsuis_CA_1 Bsuis_CA_9 stCA1 stCA2

--MKKIVLFSAAMAMLIACGNQTTQTKSDTPTAAVEGRIGEVLTQDIQQGLTPEAVLVGL -------MPKKLLIAAFLCNIFCNPSHLAYASSTEIPILGKTMTQAKQQQMTPRQALQRL ---------------------------------------MWTLTKEQQQAITPEKAIELL ---------------------------------------------------------MKA ----------------------------------------------------MADLPDSL -------------------------------------------MPMKNDHSPDQRTLSEL -----------------------------------------------------MKDIDTL MEQNQPAQPSRRAILKQTLAVSALSVTGLAALSVPTISFAASLSKEERDGMTPDAVIEHF

PgiCAb lpCA1 lpCA2 HpyCA Bsuis_CA_1 Bsuis_CA_9 stCA1 stCA2

QEGNARYVANKQLPRDLNAQAVAGLEGQFPEAIILSCIDSRVPVEYIFDKGIGDLFVGRV KDGNQRFLSNKPLARDYLKQAKQSAYGQYPFAVILNCMDSRSVPEFFFDQGLADLFTLRV KEGNKRFVSNLKLNRNLIQQVNETSQGQFPFAVILSCMDSRTPAELIFDQGLGDIFSIRV FLGALEFQENE-YEELKELYESLKT-KQKPHTLFISCVDSRVVPNLITGTKPGELYVIRN LAGYKTFMSEH-FAHETARYRDLAEKGQSPETLVVACCDSRAAPETIFNAAPGEIFVLRN FEHNRQWAAEK-QEKDPEYFSRLSS-SQRTEFLWIGCSDSRVPANVVMGLQPGEVFVHRN ISNNALWSKML-VEEDPGFFEKLAQ-AQKPRFLWIGCSDSRVPAERLTGLEPGELFVHRN KQGNLRFRENRPAKHDYLAQKRNSIAGQYPAAVILSCIDSRAPAEIVLDAGIGETFNSRV : * . : : * *** : . . .: : *

PgiCAb lpCA1 lpCA2 HpyCA Bsuis_CA_1 Bsuis_CA_9 stCA1 stCA2

AGNVV--------DDHMLGSLEYACEVSGSKVLLVLGHEDCGA------IKSAIKGVEMG AGNVL--------NDDILGSMEFATKVVGARLVVVLAHTSCGA------VAGACKDVKLG AGNIL--------NDDILGSIEFACQVVGVKLIAVVGHTQCGA------IKGACDGVKLG MGNVIPPKTSHKESLSTMASIEYAIVHVGVQNLIICGHSDCGACGSTHLINDGXTKAKTP VANLIPPYEPDGEYHAASAALEFAVQSLKVKHIVVMGHGRCGG------IKAALDTESAP VANLV-----HRADLNLLSVLEFAVGVLEIKHIIVCGHYGCGG------VRAAMDGYGHG VANLV-----IHTDLNCLSVVQYAVDVLEVEHIIICGHSGCGG------IKAAVENPELG AGNIS--------NRDMLGSMEFACAVAGAKVVLVIGHTRCGA------VRCAIDNAELG .*: . :::* : : .* **. : .

PgiCAb lpCA1 lpCA2 HpyCA Bsuis_CA_1 Bsuis_CA_9 stCA1 stCA2

N-----ITSLMEEIKPSV-EATQYTGERTYANKEFADAVVKENVIQTMDEIRRDSPILKK H-----LTDVINKIHPVVKPSMESTGIDNCSDPKLIDDMAKANALHVVKNILEQSPILNE N-----LTNLLNKINPVIQEAKKLDAKHDVHSPEFLNCVTSLNVKHTMNEITQRSDIVHQ Y-----IADWIQFLEPIK-EELKNHPQFSNHFAKRSWLTERLNVRLQLNNLLS-YDFIQE LSPSDFIGKWMSLISP---AAEAISGNALMTQSERHTALERISIRYSLANLRT-FPCVDI I-----IDNWLQPIRDIA-QANQAELDTIENTQDRLDRLCELGVSSQVESLSR-TPVLQS L-----INNWLLHIRDIWLKHSSLLGK--MPEEQRLDALYELNVMEQVYNLGH-STIMQS N-----LTGLLDEIKPAI-AKTEYSGERKGSNYDFVDAVARKNVELTIENIRKNSPVLKQ : : : . . : .: :

PgiCAb lpCA1 lpCA2 HpyCA Bsuis_CA_1 Bsuis_CA_9 stCA1 stCA2

LEEEGK-IKICGAIYEMSTGKVHFL----------------------------LVKNKQ-IGIVAGIHDIKTGKVTFFEEKRSVPE--------------------LLNEKR-IAIAGGLYQLETGEVQFFDE--------------------------RVVNNE-LKIFGWHYIIETGRIYNYNFESHFFEPIXETXKQRKSHENF-----LEKKGK-LTLHGAWFDISTGELWVMDHRTGD----------------FK-RPEL AWKDGKDIIVHGWMYNLKDGLLRDIGCDCTR------------NALQFACQPAE AWKRGQNVTIHGWAYSINDGLLRDLDVTATNRETLENGYHKGISALSLKYIPHQ LEDEKK-IKIVGSMYHLTGGKVEFFEV--------------------------: : . . : * :

90 92 94

143 146

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 the coordination 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 water molecule 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)

Table 1 Kinetic parameters for the CO2 hydration reaction catalyzed by the human cytosolic isozymes 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 done at 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

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

hCA I hCA II PgiCAb HpyCA BsuCA219 BsuCA213 LpCA1 LpCA2 PgiCA

Class

a a b b b b b b

c

kcat (s 1)

kcat/Km (M 1  s

2.0  105 1.4  106 2.8  105 7.1  105 6.4  105 1.1  106 3.4  105 8.3  105 4.1  105

5.0  107 1.5  108 1.5  107 4.8  107 3.9  107 8.9  107 4.7  107 8.5  107 5.4  107

1

)

KI (acetazolamide) (nM) 250 12 214 40 63 303 76.8 72.1 324

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

b-class enzyme, as mentioned above. Apart 14 which is a heterocyclic derivative, the other compounds in this category are benznesulfonamides incorporating hydroxyalkyl,

SO2NH2

SO2NH2

SO2NH2

SO2NH2

SO2NH2

NH2 NH2

1

2

4

3

SO2NH2

SO2NH2

SO2NH2

CH2NH2

CH2CH2NH2

NH2

SO2NH2

F 6

5

7

SO2NH2

SO2NH2

Cl

Cl

Cl

SO2NH2 NH2

10

9

N SO2NH2

HN

S

SO2NH2

NH2

(CH2)nOH

COOH

15: n = 0 16: n = 1 17: n = 2

18

N N

O H2N

N

SO2NH2

SO2NH2

14

N

12

SO2NH2 N

13

H N

SO2NH2 NH2

11

H3C

N N S

SO2NH2

CF3

NH2

H2N

8

SO2NH2

OH Br

Cl NH2

S N H

S

SO2NH2

O

19

20

O O2N

S N H O HO

O SO2NH2 H2N

21

Data of Table 2 show the inhibition data of PgiCAb with the 40 derivatives mentioned above, as obtained by a stopped-flow CO2 hydrase assay monitoring the physiologic reaction catalyzed by CAs.40 Inhibition data of the human (h), possibly off target isoforms hCA 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 presented in Table 2, for comparison reasons. The following structureactivity relationship (SAR) data can be observed for the inhibition of PgiCAb with this panel of sulfonamides/sulfamates: (i) Several sulfonamides, such as 14, 15, 17, 18, and DCP, did not significantly inhibit PgiCAb up to concentrations of 20 lM, although many of them were rather effective PgiCA inhibitors. For example 17 showed an inhibition constant of 178 nM against PgiCA, although it does not inhibit the

( )n S N H O 22: n = 0 23: n = 1 24: n = 1

SO2NH2

carboxyl or in the case of DCP, chlorine and sulfamoyl moieties. (ii) A rather large number of the investigated sulfonamides, among which 7–13, 16, 19–24, DZA, BZA, TPM, and SLPHCT, showed modest inhibitory properties against PgiCAb, with inhibition constants in the micromolar range, of 1353–9240 nM. Again the first striking feature is the difference in inhibitory constants of the b- and c-class enzymes from P. gingivalis, with the last one being more susceptible in most cases (except TPM) than PgiCAb. Indeed, only topiramate is not at all inhibitory against PgiCA, being a weak, micromolar inhibitor of PgiCAb (Table 2). It can also be observed that these ineffective PgiCAb inhibitors belong to a variety of scaffolds, such as the halogenated sulfanilamides (7–10), the 1,3-disulfamoyl-benzene derivatives (11 and 12),

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H3C

N N CH3CONH

N SO2NH2

S

CH3CON

N SO2NH2

S

AAZ

EtO

EZA NHEt

NHEt SO2NH2

SO2NH2

Me

Cl DCP

O BRZ

N N

S N H O

S

S

O

O DZA

O

SO2NH2

N

MeO(CH2)3

S

S O

SO2NH2

S

MZA

SO2NH2

Cl

N

O NH2 O S O O

O

SO2NH2

SO2NH2

S

O

O

N

O BZA

O

TPM ZNS

OMe O H N

N H

N

O O S N H

Cl

SO2NH2

SO2NH2 SLP IND

SO2NH2

SO2NH2

N CH3

N

H3C

O O

N O N

SO2NH2

S

SLT

F F F

VLX CLX

O

H N

Cl

NH HN

S O

O

O

S

SO2NH2 O

SAC HCT

the 5-substituted-1,3,4-thiadiazole-2-sulfonamides (13, 20, BZA), the sulfonylated sulfanilamides (21–24) as well as to the various heterocyclic systems to which the clinically used sulfonamides/sulfamates DZA, TPM, and SLP-HCT belong. (iii) More effective PgiCAb inhibitors were compounds 1–7, MZA, BRZ and ZNS, which showed inhibition constants ranging between 345 and 818 nM (Table 1). Most of them (except ZNS) were rather ineffective as PgiCA inhibitors, which make the two CAs from P. gingivalis rather antithetic: the effective inhibitors for the b-class enzyme are ineffective as c-CA inhibitors, and vice versa. Indeed, only ZNS has an inyteresting inhibition profile against both enzymes, with a KI of

345 nM against PgiCAb and 157 nM against PgiCA, respectively. It is interesting to note that these compounds with medium potency as PgiCAb inhibitors incorporate quite simple scaffolds of 3- or 4-substituted benzenesulfonamides with compact moieties such as amino, methyl, sulfamoyl, aminoalkyl, etc. (compounds 1–6). Among the halogenated sulfanilamides investigated here, only the fluoro-derivative 7 showed such properties, whereas the compounds with heavier halogens were less effective as PgiCAb inhibitors (e.g., compounds 8–10). (iv) Only two compounds, AAZ and EZA, showed KIs > 300 nM against PgiCAb. Indeed, with inhiibtion constants of 214

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Table 2 Inhibition 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 clinically used drugs AAZ-HCT. Inhibition data of the c-class enzyme from this pathogen (PgiCA)11 are also given Inhibitor/ enzyme class

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 AAZ MZA EZA DCP DZA BRZ BZA TPM ZNS SLP IND VLX CLX SLT SAC HCT

hCA Ia a

28,000 25,000 79c 78,500 25,000 21,000 8300 9800 6500 7300 5800 8400 8600 9300 5500 9500 21,000 164 109 6 69 164 109 95 250 50 25 1200 50,000 45,000 15 250 56 1200 31 54,000 50,000 374 18,540 328

hCA IIa a

KI* (nM)

300 240 8 320 170 160 60 110 40 54 63 75 60 19 80 94 125 46 33 2 11 46 33 30 12 14 8 38 9 3 9 10 35 40 15 43 21 9 5959 290

HpyCAb b

PgiCAbc b

PgiCA c

nt 1845 nt 2470 2360 3500 1359 1463 1235 nt 973 640 2590 768 nt 236 218 450 38 64 nt nt 87 71 40 176 33 nt 73 128 54 32 254 35 143 nt nt nt nt nt

477 715 364 710 783 475 818 4525 6620 5040 4765 3898 7100 >20,000 >20,000 8955 >20,000 >20,000 9150 7645 6450 3405 9240 7960 214 393 280 >20,000 2415 408 2675 4250 345 1470 1353 2395 4150 3140 2244 1572

4220 893 >100,000 945 3600 3840 680 662 201 218 711 1040 510 595 326 223 178 560 685 1450 3540 4100 4650 3400 324 343 613 1035 685 722 741 >100,000 157 418 131 755 169 424 273 380

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.

and 280 nM, respectively, these were the most effective PgiCAb inhibitors detected so far. AAZ is also one of the few compounds which inhibited better the b- than the c-class enzyme from this pathogen, whereas EZA was a much weaker PgiCA inhibitor (Table 1). The analyzed CAIs might be useful in the eradication of Porphyromonas gingivalis infection, but one may expect that inhibition of only one of the two CAs present in the pathogen may not be enough to eradicate the bacteria, as one enzyme might compensate the role of the other one. Thus, the best possible drugs would be the ones which most efficiently inhibit both PgiCAb as well as PgiCA, the c-class enzyme from Porphyromonas gingivalis. Among the compounds investigated so far, the best candidates would be AAZ, MZA and ZNS, which have KIs < 400 nM against both enzymes. However, the detection of inhibitors with higher efficacy in inhibiting both these CAs may be feasible, considering that only one 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 inhibitory action against these enzymes.

3. Conclusions Porphyromonas gingivalis encodes for two CAs, one belonging to the c-class (PgiCA) and another one to the b-class (PgiCAb). This last enzyme has been cloned and characterized here for its inhibition profile with the main class of CA inhibitors, the sulfonamides. PgiCAb shows a significant although smaller catalytic activity compared to other bacterial b-CAs for the hydration of CO2 to bicarbonate and protons. Many of the clinically used sulfonamides as well as simple aromatic/heterocyclic sulfonamides were ineffective as PgiCAb inhibitors whereas better inhibition was observed with simple derivatives such as sulfanilamide, metanilamide, 4-aminoalkylbenzenesulfonamides (KIs of 364–475 nM). The halogenosulfanilamides incorporating heavy halogens, 4-hydroxy- and 4-hydroxyalkyl-benzenesulfonamides, were also micromolar, ineffective as PgiCAb inhibitors. The best inhibitors of the b-class enzyme were acetazolamide and ethoxzolamide, with KIs of 214–280 nM. Interestingly, the c-class enzyme was much more sensitive to sulfonamide inhibitors compared to the b-class one PgiCAb. Identification of potent and possibly selective inhibitors of PgiCAb/PgiCA may lead to pharmacological tools useful for

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understanding the physiological role(s) of these enzymes, since this bacterium is the main causative agent of periodontitis and few treatment options are presently available. Effective, dual inhibitors 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 available or 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 using the programs PileUp (G.C.G.-Wiscon- sin)42 and ClustalW version 1.7. A most parsimonious tree was constructed with the program PhyML.43 4.3. CA activity measurements and inhibition studies A stopped-flow CO2 hydration assay with an applied photophysics instrument has been used for measuring catalytic activity and inhibition of the new enzymes reported here. Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.4) or 20 mM Tris (pH 8.3) as buffers, and 20 mM Na2SO4 or NaClO4 (for maintaining constant the ionic strength). The initial rates of the CA-catalyzed CO2 hydration reaction were followed for a period of 10–100 s.40 The concentrations of substrate (CO2) ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants, with at least six traces of the initial 5–10% of the reaction being used for determining the initial velocity, for each inhibitor. The uncatalyzed rates were determined subtracted from the total observed rates. Stock solutions of inhibitors (10 mM) were prepared in distilled-deionized water and dilutions up to 0.01 nM were done with the assay buffer. Enzyme and inhibitor solutions were preincubated prior to assay for 15 min (at room temperature), in order to allow for the formation of the E-I complex. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3 and the Cheng– Prusoff equation as reported earlier by our groups. The kinetic parameters for the uninhibited enzymes were derived from Lineweaver–Burk plots, as reported earlier,11,14 and represent the mean 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). References and notes 1. Haruki, H.; Pedersen, M. G.; Gorska, K. I.; Pojer, F.; Johnsson, K. Science 2013, 340, 987. 2. Capasso, C.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2014, 29, 379. 3. Yun, M. K.; Wu, Y.; Li, Z.; Zhao, Y.; Waddell, M. B.; Ferreira, A. M.; Lee, R. E.; Bashford, D.; White, S. W. Science 2012, 335, 1110. 4. Mockenhaupt, F. P.; Teun Bousema, J.; Eggelte, T. A.; Schreiber, J.; Ehrhardt, S.; Wassilew, N.; Otchwemah, R. N.; Sauerwein, R. W.; Bienzle, U. Trop. Med. Int. Health 2005, 10, 901. 5. Khalil, I.; Ronn, A. M.; Alifrangis, M.; Gabar, H. A.; Satti, G. M.; Bygbjerg, I. C. Am. J. Trop. Med. Hyg. 2003, 68, 586. 6. Hampele, I. C.; D’Arcy, A.; Dale, G. E.; Kostrewa, D.; Nielsen, J.; Oefner, C.; Page, M. G.; Schonfeld, H. J.; Stuber, D.; Then, R. L. J. Mol. Biol. 1997, 268, 21. 7. Shiota, T.; Disraely, M. N.; McCann, M. P. J. Biol. Chem. 1964, 239, 2259.

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