Bioorganic & Medicinal Chemistry Letters 25 (2015) 2291–2297

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Sulfonamide inhibition study of the b-class carbonic anhydrase from the caries producing pathogen Streptococcus mutans Nurcan Dedeoglu a,b, Viviana DeLuca c, Semra Isik b, Hatice Yildirim d, Feray Kockar d, Clemente Capasso c, Claudiu T. Supuran e,⇑ a

Università degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino (Firenze), Italy Department of Chemistry, Faculty of Art & Science, Balikesir University, Balikesir, Turkey Istituto di Biochimica delle Proteine—CNR, Via P. Castellino 111, 80131 Napoli, Italy d Department of Biology, Faculty of Art & Science, Balikesir University, Balikesir, Turkey e Università degli Studi di Firenze, Polo Scientifico, Dipartimento NEUROFABA, Sezione di Scienze Farmaceutiche e Nutraceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino (Firenze), Italy b c

a r t i c l e

i n f o

Article history: Received 10 March 2015 Revised 9 April 2015 Accepted 10 April 2015 Available online 17 April 2015 Keywords: Streptococcus mutans Carbonic anhydrase b-Class enzyme Inhibitor Sulfonamide

a b s t r a c t Streptococcus mutans, the oral pathogenic bacterium provoking dental caries formation, encodes for a b-class carbonic anhydrase (CA, EC 4.2.1.1), SmuCA. This enzyme was cloned, characterized and investigated for its inhibition profile with the major class of CA inhibitors, the primary sulfonamides. SmuCA has a good catalytic activity for the CO2 hydration reaction, with a kcat of 4.2  105 s1 and kcat/Km of 5.8  107 M1  s1, and is efficiently inhibited by most sulfonamides (KIs of 246 nM–13.5 lM). The best SmuCA inhibitors were bromosulfanilamide, deacetylated acetazolamide, 4-hydroxymethylbenzenesulfonamide, a pyrimidine-substituted sulfanilamide derivative, aminobenzolamide and compounds structurally similar to it, as well as acetazolamide, methazolamide, indisulam and valdecoxib. These compounds showed inhibition constants ranging between 246 and 468 nM. Identification of effective inhibitors of this enzyme may lead to pharmacological tools useful for understanding the role of S. mutans CAs in dental caries formation, and eventually the development of pharmacological agents with a new mechanism of antibacterial action. Ó 2015 Elsevier Ltd. All rights reserved.

Bacteria encode carbonic anhydrases (CAs, EC 4.2.1.1) belonging to three of the six genetic families discovered so far, that is, a-, b-, and c-CAs.1 These enzymes catalyze a simple but physiologically relevant reaction in all life kingdoms, the hydration of carbon dioxide to bicarbonate and protons.2–5 In organisms all over the phylogenetic tree, CAs are involved in crucial physiologic processes connected with pH regulation, CO2 sensing, photosynthesis, respiration, CO2 transport, as well as metabolism of xenobiotics (e.g., cyanate in Escherichia coli).6–9 Among the six genetically distinct families (a-, b-, c-, d-, f- and g-CAs), the Zn(II), Cd(II), or Fe(II) cofactors are essential for the catalytic activity of these metalloenzymes, playing a crucial role in the catalytic process.9–11 Ultimately, a large number of CA enzymes have been investigated in detail in pathogenic as well as nonpathogenic bacteria such as Helicobacter pylori, Brucella suis, Mycobacterium tuberculosis, Streptococcus pneumoniae, Salmonella enterica, Sulfurihydrogenibium yellowstonense, Sulfurihydrogenibium azorense, Vibrio cholerae, Legionella

⇑ Corresponding author. Tel.: +39 055 4573005; fax: +39 055 4573385. E-mail address: claudiu.supuran@unifi.it (C.T. Supuran). http://dx.doi.org/10.1016/j.bmcl.2015.04.037 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

pneumophila, Porphyromonas gingivalis, and others.3,8–11 The presence of CAs in pathogenic microorganisms was proposed as a new strategy for the development of antiinfectives with a new, less explored mechanism of action.3 Streptococcus mutans is considered the principal causative agent of human dental caries, being a major public health problem worldwide.12,13 Furthermore, untreated dental caries favor this pathogenic bacterium to enter the bloodstream and cause severe infections, some of which even fatal if untreated, such as, for example, bacteremia, infective endocarditis or infection of the heart valve.13 A feature of this bacterium is that it is both acidogenic and aciduric, with many of its strains being able to produce large quantities of intra- and extracellular polysaccharides, which enhance the cariogenicity of the pathogen.13,14 Although conventional antibiotic drugs usually provide effective therapy for such bacterial infections, there is an increasing problem of antibiotic resistance and a stringent need for new such agents.15 In recent years, many isolates of S. mutans started to show considerable resistance to commonly used antibiotics, with serious costs for the healthcare system and unpleasant consequences for the affected patients.14–16

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Primary sulfonamides and their bioisosteres (sulfamates, sulfamides) are the most investigated types of CA inhibitors (CAIs), having various biomedical applications as diuretics, antiglaucoma, anticonvulsant, antiobesity, anticancer, antipain and antiinfective agents.17–21 They bind as sulfonamidate anions (RSO2NH) to the metal ion within the CA active site, replacing the coordinated water molecule/hydroxide anion acting as nucleophile in the catalytic cycle.1–3 Other anions, such as the inorganic metal-complexing ones or more complicated species such as the carboxylates, are also known to bind to the CAs, but generally with less efficiency compared to sulfonamides.22,23 In the last years, however, CA anion inhibitors received great importance and, in fact, all catalytically active mammalian isoforms have been investigated for their inhibition with a large number of such inorganic or organic anions, including sulfonamides, sulfamates, sulfamides, dithiocarbamates, xanthates, carboxylates, etc.3,16–20 Bacterial CAs have been included in such studies only recently, above all in order to develop new types of CAIs with potential applications as antiinfectives but also for discovering lead compounds.8,22,23 In this Letter we report the cloning, characterization and sulfonamide inhibition studies of a new b-CA from the oral cariogenic pathogen S. mutans, denominated SmuCA. We have identified one b-CA-encoding gene in the genome of S. mutans,13b denominated SMU_328 (accession number NP_720781), which encodes a protein of 166 amino acid residues, and has a molecular weight, as a monomer, of 18096 Da.24 The amino acid sequence of the new b-CA, SmuCA, was aligned with that of other such enzymes from bacteria (B. suis—BsuCA; P. gingivalis— PgiCA,)3–11 and the plant Flaveria bidentis (FbiCA)—Figure 1. Data of Figure 1 show that SmuCA possesses all the typical features of an active b-CA, including the three residues that coordinate the

Table 1 Kinetic parameters for the CO2 hydration reaction catalysed by the human cytosolic isozymes hCA I and II (a-class CAs); bacterial b-CAs: PgiCAb, HpyCA, BsuCA (isoform 1) and SmuCA Enzyme hCA I hCA II PgiCAb HpyCA BsuCA SmuCA

Class

a a b b b b

kcat (s1) 5

2.0  10 1.4  106 2.8  105 7.1  105 6.4  105 4.2  105

kcat/Km (M1  s1)

KI (acetazolamide) (nM)

5.0  107 1.5  108 1.5  107 4.8  107 3.9  107 5.8  107

250 12 214 40 63.0 344

All the measurements have been made at 20 °C, pH 7.5 (a-enzymes) and pH 8.3 (bclass enzymes) by a stopped flow CO2 hydrase assay method.25

catalytically crucial Zn(II) ion (two cysteines and one histidine, more precisely Cys160, His220 and Cys223, see Fig. 1). Furthermore, the catalytic dyad (Asp162–Arg164) involved in the activation of the water molecule coordinated to the zinc ion from the enzyme active site is also conserved in SmuCA, as for other b-CAs investigated earlier and shown in the alignment of Figure 1.3–11 We measured the catalytic activity of the new CA by using a stopped-flow CO2 hydrase assay,25 monitoring the physiologic reaction catalyzed by a- (human, hCA I and II) or b-class (PgiCAb, HpyCA, and BsuCA) enzymes. As seen from data of Table 1, SmuCA possess a good catalytic activity for the physiologic reaction that converts the CO2 to bicarbonate and protons, with the following kinetic parameters: kcat of 4.2  105 s1 and kcat/Km of 5.8  107 M1  s1. In fact the catalytic activity of the S. mutans enzyme is slightly better than those of other b-CAs investigated earlier, such as those from P. gingivalis (PgiCAb), H. pylori (HpyCA), or B. suis (BsuCA), recently characterized by this group.3–11 The activity of SmuCA was inhibited by the sulfonamide

SmuCA_beta BsuCA_beta PgiCA_beta FbiCA_beta VraCA_beta

-------------------------------------------------------------------------------------------------------------------------------------------MKKIVLFSAAMAMLIACGNQTTQTKSDTPTAAVEGR--MSAASAFAMNAPSFVNASSLKKASTSARSGVLSARFTCNSSSSSSSSATPPSLIRNEPVF -MSSSSINGWCLSSISPAKTSLKKATLRPSVFATLTT---PSSPSSSSSFPSLIQDKPVF

SmuCA_beta BsuCA_beta PgiCA_beta FbiCA_beta VraCA_beta

------------------------MVMSYFDN---------------------------------------MPMKNDHSPDQRTLSELFEHNRQ------------------------------IGEVLTQDIQQGLTPEA-VLVGLQEGNAR------------------------AAPAPIITPNWTEDGNESYEEAIDALKKTLIEKGELEPVAATRIDQITAQ--AAAPDTKA AAPSHIITPTVREDMAKDYEQAIEELQKLLREKTELKATAAEKVEQITASLGTSSSDSIP : 160 -----------FIKANQAYVDLHGTAHLPL--KPKTRVAIVTCMDSRLHVAPALGLALGD -----------WAAEKQ---EKDPEYFSRLLSSQRPEFLWIGCSDSRVPANVVTGLQPGE -----------YVANKQLPRDLNAQAVAGL-EGQFPEAIILSCIDSRVPVEYIFDKGIGD PFDPVERIKSGFVKFKTEKFVTNPALYDELAKGQSPKFMVFACSDSRVCPSHVLDFQPGE SSEASDRIKSGFLYFKKEKYDKNPALYGELAKGQSPKFMVFACSDSRVCPSHVLDFQPGE : : * . . * ***: . *: 220 223 AHILRNAGGRV---TDDVIR----SLVISEQQLGTSEIVVLHHTDCGAQTFT-------VFVHRNVANLVHRADLNLLS----VLEFAVGVLEIKHIIVCGHYGCGGVRAAMDGYGHGI LFVGRVAGNVV---DDHMLG----SLEYACEVSGSKVLLVLGHEDCGAIKSAIKGVEMGN AFVVRNVANMVPPFDKTKYSGVGAAVEYAVLHLKVQEIFVIGHSRCGGIKGLMTFPDEGP AFVVRNVANIVAPYDQSKYSGTGAAIEYAVLHLKVSNIVVIGHSACGGIKGLLSFPFDGT .: * ... * : : . :.* ***.

SmuCA_beta BsuCA_beta PgiCA_beta FbiCA_beta VraCA_beta SmuCA_beta BsuCA_beta PgiCA_beta FbiCA_beta VraCA_beta SmuCA_beta BsuCA_beta PgiCA_beta FbiCA_beta VraCA_beta

-NAEFTEQLKR-------DLAVDAGDQDFLPF-TDIE-ESVREDIALLKN-SPLIPE---IDNWLQPIRDIAQANQAELDTIENTQDRLD--RLCE-LSVSSQVESLSR-TPVLQSAWK -ITSLMEEIKPSVEATQYTGERTYANKEFAD--AVVK-ENVIQTMDEIRRDSPILKKLEE HSTDFIEDWVKVCLPAKSKVVAEHNGTHLDDQCVLCEKEAVNVSLGNLLT-YPFVRDGLR YSTDFIEEWVKIGLPAKAKVKTQHGDAPFAELCTHCEKEAVNVSLGNLLT-YPFVRDGLV . : : * : : *.: .

SmuCA_beta BsuCA_beta PgiCA_beta FbiCA_beta VraCA_beta : : * *:.

---DIIISGAIYDVDTGRVREVN---------------DGKDIIVHGWMYNLKDGLLRDIGCDCTRNALQFACQPAE EG-KIKICGAIYEMSTGKVHFL----------------NK-TLALKGGHYDFVNGTFELWALDFGLSSPTSV----NK-TLALKGGYYDFVKGTFELWSLNFGLASSFSV----* .

Figure 1. Alignment of the amino acid sequences from selected b-CAs belonging to prokaryotic microorganisms. Pisum sativum numbering system was used. Zinc ligands are indicated in red; amino acids involved in the enzyme catalytic cycle are indicated in blue; amino acids forming the continuous hydrophobic surface in the binding pocket are indicated in bold. Legend: SmuCA_beta, S. mutans; BsuCA, Brucella suis; PgiCA_beta, Porphyromonas gingivalis; FbiCA, Flaveria bidentis; VraCA, Vigna radiata. The asterisk (⁄) indicates identity at a position; the symbol (:) designates conserved substitutions, while (.) indicates semi-conserved substitutions. Multiple alignment was performed with the program Muscle, version 3.7.

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inhibitor acetazolamide, with a KI of 344 nM, in the same range as that of other a- (hCA I) or b-CAs (PgiCAb) (Table 1). A phylogenetic analysis of SmuCA was performed (Fig. 2), comparing its sequence with those of b-CAs described in various other bacteria (see caption to Fig. 2 for the organisms from which these enzymes were isolated). SmuCA clustered on a branch with the bacterial enzymes from P. gingivalis, Myroides injenensis and Cab (from the anaerobic bacterium Methanobacterium thermoautotrophicum).26–29 It is interesting to note that the most similar enzyme to SmuCA was just Cab, the first ever bacterial b-CA investigated in detail,26 whereas the next related enzymes were those from P. gingivalis and M. injenensis. One should also note that the b-CAs from other bacteria (H. pylori, L. pneumophila, E. coli, B. suis) clustered on different branches of this tree, proving that they are evolutionarily more distant from the bacterial enzymes found on the branch to which SmuCA belongs. It is challenging to explain why the CA from an oral cavity pathogen (S. mutans) is more similar to Cab, an enzyme from an anaerobic, methane producing organism.

A library of 40 compounds, comprising 39 sulfonamides and one sulfamate investigated earlier as inhibitors of a-, b-or cclass CAs, were 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,30 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).1 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.1,30 Sulpiride SLP and indisulam IND were also shown by our group to belong to this class of pharmacological agents,30 together with the COX2 ‘selective’ inhibitors celecoxib CLX and valdecoxib VLX. Saccharin and the diuretic hydrochlorothiazide HCT are also known to act as CAIs.30

SO2NH2

SO2NH2

SO2NH2

SO2NH2

SO2NH2

NH2

NH2

1

2

4

3

SO2NH2

SO2NH2

SO2NH2

CH2NH2

CH2CH2NH2

NH2

SO2NH2

F 6

5

7

SO2NH2

SO2NH2

OH Cl

Br

Cl SO2NH2 NH2

10 H3C

N N SO2NH2

HN

N S

SO2NH2

NH2

19

S N H O

O O2N

S N H

O HO

21

SO2NH2

(CH2)nOH

COOH

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

18

SO2NH2

O H2N

N

12

SO2NH2 N

14

H N

SO2NH2 NH2

11

13

N

SO2NH2

CF3

Cl

9

S

8

SO2NH2

NH2

H2N

Cl NH2

N N S

SO2NH2

20 O

SO2NH2 H2N

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

SO2NH2

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H3C

N N CH3CONH

SO2NH2

S

N

CH3CON

N SO2NH2

S

AAZ

EtO

EZA NHEt

NHEt

SO2NH2

Me

Cl

O

DCP

S

SO2NH2

S

N

MeO(CH2)3

O

O DZA

S N H O

S

SO2NH2

O

O NH2 O S O O

O SO2NH2

S

S

BRZ

N N

O

SO2NH2

S

MZA

SO2NH2

Cl

N

SO2NH2

O

O O

BZA

N O

TPM

ZNS

OMe O H N

N H

N

O O S N H

Cl

SO2NH2

SO2NH2 SLP

IND SO2NH2

SO2NH2

H3C

N O N

CH3

N

S

N

SO2NH2

O O SLT

F F F

VLX

CLX

O NH O

S

O

SAC

Data of Table 2 show the SmuCA inhibition data with these sulfonamides/sulfamate, as determined by a stopped-flow CO2 hydrase assay. Inhibition data of the two widely spread human (h) isoforms hCA I and II (belonging to the a-class) as well as the bacterial b-CAs from H. pylori and P. gingivalis are also shown in Table 2, for comparison reasons. The following structure–activity relationship (SAR) can be observed from data of Table 2: (i) A number of compounds, such as 18, 22, BRZ and TPM did not significantly inhibit SmuCA up to concentrations of 50 lM, whereas 10 and 17 were also weakly effective CAIs,

H N HN O

Cl

S O

SO2NH2

HCT

with inhibition constants ranging between 12 and 13.5 lM. It may be observed that these derivatives incorporate rather diverse scaffolds in order to draw a clear-cut SAR conclusion. (ii) The largest majority of the tested compounds showed micromolar inhibitory effects against SmuCA, with KIs in the range of 1.58–4.35 lM. They include sulfonamides 1–8, 11, 12, 14, 15, EZA, DCP, DZA, BZA, ZNS, SLP, CLX, SLT, SAC and HCT (Table 2). Thus, simple benzenesulfonamides incorporating small, compact moieties (amino, aminoalkyl, halogeno, hydroxyalkyl), as well as the heterocyclic mono-

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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), P. gingivalis (PgiCAb) and S. mutans (SmuCA) with sulfonamides 1–24 and the clinically used drugs AAZ–HCT KI* (nM)

Inhibitor/enzyme class a

Figure 2. Phylogenetic tree of the b-CAs from selected prokaryotic and eukaryotic species. The tree was constructed using the program PhyML 3.0. Legend: SmuCA, Streptococcus mutans (Accession number: NP_720781.1); HpyCA, Helicobacter pylori (Accession number: BAF34127.1); PgiCA, Porphyromonas gingivalis (Accession number: YP_001929649.1); MinCA, Myroides injenensis M09-0166 (Accession number: ZP_10784819); Cab, Methanobacterium thermoautotrophicum (Accession number: GI: 13786688); EcoCa, Escherichia coli (Accession number: ACI70660); LpnCA, Legionella pneumophila (Accession number: YP_003619232); DbrCA, Dekkera bruxellensis AWRI1499 (Accession number: EIF49256); BsuCA, Brucella suis (Accession number: NP_699962.1); BthCA, Burkholderia thailandensis (Accession number: ZP_02386321).

or bicyclic ring systems (1,3,4-thiadiazoline present in deacetylated MZA; benzothiazole, present in EZA, etc.; the acylated sulfonamide possessing the SO2NHCO moiety (SAC) are among these derivatives. SAR is not easily understandable, since apparently small structural changes lead to very different inhibition profiles. For example, for the halogenosulfanilamides 7–9, the inhibition worsened from the fluoro (7) to the chloro (8) derivatives, whereas the bromo-substituted compound 9 was the best SmuCA inhibitor detected so far, with a KI of 246 nM (Table 1). The inhibitory power is also quite diverse between CLX and VLX, although the shape of the two molecules is rather similar. This is a clear information that very small structural changes in the molecule of the inhibitor leads to dramatic changes in the affinity for the enzyme, a situation observed for many other CAIs in their interaction with various classes of such enzymes.1–8 (iii) The most effective SmuCA inhibitors were the following sulfonamides: bromosulfanilamide 9, deacetylated acetazolamide 13, 4-hydroxymethylbenzenesulfonamide 16, the pyrimidine-substituted sulfanilamide 19, aminobenzolamide 20 and the structurally similar compounds 21, 23 and 24, as well as AAZ, MZA, IND and VLX. These compounds showed inhibition constants ranging between 246 and 468 nM (Table 2). All these compounds incorporate either benzenesulfonamide or 1,3,4-thiadiazole-2-sulfonamide as zinc coordinating scaffold, and generally either small groups (bromine, hydroxymethyl) or bulkier linear moieties of the sulfanilyl type (present in aminobenzolamide and the structurally similar compounds to it, such as 21, 23 and 24). However, 22, which is also structurally similar to aminobenzolamide and the other derivatives 23 and 24, were not efficient CAIs. (iv) It is obvious from data of Table 2 that SmuCA is not very sensitive to sulfonamide inhibition, compared to other enzymes such as hCA II, HpyCA and even PgiCAb. Among these enzymes, the Porphyromonas one has the most similar inhibition profile with SmuCA, although notable differences between the two are obvious. The Helicobacter enzyme was

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

a

hCA I

hCA II

a

a

HpyCAb b

PgiCAbc b

SmuCAd b

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

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

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

2750 1975 1710 2520 2200 1580 2155 3840 246 12,000 1870 3430 414 2050 468 4355 13500 >50,000 250 455 307 >50,000 438 430 344 445 2430 2700 4315 >50,000 4040 >50,000 3620 2710 412 444 2425 1865 3320 2615

nt = not tested * Errors in the range of 5–10% of the shown data, from 3 different assays.25 a Human recombinant isozymes, stopped flow CO2 hydrase assay method, from Ref. 27. b,c Recombinant bacterial enzyme, stopped flow CO2 hydrase assay method, from Refs. 27,28. d Recombinant bacterial enzyme, this work.

much more sensitive to this class of inhibitors, with many compounds showing KIs < 100 nM (situation not observed for SmuCA or PgiCAb). SmuCA is an enzyme which probably has to work in the rather acidic environment within the oral cavity generated during the initial phases of digestion, and this fact makes it rather similar to hCA VI, a human a-CA isoform secreted into saliva, which is on the other hand inhibited in the nanomolar range31 by many sulfonamides as those investigated here. Recently, Parkkila’s group32 reported that CA VI-deficient mice have a much lower propensity to develop caries compared to the normal animals possessing CA VI in their saliva, which represents a strong indication in favor of our proposal of saliva CA VI inhibition as an anticariogenic strategy.31 In addition, it has been shown that oral colonization by S. mutans and caries development is reduced upon deletion of CA VI expression in saliva.32b This phenomenon may be probably also achieved by using strong sulfonamide CAIs identified by our

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group, which should thus inhibit both the human (CA VI) and bacterial enzymes (SmuCA) present in saliva, representing thus a new strategy for the control of caries formation. In conclusion, we report here the cloning, purification and characterization of the S. mutans b-class enzyme SmuCA, and its inhibition profile with sulfonamides. The enzyme showed a significant catalytic activity compared to other bacterial b-CAs for the hydration of CO2 to bicarbonate and protons, with a kcat 4.2  105 s1 and a kcat/Km of 5.8  107 M1  s1. SmuCA was inhibited efficiently by most sulfonamides (KIs of 246 nM– 13.5 lM). The best SmuCA inhibitors were bromosulfanilamide, 5-amino-1,3,4-thiadiazole-2-sulfonamide, 4-hydroxymethyl-benzenesulfonamide, a pyrimidine-substituted sulfanilamide, aminobenzolamide and compounds structurally similar to it, as well as acetazolamide, methazolamide, indisulam and valdecoxib. These sulfonamides showed inhibition constants ranging between 246 and 468 nM. Identification of effective inhibitors of this new enzyme may lead to pharmacological tools useful for understanding the role of S. mutans CAs in dental caries formation, and eventually the development of pharmacological agents with a new mechanism of antibacterial action.

11.

12.

13.

14. 15.

16.

References and notes 1. (a) Alterio, V.; Di Fiore, A.; D’Ambrosio, K.; Supuran, C. T.; De Simone, G. Chem. Rev. 2012, 112, 4421; (b) Supuran, C. T. Nat. Rev. Drug Disc. 2008, 7, 168; (c) Harju, A. K.; Bootorabi, F.; Kuuslahti, M.; Supuran, C. T.; Parkkila, S. J. Enzyme Inhib. Med. Chem. 2013, 28, 231; (d) Neri, D.; Supuran, C. T. Nat. Rev. Drug Disc. 2011, 10, 767; (e) Supuran, C. T.; Vullo, D.; Manole, G.; Casini, A.; Scozzafava, A. Curr. Med. Chem.-Cardiovasc. Hematol. Agents 2004, 2, 49. 2. (a) Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 759; (b) Supuran, C. T. Curr. Pharm. Des. 2008, 14, 603; (c) Supuran, C. T. Curr. Pharm. Des. 2008, 14, 601; (d) Supuran, C. T. Med. Chem. 2011, 3, 1165. 3. (a) Supuran, C. T. Front. Pharmacol. 2011, 2, 34; (b) Supuran, C. T. Bioorg. Med. Chem. 2013, 21, 1377; (c) Capasso, C.; Supuran, C. T. Expert Opin. Ther. Pat. 2013, 23, 693. 4. (a) Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 229; (b) Fabrizi, F.; Mincione, F.; Somma, T.; Scozzafava, G.; Galassi, F.; Masini, E.; Impagnatiello, F.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 138; (c) Ekinci, D.; Karagoz, L.; Ekinci, D.; Senturk, M.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 283. 5. (a) Nishimori, I.; Vullo, D.; Minakuchi, T.; Scozzafava, A.; Osman, S. M.; AlOthman, Z.; Capasso, C.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2014, 24, 1127; (b) Nishimori, I.; Vullo, D.; Minakuchi, T.; Scozzafava, A.; Capasso, C.; Supuran, C. T. Bioorg. Med. Chem. 2014, 22, 2939. ˇ , F.; Remko, M. J. Enzyme Inhib. Med. 6. (a) Supuran, C. T.; Maresca, A.; Gregán Chem. 2013, 28, 289; (b) Alp, C.; Maresca, A.; Alp, N. A.; Gültekin, M. S.; Ekinci, D.; Scozzafava, A.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 294; (c) Carta, F.; Scozzafava, A.; Supuran, C. T. Expert Opin. Ther. Pat. 2012, 22, 747; (d) Supuran, C. T. Expert Opin. Ther. Pat. 2013, 23, 677; (e) Wilkinson, B. L.; Bornaghi, L. F.; Houston, T. A.; Innocenti, A.; Vullo, D.; Supuran, C. T.; Poulsen, S. A. J. Med. Chem. 2007, 50, 1651; (f) Chohan, Z. H.; Scozzafava, A.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2002, 17, 261. 7. (a) Migliardini, F.; De Luca, V.; Carginale, V.; Rossi, M.; Corbo, P.; Supuran, C. T.; Capasso, C. J. Enzyme Inhib. Med. Chem. 2014, 29, 146; (b) Bonneau, A.; Maresca, A.; Winum, J. Y.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 397; (c) Supuran, C. T.; Carta, F.; Scozzafava, A. Expert Opin. Ther. Pat. 2013, 23, 777; (d) McDonald, P. C.; Winum, J. Y.; Supuran, C. T.; Dedhar, S. Oncotarget 2012, 3, 84; (e) Scozzafava, A.; Menabuoni, L.; Mincione, F.; Mincione, G.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2001, 11, 575. 8. (a) Buchieri, M. V.; Riafrecha, L. E.; Rodriguez, O. M.; Vullo, D.; Morbidoni, H. R.; Supuran, C. T.; Colinas, P. A. Bioorg. Med. Chem. Lett. 2013, 23, 740; (b) Vullo, D.; De Luca, V.; Scozzafava, A.; Carginale, V.; Rossi, M.; Supuran, C. T.; Capasso, C. Bioorg. Med. Chem. Lett. 2012, 22, 6324; (c) Del Prete, S.; De Luca, V.; Scozzafava, A.; Carginale, V.; Supuran, C. T.; Capasso, C. J. Enzyme Inhib. Med. Chem. 2014, 29, 23; (d) Vullo, D.; Isik, S.; Del Prete, S.; De Luca, V.; Carginale, V.; Scozzafava, A.; Supuran, C. T.; Capasso, C. Bioorg. Med Chem. Lett. 2013, 23, 1636. 9. (a) Del Prete, S.; Isik, S.; Vullo, D.; De Luca, V.; Carginale, V.; Scozzafava, A.; Supuran, C. T.; Capasso, C. J. Med. Chem. 2012, 55, 10742; (b) Supuran, C. T. Curr. Med. Chem. 2012, 19, 831; (c) Maresca, A.; Scozzafava, A.; Kohler, S.; Winum, J. Y.; Supuran, C. T. J. Inorg. Biochem. 2012, 110, 36; (d) Vullo, D.; Nishimori, I.; Minakuchi, T.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2011, 21, 3591; (e) Nishimori, I.; Minakuchi, T.; Morimoto, K.; Sano, S.; Onishi, S.; Takeuchi, H.; Vullo, D.; Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2006, 49, 2117. 10. (a) Nishimori, I.; Minakuchi, T.; Vullo, D.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. 2011, 19, 5023; (b) Davis, R. A.; Hofmann, A.; Osman, A.; Hall, R. A.; Muhlschlegel, F. A.; Vullo, D.; Innocenti, A.; Supuran, C. T.; Poulsen, S. A. J. Med. Chem. 2011, 54, 1682; (c) Guzel, O.; Maresca, A.; Scozzafava, A.; Salman, A.;

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Balaban, A. T.; Supuran, C. T. J. Med. Chem. 2009, 52, 4063; (d) Vullo, D.; Del Prete, S.; Osman, S. M.; De Luca, V.; Scozzafava, A.; Alothman, Z.; Supuran, C. T.; Capasso, C. Bioorg. Med. Chem. Lett. 2014, 24, 240; (e) Nishimori, I.; Minakuchi, T.; Onishi, S.; Vullo, D.; Cecchi, A.; Scozzafava, A.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2009, 24, 70. (a) Supuran, C. T.; Mincione, F.; Scozzafava, A.; Briganti, F.; Mincione, G.; Ilies, M. A. Eur. J. Med. Chem. 1998, 33, 247; (b) Innocenti, A.; Vullo, D.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2008, 18, 1583; (c) Vullo, D.; Kupriyanova, E. V.; Scozzafava, A.; Capasso, C.; Supuran, C. T. Bioorg. Med. Chem. 2014, 22, 1667; (d) Supuran, C. T.; Ilies, M. A.; Scozzafava, A. Eur. J. Med. Chem. 1998, 33, 739; (e) Thiry, A.; Dognè, J. M.; Masereel, B.; Supuran, C. T. Curr. Top. Med. Chem. 2007, 7, 855. (a) Simón-Soro, A.; Mira, A. Trends Microbiol. 2015, 23, 76; (b) Ajdic, D.; McShan, W. M.; McLaughlin, R. E.; Savic´, G.; Chang, J.; Carson, M. B.; Primeaux, C.; Tian, R.; Kenton, S.; Jia, H.; Lin, S.; Qian, Y.; Li, S.; Zhu, H.; Najar, F.; Lai, H.; White, J.; Roe, B. A.; Ferretti, J. J. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 14434; (c) Ajdic´, D.; Pham, V. T. J. Bacteriol. 2007, 189, 5049. (a) Herzberg, M. C. In Streptococcal Infections; Stevens, D. L., Kaplan, E. L., Eds.; Oxford Univ. Press: New York, 2000; pp 333–370; (b) Kazuhiko, N.; Ryota, N.; Takashi, O. Jpn. Dent. Sci. Rev. 2008, 44, 29; (c) Loesche, W. J. Microbiol. Rev. 1986, 50, 353; (d) Eckert, R.; Sullivan, R.; Shi, W. Adv. Dent. Res. 2012, 24, 94. Dhamodhar, P.; Murthy, S.; Channarayappa, R. K.; Gopinath, N.; Kumar, S. S.; Varuvelil, G. J. J. Biochem. Technol. 2012, 3, 155. (a)Novel Antimicrobial Agents and Strategies; Phoenix, D. A., Harris, F., Dennison, S. R., Eds.; Wiley VCH: Weinheim, 2015. pp 1–413; (b) Capasso, C.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2014, 29, 379; (c) Capasso, C.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2014, 29, 693; (d) Bush, K.; Macielag, M. J. Expert Opin. Ther. Pat. 2010, 20, 1277. (a) Capasso, C.; Supuran, C. T. Developing Novel Bacterial Targets: Carbonic Anhydrases as Antibacterial Drug Targets. In Novel Antimicrobial Agents and Strategies, Phoenix, D. A.; Harris, F.; Dennison, S. R. Eds.; Weinheim, 2015, pp 31–45; (b) Capasso, C.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2015, 30, 325; (c) Furtado, G. H.; Nicolau, D. P. Expert Opin. Ther. Pat. 2010, 20, 1273. (a) Vullo, D.; Flemetakis, E.; Scozzafava, A.; Capasso, C.; Supuran, C. T. J. Inorg. Biochem. 2014, 136, 67; (b) Vullo, D.; Del Prete, S.; Osman, S. M.; De Luca, V.; Scozzafava, A.; Alothman, Z.; Supuran, C. T.; Capasso, C. Bioorg. Med. Chem. Lett. 2014, 24, 275. (a) Del Prete, S.; Vullo, D.; Scozzafava, A.; Capasso, C.; Supuran, C. T. Bioorg. Med. Chem. 2014, 22, 531; (b) Maresca, A.; Vullo, D.; Scozzafava, A.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 388; (c) Alp, C.; Özsoy, S.; Alp, N. A.; _ S Erdem, D.; Gültekin, M. S.; Küfreviog˘lu, Ö. I.; ß entürk, M.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 818. Alafeefy, A. M.; Abdel-Aziz, H. A.; Vullo, D.; Al-Tamimi, A. M.; Al-Jaber, N. A.; Capasso, C.; Supuran, C. T. Bioorg. Med. Chem. 2014, 22, 141. (a) Vullo, D.; Luca, V. D.; Scozzafava, A.; Carginale, V.; Rossi, M.; Supuran, C. T.; Capasso, C. Bioorg. Med. Chem. 2013, 21, 1534; (b) Vullo, D.; Leewattanapasuk, W.; Muhlschlegel, F. A.; Mastrolorenzo, A.; Capasso, C.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2013, 23, 2647. (a) Kazancıog˘lu, E. A.; Güney, M.; S ß entürk, M.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 880; (b) Liu, F.; Martin-Mingot, A.; Lecornué, F.; Maresca, A.; Thibaudeau, S.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 886; (c) Maresca, A.; Scozzafava, A.; Vullo, D.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 384; (d) Koz, O.; Ekinci, D.; Perrone, A.; Piacente, S.; Alankus-Caliskan, O.; Bedir, E.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 412; (e) Winum, J. Y.; Kohler, S.; Supuran, C. T. Curr. Pharm. Des. 2010, 16, 3310. (a) Vullo, D.; De Luca, V.; Scozzafava, A.; Carginale, V.; Rossi, M.; Supuran, C. T.; Capasso, C. Bioorg. Med. Chem. Lett. 2012, 22, 7142; (b) De Luca, V.; Vullo, D.; Scozzafava, A.; Carginale, V.; Rossi, M.; Supuran, C. T.; Capasso, C. Bioorg. Med. Chem. Lett. 2012, 22, 5630; (c) Maresca, A.; Carta, F.; Vullo, D.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 407; (d) Monti, S. M.; De Simone, G.; Dathan, N. A.; Ludwig, M.; Vullo, D.; Scozzafava, A.; Capasso, C.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2013, 23, 1626; (e) Del Prete, S.; Vullo, D.; De Luca, V.; Carginale, V.; Scozzafava, A.; Supuran, C. T.; Capasso, C. Bioorg. Med. Chem. Lett. 2013, 23, 4067; (f) Burghout, P.; Vullo, D.; Scozzafava, A.; Hermans, P. W. M.; Supuran, C. T. Bioorg. Med. Chem. 2011, 19, 243. De Simone, G.; Supuran, C. T. J. Inorg. Biochem. 2012, 111, 117. Genomic DNA of Streptococcus mutans strain UA159 was kindly provided by Prof. Georg Conrads, RWTH Aachen University Hospital, Aachen, Germany. The b-CA-encoding gene SMU_328 (accession number NP_720781) encodes a protein of 166 amino acid residues, and has a molecular weight (as a monomer) of 18096 Da. The full length gene was identified and amplified by PCR from genomic DNA of S. mutans strain UA159. Addition of KpnI recognition sequence (underlined in the following sequence) resulted in the forward primer sequence: 50 -GGT ACCATGGTAATGTCTTATTTTGAT-30 . The reverse primer sequence including the BamHI recognition site (underlined in the following sequence) was 50 -GGATCCTTAATTGACTTCTCTTACTCG-30 . PCR conditions were: 94 °C for 5 min, 35 cycles of 94 °C for 45 s, 58 °C for 45 s and 72 °C for 1 min and a final step of 72 °C for 5 min. The PCR product was run on an agarose gel, and the obtained band was purified using a Thermo Scientific GeneJET Gel Extraction Kit. The amplified band containing SMU_328 ORF was inserted into the pGEM-T Easy (PROMEG (A) vector with T:A strategy.1 The ligated product was transformed into DH10B™ competent cells. Overnight cultures (10 mL) were made from these colonies, and plasmids were purified using a QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany). In order to confirm the integrity of the SMU_328 gene and the fact that no errors occurred at the ligation sites, the vector containing the fragment (pGEM-T

N. Dedeoglu et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2291–2297 Easy/SMU_328) was sequenced. Purified plasmids, including SMU_328 gene, and pET30a(+) were digested at 37 °C overnight with KpnI and BamHI restriction enzymes (Fermentas). Digested SMU_328 gene and pET30a(+) vector were separated on 0.8% agarose gel. The recovered SMU_328 gene and the linearized expression vector (pET30a(+)) were ligated by T4 DNA ligase to form the expression vector pET30a(+)/SMU_328. The vectors were transformed into E. coli Arctic cells, grown at 4 °C and induced with 1 mM IPTG. Arctic Express competent cells have been engineered for improved protein processing at low temperatures, which represents one strategy for increasing the recovery of soluble protein. After additional growth for 6 h, the cells were harvested and resuspended in the following buffer: 10 mM Tris HCl pH 8.3. The cells were then disrupted by sonication at 4 °C. Following centrifugation, the supernatant was loaded into His-select HF Nickel affinity gel, eluted with 250 mM imidazole and dialyzed against 10 mM Tris HCl pH 8.3 buffer. At this stage of purification the enzyme was at least 90% pure and the obtained recovery was of 2.8 mg of the recombinant protein as shown by SDS–Page (data not shown). 25. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561. An Applied Photophysics stoppedflow instrument has been used for assaying the CA catalyzed CO2 hydration activity. 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/Tris (pH 7.4–8.3) as buffer, and 20 mM NaClO4 (for maintaining constant the ionic strength), following the initial rates of the CA-catalyzed CO2 hydration reaction for a period of 10–100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor at least six traces of the initial 5–10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled–deionized water and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay, in order to allow for the formation of the E–I complex. The inhibition constants were obtained by non-linear least-squares methods using

26.

27. 28.

29.

30.

31. 32.

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PRISM 3 and the Cheng–Prusoff equation, as reported earlier,26–29 and represent the mean from at least three different determinations. All CA isoforms were recombinant ones obtained in-house and the sulfonamides were from Sigma–Aldrich (Milan, Italy) highest purity available reagents or were prepared as reported earlier by us.30. (a) Zimmerman, S.; Innocenti, A.; Casini, A.; Ferry, J. G.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2004, 14, 6001; (b) Innocenti, A.; Zimmerman, S.; Ferry, J. G.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2004, 14, 4563; (c) Zimmerman, S. A.; Ferry, J. G.; Supuran, C. T. Curr. Top. Med. Chem. 2007, 7, 901. Vullo, D.; Del Prete, S.; Osman, S. M.; Scozzafava, A.; AlOthman, Z.; Supuran, C. T.; Capasso, C. Bioorg. Med. Chem. Lett. 2014, 24, 4402. (a) Nishimori, I.; Innocenti, A.; Vullo, D.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2007, 17, 1037; (b) Maresca, A.; Vullo, D.; Scozzafava, A.; Supuran, C. T J. Enzyme Inhib Med. Chem. 2013, 28, 388; (c) Çavdar, H.; Ekinci, D.; Talaz, O.; Saraçog˘lu, N.; Sßentürk, M.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 148; (d) Chohan, Z. H.; Scozzafava, A.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2003, 18, 259. (a) Innocenti, A.; Muhlschlegel, F. A.; Hall, R. A.; Steegborn, C.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2008, 18, 5066; (b) Del Prete, S.; De Luca, V.; Vullo, D.; Scozzafava, A.; Carginale, V.; Supuran, C. T.; Capasso, C. J Enzyme Inhib Med Chem 2014, 29, 23. (a) Supuran, C. T.; Scozzafava, A. Bioorg. Med. Chem. 2007, 15, 4336; (b) Nishimori, I.; Onishi, S.; Takeuchi, H.; Supuran, C. T. Curr. Pharm. Des. 2008, 14, 622; (c) McKenna, R.; Supuran, C. T. Subcell. Biochem. 2014, 75, 291. Nishimori, I.; Minakuchi, T.; Onishi, S.; Vullo, D.; Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2007, 50, 381. (a) Aidar, M.; Marques, R.; Valjakka, J.; Mononen, N.; Lehtimäki, T.; Parkkila, S.; de Souza, A. P.; Line, S. R. Caries Res. 2013, 47, 414; (b) Culp, D. J.; Robinson, B.; Parkkila, S.; Pan, P. W.; Cash, M. N.; Truong, H. N.; Hussey, T. W.; Gullett, S. L. Biochim. Biophys. Acta 2011, 1812, 1567.