Bioorganic & Medicinal Chemistry 22 (2014) 2939–2946

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Sulfonamide inhibition studies of two b-carbonic anhydrases from the bacterial pathogen Legionella pneumophila Isao Nishimori a, Daniela Vullo b, Tomoko Minakuchi a, Andrea Scozzafava b, Clemente Capasso c, Claudiu T. Supuran b,d,⇑ a

Department of Gastroenterology, Kochi Medical School, Kochi, Japan Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy Istituto di Biochimica delle Proteine—CNR, Via P. Castellino 111, 80131 Napoli, Italy d Università degli Studi di Firenze, Polo Scientifico, Dipartimento di Scienze Farmaceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino (Florence), Italy b c

a r t i c l e

i n f o

Article history: Received 21 February 2014 Accepted 6 April 2014 Available online 13 April 2014 Keywords: Carbonic anhydrase Sulfonamide Legionella pneumophila Antibacterial agent

a b s t r a c t Two b-carbonic anhydrases (CAs, EC 4.2.1.1) were identified, cloned and purified in the pathogenic bacterium Legionella pneumophila, denominated LpCA1 and LpCA2. They efficiently catalyze CO2 hydration to bicarbonate and protons, with kcat in the range of (3.4–8.3)  105 s1 and kcat/Km of (4.7– 8.5)  107 M1 s1, and are inhibited by sulfonamides and sulfamates. The best LpCA1 inhibitors were aminobenzolamide and structurally similar sulfonylated aromatic sulfonamides, as well as acetazolamide and ethoxzolamide(KIs in the range of 40.3–90.5 nM). The best LpCA2 inhibitors belonged to the same class of sulfonylated sulfonamides, together with acetazolamide, methazolamide and dichlorophenamide (KIs in the range of 25.2–88.5 nM). As these enzymes may be involved in pH regulation in the phagosome during Legionella infection, their inhibition may lead to antibacterials with a novel mechanism of action. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Legionella pneumophila is a Gram-negative bacterium causing Legionnaires’ disease or legionellosis, an often fatal pneumonia (mortality rate of 20–50%), which has been observed for the first time in July 1976, among the attendees of the 58th Annual Convention of the American Legion in Philadelphia.1–4 There are many species of this bacterium, but only L. pneumophila and to a lower extent Legionella longbeachae provoke disease in humans (the last species is prevalent in Australia and New Zealand, whereas the first one in the other continents).1 Legionellae are environmental bacteria and their natural host is the amoeba in which they replicate, but by accidentally infecting human macrophages they cause oportunistic infections.4–6 The spread of legionellosis was favored ultimately by the development of artificial water systems for air conditioning, cooling towers, aerosolizing devices, etc.5 It seems that many of the biochemical pathways used by Legionella to infect Amoeba are also used when infecting human macrophages, suggesting thus a co-evolution of these organisms.4 Among such mechanisms are for example the formation of the so-called Legionella-containing vacuoles (LCVs) within the host cytoplasm, which requires the remodeling of the LCV surface and ⇑ 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.bmc.2014.04.006 0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

the hijacking of vesicles and organelles for effective infection.4 These processes are highly influenced by the pH of these orgnelles. This pathogen, similar to bacteria, fungi or protozoa, evolved mechanisms of interfering with the pH.7–9 Both macrophages and amoebae use phagosomes to destroy foreign particles/pathogens, in which a highly acidic pH is crucial for the outcome of the process.5,10 Intracellular pathogens, such as L. pneumophila thus developed sophisticated strategies to evade their destruction by the acidic phagolysosomes.5 Indeed, it has been demonstrated that L. pneumophila is able to maintain a neutral pH in its phagosome for at least 6 h, whereas vacuoles which did not contain the bacterium became highly acidic in 15 min after their formation.10 One of the proteins involved in this process is a vacuolar V-ATPase,5 but as for other organisms7–9 (or even as for the human hypoxic tumor cells)11 investigated earlier, the pH regulation is a complex process in which many other proteins are involved. In fungal,8 bacterial,7,9 and protozoan9c pathogens, as well as in hypoxic tumor cells,11 it has been proven that one or more enzymes belonging to the CA family are also involved in the pH regulation processes, and that interference with these enzymes constitutes a new strategy to treat infections12 or tumors.11,13 CAs catalyze the simple but crucial reaction between CO2 and water leading to the formation of protons and bicarbonate,11–14 and these enzymes are widespread in all organisms, from very simple to complex ones on the phylogenetic tree of life. Furthermore,

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five distinct genetic families, the a-, b-, c-, d- and f-CAs are known to date, which constitute an interesting example of convergent evolution at the molecular level.11–14 Considering our interest in the cloning and characterization of CAs from pathogenic organisms,7 we investigated whether in the genome of L. pneumophila6 there are putative such enzymes. We found out that two b-CAs are present, with the following accession numbers: NC_002942, which we have designated as LpCA1 (locus tag lpg2500, NCBI reference sequence WP_014844650.1), and LpCA2 (locus tag lpg2194; NCBI reference sequence WP_014842179.1). In a recent brief communication we reported that these two enzymes are catalytically active and that they are inhibited by some inorganic anions.4b Here we describe in detail the cloning, characterization and sulfonamide inhibition studies of these two enzymes, LpCA1 and LpCA2, which we propose as possible new targets for anti-legionella drugs. Indeed, although this bacterium generally responds well to the antibiotics in clinical use, cases of drug resistance have also been reported,15 which makes the search of new targets of great interest. 2. Results and discussion 2.1. Cloning and purification of LpCA1 and LpCA2 The genomic DNA from Legionella pneumophilia subsp. pneumophila strain Philadelphia-1 has been used for cloning of the CAs reported in this paper. As mentioned above, inspection of the genome of this pathogen revealed the presence of two putative such enzymes belonging to the b-class, denominated LpCA1 and LpCA2. They have have been cloned as glutathione S-transferase (GST)fusion proteins as described earlier by our groups for other bacterial/mammalian a- and b-CAs.9,16 LpCA1 contains 245 amino acid residues and has a calculated molecular weight of 26.994 kDa, whereas LpCA2 is shorter, comprising 208 amino acid residues, with a theoretical molecular weight of 22.997 kDa. GST-LpCA1 and -2 fusion proteins have been obtained as described in the experimental protocols, and the recombinant proteins were induced by addition of IPTG. Glutathione Sepharose 4B columns were used for the first step purification of the two fusion proteins. After the cleavage of the GST part of the fusion protein with thrombin, as reported earlier for other bacterial/mammalian CAs,9,16 the obtained LpCA1 and -2 recombinant enzymes were further purified by sulfonamide affinity chromatography leading to pure LpCA1 and LpCA2 with the subunit molecular weights of 27 and 23 kDa, respectively (Supplementary Fig. 1). 2.2. Catalytic activity, amino acid sequences and phylogeny of LpCA1 and LpCA2 We measured the CO2 hydrase activity of the two new enzymes, LpCA1 and LpCA2 by a stopped-flow assay.17 As seen from data of Table 1, where the activity of LpCA1 and LpCA2 was compared to that of other a- and b-CAs from human (h),12 fungal (Cryptococcus neoformans,8a,b Candida albicans,16a Saccharomyces cerevisiae),16b plant (Flaveria bidentis),16c and bacterial (Helicobacter pylori16d and Brucella suis9b) sources, both these new enzymes possess a significant catalytic activity for the physiologic reaction catalyzed by CAs, that is, hydration of carbon dioxide with formation of protons and bicarbonate. The first isoform, LpCA1 showed a moderate degree of activity with a kcat of 3.4  105 s1 and kcat/Km of 4.7  107 M1  s1. This activity is in fact comparable to that of other a- and b-CAs, such as hCA I (a physiologically relevant human isoform),12 Can2 from the fungal pathogen C. neoformans,8 and the bacterial pathogenic enzymes HpyCA16d and BsuCA219,9b from H. pylori and B. suis, respectively. The second isoform, LpCA2,

was on the other hand more active than LpCA1, with the folllowing kinetic parameters: kcat of 8.3  105 s1 and kcat/Km of 8.5  107 M1  s1 (Table 1). This is a level of activity similar to that of the C. albicans and S. cerevisiae enzymes or the BsuCA213 isoform (all belonging to the b-CA family) and is only slightly lower compared to hCA II, a highly effective catalyst for the CO2 hydration reaction (Table 1).12 It may also be observed that acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide), a clinically used drug,6 inhibited efficiently the two Legionella enzymes, with KIs in the range of 36–95 nM. The moderately active enzyme (LpCA1) was less sensitive to this compound compared to LpCA2 which showed a higher catalytic activity and also higher affinity for the sulfonamide inhibitor (Table 1). In order to explain the efficient catalytic properties of the two new enzymes, we have aligned the amino acid sequences of LpCA1 and LpCA2 with those of other such enzymes characterized earlier (kinetically, and in some cases also by means of X-ray crystallography), such as HpyCA,16d BsuCA213,9a BsuCA2199a and stCA1 and stCA2 from the bacterial pathogen Salmonella typhimurium18 (Fig. 1). Data of Figure 1 show that as all other bacterial b-CAs investigated so far, the two Legionella proteins LpCA1 and LpCA2 possess the amino acid residues crucial in the catalytic cycle of CO2 conversion to bicarbonate and protons: (i) the zinc coordinating amino acids, constituted by two Cys and one His residues, more precisely Cys90, His143 and Cys146 (LpCA1 numbering system); and (ii) the catalytic dyad constituted by residues Asp92 and Arg94, which is involved in the activation of the zinc-coordinated water molecule which leads to the formation of the nucleophilic, zinc-hydroxide species of the enzyme.7–9,16,18 We have also examined the phylogenetic relationship of the two new bacterial b-CAs, LpCA1 and LpCA2, with that of other such enzymes characterized earlier from various bacterial and fungal pathogens, as well as algae, plants and Archaea (see Table 2 for the organisms included in the phylogenetic analysis). As seen from the dendrogram of Figure 2, the two Legionella enzymes are highly similar, clustering on their own branch at the upper part of the tree, with the next most similar enzyme being MinCA from the recently identified bacterial pathogen resistant to all known antibiotics Myroides injenesis19 (this b-CA was not yet cloned and characterized). On the other hand the other evolutionarily most related enzyme to LpCA1 and LpCA2 is AbaCA, from Acinetobacter baumanii another not yet cloned or characterized bCA. The remaining b-CAs from Figure 2 were less related to LpCA1 and LpCA2 compared to the previously discussed ones, and clustered all together on the lower branches of the tree. This includes two main branches, one constituted only by the archaeal enzyme cab (from Methanobacterium thermoautotrophicum),20 whereas the remaining, rather ramificated branch includes yeast/fungal (SceCA, DbrCA and SpoCA), bacterial (HpyCA, BsuCA), algal (CspCA and CreCA) and plant enzymes (VraCA, FbiCA, ZmaCA and AthCA), which are all more distantly related to the bacterial CAs from Legionella examined here (Fig. 2). 2.3. Sulfonamide/sulfamate inhibition of LpCA1 and LpCA2 Sulfonamides and their isosteres (sulfamates, sulfamides, etc.) constitute the main and most investigated family of CA inhibitors (CAIs), with several drugs used clinically for the last 60 years.11,12,21 Among them are first generation compounds such as acetazolamide AAZ, methazolamide MZA, ethoxzolamide EZA and dichlorophenamide DCP.12,14,21 Dorzolamide DZA and brinzolamide BRZ are topically-acting antiglaucoma agents belonging to the second generation of such drugs, whereas benzolamide BZA is an orphan drug belonging to this class of pharmacological agents.12

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Table 1 Kinetic parameters for the CO2 hydration reaction catalysed by the human cytosolic isozymes hCA I and II (a-class CAs) at 20 °C and pH 7.5 in 10 mM HEPES buffer and 20 mM Na2SO4, and the b-CAs Can2, CalCA (from C. neoformans and C. albicans, respectively), SceCA (from S. cerevisiae), the Flaveria bidentis CA (FbiCA 1), H. pylori b-CA (HpyCA), B. suis (BsuCA219 and BsuCA213) and the two L. pneumophila enzymes LpCA1 and LpCA2, measured at 20 °C, pH 8.3 in 20 mM TRIS buffer and 20 mM NaClO4,17 by a stopped flow CO2 hydrase assay method17 Isozyme

Activity level

Class

kcat (s1)

kcat/Km (M1  s1)

KI (acetazolamide) (nM)

Ref.

hCA I hCA II Can2 CalCA SceCA FbiCA 1 HpyCA BsuCA219 BsuCA213 LpCA1 LpCA2

Moderate Very high Moderate High High Low Moderate Moderate High Moderate High

a a

2.0  105 1.4  106 3.9  105 8.0  105 9.4  105 1.2  105 7.1  105 6.4  105 1.1  106 3.4  105 8.3  105

5.0  107 1.5  108 4.3  107 9.7  107 9.8  107 7.5  106 4.8  107 3.9  107 8.9  107 4.7  107 8.5  107

250 12 10.5 132 82 27 40 63 303 76.8 72.1

12 12 8b 16a 16b 16c 16d 9b 9b This work This work

b b b b b b b b b

Inhibition data with the clinically used sulfonamide acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide) are also provided.

LpCA1 LpCA2 HpyCA BsuCA213 BsuCA219 stCA1 stCA2

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

LpCA1 LpCA2 HpyCA BsuCA213 BsuCA219 stCA1 stCA2

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

LpCA1 LpCA2 HpyCA BsuCA213 BsuCA219 stCA1 stCA2

AGNVLNDD--------ILGSMEFATKVVGARLVVVLAHTSCGA------VAGACKDVKLG AGNILNDD--------ILGSIEFACQVVGVKLIAVVGHTQCGA------IKGACDGVKLG MGNVIPPKTSHKESLSTMASIEYAIVHVGVQNLIICGHSDCGACGSTHLINDGXTKAKTP VANLIPPYEPDGEYHAASAALEFAVQSLKVKHIVVMGHGRCGG------IKAALDTESAP VANLVHRADLN-----LLSVLEFAVGVLEIKHIIVCGHYGCGG------VRAAMDGYGHG VANLVIHTDLN-----CLSVVQYAVDVLEVEHIIICGHSGCGG------IKAAVENPELG AGNISNRD--------MLGSMEFACAVAGAKVVLVIGHTRCGA------VRCAIDNAELG .*: . :::* : : .* **. : .

LpCA1 LpCA2 HpyCA BsuCA213 BsuCA219 stCA1 stCA2

H-----LTDVINKIHPVVKPSMESTGIDNCSDPKLIDDMAKANALHVVKNILEQSPILNE N-----LTNLLNKINPVIQEAKKLDAKHDVHSPEFLNCVTSLNVKHTMNEITQRSDIVHQ Y-----IADWIQFLEPIKEEL-KNHPQFSNHFAKRSWLTERLNVRLQLNNLLSYDFIQER LSPSDFIGKWMSLISPAAEAI---SGNALMTQSERHTALERISIRYSLANLRTFPCV-DI I-----IDNWLQPIRDIAQAN-QAELDTIENTQDRLDRLCELGVSSQVESLSRTPVLQSA L-----INNWLLHIRDIWLKH--SSLLGKMPEEQRLDALYELNVMEQVYNLGHSTIMQSA N-----LTGLLDEIKPAIAKT-EYSGERKGSNYDFVDAVARKNVELTIENIRKNSPVLKQ : : : . . : .: :

LpCA1 LpCA2 HpyCA BsuCA213 BsuCA219 stCA1 stCA2

LVKNKQIGIVAGIHDIKTGKVTFFEEKRSVPE--------------------LLNEKRIAIAGGLYQLETGEVQFFDE--------------------------VVNN-ELKIFGWHYIIETGRIYNYNFESHFFEPIXETXKQRKSHENF-----LEKKGKLTLHGAWFDISTGELWVMDHRTGD----------------FK-RPEL WKDGKDIIVHGWMYNLKDGLLRDIGCDCTR------------NALQFACQPAE WKRGQNVTIHGWAYSINDGLLRDLDVTATNRETLENGYHKGISALSLKYIPHQ LEDEKKIKIVGSMYHLTGGKVEFFEV--------------------------: : . . : * :

90

143 146

Figure 1. Amino acid sequences alignment of selected b-CAs from four bacterial species. LpCA1 numbering system was used. Amino acid residues participating in the coordination of metal ion are indicated in blue, whereas the catalytic dyad involved in the activation of the metal ion coordinated water molecule (Asp92–Arg94) is shown in red. 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. Organisms, NCBI sequence numbers and cryptonyms are indicated in Table 2.

Topiramate TPM (a sulfamate) and zonisamide ZNS (an aliphatic sulfonamide) are widely used antiepileptic drugs.12,14 Other compounds incorporating primary/secondary sulfamoyl moieties such as sulpiride SLP and indisulam IND were also shown to belong to this class of pharmacological agents together with the COX2 selective inhibitors celecoxib CLX and valdecoxib VLX, the antiepileptic sulthiame SLT, the sweetener saccharin SAC and hydrochlorothia-

zide HCT, a diuretic agent.12,14,21 Compound 1–24 used in the assay as well as the clinically used drugs mention3d above were commercially available, or prepared as reported earlier by our group.22 In this way it is possible to explore a wider chemical space for detecting effective inhibitors of the new enzymes reported here, considering the fact that the 40 CAIs tested in our study possess a range of scaffolds and various substitution patterns (Table 3).

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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

H3C

N N S

SO2NH2

HN

N S

SO2NH2

(CH2)nOH

COOH

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

18

SO2NH2

SO2NH2

NH2

N N

O H2N

N

S N H O

19 O

O2N

S N H

O HO

12

SO2NH2 N

14

H N

SO2NH2 NH2

11

13

N

SO2NH2

CF3

NH2

H2N

8

SO2NH2

OH Br

Cl NH2

S

SO2NH2

20 O

SO2NH2 H2N

21

Data of Table 3 show the inhibition of the two Legionella enzymes described here, LpCA 1 and LpCA2 with this set of sulfonamides/sulfamates. For comparison reasons, the inhibition of the two human (h) offtarget isoforms hCA I and II (belonging to the a-CA family) as well as the bacterial b-CA from the pathogen H. pylori (HpyCA) with this set of 40 compounds, are also presented in Table 3. The following structure–activity relationship (SAR) can be observed for the inhibition of LpCA1 and LpCA 2 with these compounds: (i) In the case of the slow isoform LpCA1, the less effective inhibitors were SAC and HCT which showed affinities in the micromolar range (KIs of 15.8–20.5 lM), whereas quite a number of other tested derivatives were poorly effective as CAIs against LpCA1. They include compounds 1–3, 5–11, 14–18, DCP, DZA, ZNS, IND and CLX (KIs in the range of 734–3540 nM, Table 3). They belong to quite heterogeneous families of sulfonamides. Thus, mono-, di- or trisubstituted benzene-sulfonamides (1–3, 5–11, 15–18) incorporating various simple functionalities in para, meta or 3,4-positions

( )n S N H O 22: n = 0 23: n = 1 24: n = 2

SO2NH2

(among which amino, aminoalkyl; hydroxy, hydroxyalkyl, halogens, sufamoyl, etc.) were not particularly effective LpCA1 inhibitors, although some of them act as rather efficient hCA II inhiibtors (but are much weaker hCA I or HpyCA inhibitors, see Table 3). Benzenesulfonamides with a more complicated substitution pattern (DCP, IND or CLX) were also weak—medium potency inhibitors of this enzyme, together with heterocyclic sulfonamides such as 14, DZA, and ZNS. (ii) A better inhibitory power against LpCA1, with inhibition constants ranging between 101 and 665 nM was observed for the following derivatives: 12, 13, 19, 21, 22, MZA, BRZ, BZA, TPM, SLP, VLX, and SLT. As the category above, also these compounds are quite heterogeneous but several SAR data can be drawn. For example the methyl moiety in para to the sufamoyl group, as in compound 4, leads to an enhanced inhibitory power of 4 compared to the corresponding amino, aminoalkyl or hydroxyalkyl derivatives (1, 2, 5, 6, and 15–17), which is difficult to explain. An increase of LpCA1 inhibitory activity was also seen for 12 (compared to

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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

the structurally related 11) or 13 over the structurally related 14. In fact the two pairs of compounds are rather similar: 12 has a chlorine instead the CF3 moiety of 11, but their KIs differ by a factor of 1.44. For the pair 13–14, the second compound has a CH2 moiety less compared to 13, and the difference in the inhibitory power is by a factor of 1.68. This means that rather small structural changes in the scaffold of the inhibitors lead to important changes in the inhibitory power of the compound against the LpCA1 enzyme. (iii) The best LpCA1 inhibitors were 20, 23, 24, AAZ and EZA, which showed KIs in the range of 40.3–90.5 nM (Table 1). Here, the SAR is very interesting. In addition to the

H N HN O

Cl

S O

SO2NH2

HCT

heterocyclic derivatives AAZ and EZA, which are usually highly potent CAIs of most investigated enzymes belonging to all five CA families,21,22 acting thus as promiscuous inhibitors, the remaining potent LpCA1 inhibitors possess a rather similar structure of elongated, sulfonylated sulfonamide type. Aminobenzolamide 20 (and benzolamide BZA) are the prototype of these compounds, but interestingly, the aromatic compounds 23 and 24 were the best inhibitors. Furthermore, the LpCA1 inhibition clearly increased with an increase in the spacer between the two aminobenzenesulfonyl fragments from the molecules. The compound with the shortest spacer 22, was a weak inhibitor

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Table 2 Domain, species, accession numbers and cryptonyms of the b-CA amino acid sequences used to construct the phylogenetic tree Domain

Species

Accession number

Cryptonym

Bacteria

Legionella pneumophila Legionella pneumophila Myroides injenensis Porphyromonas gingivalis Acinetobacter baumannii Escherichia coli Helicobacter pylori Burkholderia thailandensis Bt4 Brucella suis 1330

WP_014844650.1 WP_014842179.1 ZP_10784819.1 YP_001929649.1 YP_002326524 ACI70660 BAF34127.1 ZP_02386321.1 NP_699962.1

LpCA1 LpCA2 MinCA PgiCA AbaCA EcoCa HpyCA BthCA BsuCA

Archaea

Methanobacterium thermoautotrophicum

GI:13786688

Cab

Eukaryota (fungus)

Saccharomyces cerevisiae Dekkera bruxellensis AWRI1499 Schizosaccharomyces pombe

GAA26059 EIF49256 CAA21790

SceCA DbrCA SpoCA

Eukaryota (green alga)

Coccomyxa sp. Chlamydomonas reinhardtii

AAC33484.1 XP_001699151.1

CspCA CreCA

Eukaryota (green plant)

Vigna radiata Flaveria bidentis, isoform I Zea mays Arabidopsis thaliana

AAD27876 AAA86939.2 NP_001147846.1 AAA50156

VraCA FbiCA ZmaCA AthCA

Table 3 (continued) Inhibitor/enzyme class

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

Figure 2. Phylogenetic analysis carried out on the b-CA amino acid sequences of the organisms reported in Table 2. 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.

Table 3 Inhibition of human isoforms hCA I and hCA II, and of the b-class bacterial enzymes from H. pylori (HypCA) and L. pneumophila (LpCA1 and LpCA2) with sulfonamides 1– 24 and the clinically used drugs AAZ–HCT

KI* (nM) hCA Ia

hCA IIa

a

a

HpyCAb b

LpCA1c b

LpCA2c b

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

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

2590 768 nt 236 218 450 38 64 nt nt 87 71 40 176 33 105 73 128 54 32 254 35 143 nt nt nt nt nt

541 913 969 2260 3540 2390 472 90.5 101 319 59.8 40.3 76.8 201 71.4 1670 2070 648 159 665 831 253 1090 536 990 485 20,500 15,800

382 391 280 631 721 476 321 45.1 78.9 52.3 50.1 25.2 72.1 88.5 103 64.1 336 467 148 882 820 245 525 879 421 463 441 745

*

Inhibitor/enzyme class

1 2 3 4 5 6 7 8 9 10 11 12

KI* (nM) hCA Ia

hCA IIa

a

a

28,000 25,000 79c 78,500 25,000 21,000 8300 9800 6500 7300 5800 8400

Errors in the range of 5–10% of the shown data, from 3 different assays. Human recombinant isozymes, stopped flow CO2 hydrase assay method, from Ref. 12 b Recombinant bacterial enzyme, stopped flow CO2 hydrase assay method, from Ref. 16d c Recombinant bacterial enzyme, this work; nt = not tested a

300 240 8 320 170 160 60 110 40 54 63 75

HpyCAb b nt 1845 nt 2470 2360 3500 1359 1463 1235 nt 973 640

LpCA1c b 939 946 1060 556 757 734 770 866 988 913 929 642

LpCA2c b 455 277 933 624 516 375 592 396 181 622 593 496

(KI of 319 nM) but the increase of the spacer to one and respectively two carbon atoms led to a drastic increase of the potency. Indeed, 23 and 24 showed KIs of 59.8 and 40.3 nM, respectively (Table 3). (iv) The fast Legionella isoform, LpCA2 was also inhibited by all sulfonamides/sulfamates investigated here (Table 3). However, the inhibition range was not as wide as for the previous isoform discussed above (LpCA1), as the best LpCA2 inhibitor

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(v)

(vi)

(vii)

(viii)

showed a KI of 25.2 nM and the worst one of 933 nM (the range for LpCA1 was between 40.3 nM and 20.5 lM). A small number of the investigated sulfonamides/sulfamates were quite weak LpCA2 inhibitors. They include 3, TPM, ZNS, VLX and HCT, with KIs in the range of 745–933 nM (Table 3). Most of the investigated sulfonamides from our study showed medium potency for the inhibition of LpCA2, with KIs in the range of 103–721 nM. They include the following derivatives: 1, 2, 4–19, EZA, DZA, BRZ, BZA, SLP, IND, CLX, SLT, and SAC (Table 3). SAR is again not easy to delineate but several interesting observations are as follows: as for LpCA1, simple benzenesulfonamide incorporating one or two substituents of the amino, aminoalkyl, hydroxy, hydroxyalkyl, halogens, sufamoyl etc. type, were not very different in their behavior as LpCA2 inhibitors, all of them leading to the medium potency profile mentioned above. The various aromatic/heterocyclic scaffolds present in the clinically used drugs/sweetener EZA, DZA, BRZ, BZA, SLP, IND, CLX, SLT, and SAC were also comparable to the simple scaffolds present in the compounds 1, 2 and 3–19 mentioned above. The best LpCA2 inhibitors were 20–24, AAZ, MZA and DCP, with KIs in the range of 25.2–88.5 nM (Table 3). SAR is highly interesting here. Except the clinically used drugs (AAZ, MZA and DCP) which have not much in common, all the other effective LpCA2 inhibitors possess the same scaffold, of the arylsulfonylated aminosulfonamide type. Thus, aminobenzolamide 20 is 3.3 times more effective as LpCA2 inhibitor compared to benzolamide BZA (only an amino group constitutes the difference between the two compounds), whereas for the aromatic componds 22–24, as for LpCA1, the activity increases with the increase of the molecule length, the best LpCA2 inhibitor being 24 (this was also the best LpCA1 inhibitor detected so far). Except for some of the effective CAIs detected here, which showed a good activity against both LpCA1 and LpCA2 (e.g., 22–24 and AAZ), generally the two isoforms had a rather different affinity for these inhibitors. For example SAC was a very weak LpCA1 inhibitor but a medium potency LpCA2 inhibitor. The same behavior was observed for DCP. Most of the time, these compounds showed an enhanced inhibition of LpCA2 over LpCA1, although several compounds with the reverse profile (e.g. 4, EZA, VLX) were also detected. The inhibition profiles of the two Legionella enzymes is very different compared to that of other bacterial b-CAs (such as HypCA) or the off-target, human isoforms hCA I and II (Table 3). This is of interest in case some of these compounds should be used for targeting the bacterial over the human isofoms in experimental or clinical settings.

3. Conclusions Legionella pneumophila is a Gram-negative bacterium causing Legionnaires’ disease and infections caused by it are increasing all over the world. Two b-CAs were identified, cloned and purified in this pathogen in the present study, which were denominated LpCA1 and LpCA2. We show that they efficiently catalyze CO2 hydration to bicarbonate and protons, with kcat in the range of (3.4–8.3)  105 s1 and kcat/Km of (4.7–8.5)  107 M1 s1, and are inhibited by sulfonamides and sulfamates, the main class of CAIs (some of which are clinically used drugs for 60 years). The best LpCA1 inhibitors detected in this study were aminobenzolamide and structurally similar sulfonylated aromatic sulfonamides, as

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well as acetazolamide and ethoxzolamide (KIs in the range of 40.3–90.5 nM). The best LpCA2 inhibitors belonged to the same class of sulfonylated sulfonamides, together with acetazolamide, methazolamide and dichlorophenamide (KIs in the range of 25.2– 88.5 nM). As these enzymes may be involved in pH regulation in the phagosome during Legionella infection, their inhibition may lead to antibacterials with a novel mechanism of action 4. Experimental protocols 4.1. Chemistry Sulfonamides 1–24 and AAZ-HCT were commercially available or reported earlier by us.23 All compounds were >95% purity, as assessed by HPLC. 4.2. LpCA1 and LpCA2 cloning and purification We purchased the genomic DNA, which was isolated from Legionella pneumophila subsp. pneumophila strain Philadelphia-1 from American Type Culture Collection (ATCC 33152D-5™, Manassas, VA), which is the principal laboratory strain for many important biochemical and genetic studies.6 GenBank searching allowed us to identify two sequences encoding putative ß-CAs in this pathogen, with the accession No. NC_002942, designated as LpCA1 (locus tag lpg2500) and LpCA2 (locus tag lpg2194). LpCA1 comprises 738 bp, encoding a 246 amino acid polypeptide, whereas LpCA2 comprises 627 bp, encoding a 208 amino acid polypeptide. Based on the reported sequences, primer pairs were synthesized for polymerase chain reaction (PCR) in order to obtain the full length CAs. The sequences of the adopter primer pairs were as follows; 50 -CGGGTCGACTGCCGCCACCATGCCAAAAAAACTGCTC AT-30 and 50 -CGCGGCCGCTTACTCTGGGACAGATCGCTT-30 for LpCA 1 (SalI and NotI recognition sequences were underlined and the extended Kozac sequence was double-underlined), and 50 -CGGGAATTCCCATGTGGACTTTAACCA-30 and 50 -CGGTCGACTCACTCATCAA AAAATTGA-30 for LpCA 2 (EcoRI and SalI recognition sequences were underlined). Initially, we set a 50 -primer based on the wildtype sequence, but no soluble protein product could be obtained (for LpCA1 cloning). Thus, a consensus sequence for translation initiation (extended Kozac sequence) was added to the 50 -end, which was obtained by a survey of 699 vertebrate mRNAs (Protocol Online; http://www.protocol-online.org), allowing us to successfully obtain the soluble, cloned protein. The PCR reaction was started at 94 °C with an incubation time of 3 min. It consisted of 40 cycles of 30 s at 94 °C, followed by 30 s at 55 °C and then 1.5 min at 72 °C. After this, a 5 min incubation at 72 °C followed. The direct sequencing of the PCR products allowed us to confirm the correct DNA sequences of the desired genes. An ABI PRISM Dye Termination Cycle Sequencing Kit (Perkin-Elmer, Foster City, CA) and an ABI 370A DNA sequencer (ABI, Foster City, CA) were used for this purpose. The following restriction enzymes were used to cleave the PCR products: SalI and NotI for LpCA1, and EcoRI and SalI for LpCA2, respectively. They were thereafter ligated in-frame into the pGEX-4T2 vector (Amersham, Tokyo, Japan). The sub-cloned plasmid vectors were then transfected into Escherichia coli strain BL21, in order to obtain the glutathione S-transferase (GST)-CA fusion proteins, as reported previously for other mammalian, bacterial or fungal CAs.8,9,16 Protein expression was induced by addition of 1 mM isopropyl-cD-thiogalactopyranoside (IPTG). Bacteria were grown at 37 °C for 24 h, then harvested and sonicated in PBS. The sonicated cell extracts were homogenized twice with a Polytron (Brinkmann) at 4 °C, for 30 s each. Centrifugation at 30,000g for 30 min afforded the supernatant containing the soluble proteins, which were

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applied to prepacked Glutathione Sepharose 4B columns (Amersham). These columns were extensively washed with buffer and then the GST-CA fusion protein was eluted with a buffer consisting of 5 mM reduced glutathione in 50 mM Tris–HCl pH 8.0. Finally, the GST parts of the fusion proteins were cleaved with thrombin, as reported earlier for othet bacterial/mammalian CAs.8,9,16 The obtained LpCA recombinant proteins were further purified by sulfonamide affinity chromatography, as reported previously.8,9,16 4.3. SDS–PAGE Sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) was performed as described previously (17) using 12% gels. 4.4. Sequence and phylogenetic analysis Multialignment of nucleotide sequences was performed using the programs PileUp (G.C.G.-Wiscon-sin)24 and ClustalW version 1.7. A most parsimonious tree was constructed with the program PhyML.25 4.5. 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.17 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 ChengPrusoff equation as reported earlier by our groups. The kinetic parameters for the uninhibited enzymes were derived from Lineweaver–Burk plots, as reported earlier,9,16,18 and represent the mean from at least three different determinations. Supplementary data Supplementary data (a SDS PAGE showing the purification of LpCA1 and LpCA2) associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.2014.04.006. References and notes 1. Fraser, D. W.; Tsai, T. R.; Orenstein, W.; Parkin, W. E.; Beecham, H. J.; Sharrar, R. G.; Harris, J.; Mallison, G. F.; Martin, S. M.; McDade, J. E.; Shepard, C. C.; Brachman, P. S. N. Engl. J. Med. 1977, 297, 1189.

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