Bioorganic & Medicinal Chemistry Letters 24 (2014) 1127–1132

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

Anion inhibition studies of two new b-carbonic anhydrasesfrom the bacterial pathogen Legionella pneumophila

0960-894X/$ - see front matter � 2014 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.bmcl.2013.12.124

⇑ Corresponding author. Tel.: +39 055 4573005; fax: +39 055 4573385.E-mail address: claudiu.supuran@unifi.it (C.T. Supuran).

Isao Nishimori a, Daniela Vullo b, Tomoko Minakuchi a, Andrea Scozzafava b, Sameh M. Osman c,Zeid AlOthman c, Clemente Capasso d, Claudiu T. Supuran b,c,e,⇑a Department of Gastroenterology, Kochi Medical School, Kochi, Japanb Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italyc Department of Chemistry, College of Science, King Saud University, PO Box 2455, Riyadh 11451, Saudi Arabiad Istituto di Biochimica delle Proteine—CNR, Via P. Castellino 111, 80131 Napoli, Italye Università degli Studi di Firenze, Polo Scientifico, Dipartimento di Scienze Farmaceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino (Florence), Italy

a r t i c l e i n f o

Article history:Received 27 November 2013Revised 30 December 2013Accepted 31 December 2013Available online 8 January 2014

Keywords:Carbonic anhydraseb-Class enzymeLegionella pneumophilaAnionDithiocarbamateSulfamide

a b s t r a c t

We investigated the cloning, catalytic activity and anion inhibition of the b-class carbonic anhydrases(CAs, EC 4.2.1.1) from the bacterial pathogen Legionella pneumophila. Two such enzymes, lpCA1 andlpCA2, were found in the genome of this pathogen. These enzymes were determined to be efficientcatalysts for CO2 hydration, with kcat values in the range of (3.4–8.3) � 105 s�1 and kcat/KM values of(4.7–8.5) � 107 M�1 s�1. A set of inorganic anions and small molecules was investigated to identify inhib-itors of these enzymes. Perchlorate and tetrafluoroborate were not acting as inhibitors (KI >200 mM),whereas sulfate was a very weak inhibitor for both lpCA1 and lpCA2 (KI values of 77.9–96.5 mM). Themost potent lpCA1 inhibitors were cyanide, azide, hydrogen sulfide, diethyldithiocarbamate, sulfamate,sulfamide, phenylboronic acid and phenylarsonic acid, with KI values ranging from 6 to 94 lM. The mostpotent lpCA2 inhibitors were diethyldithiocarbamate, sulfamide, sulfamate, phenylboronic acid andphenylarsonic acid, with KI values ranging from 2 to 13 lM. As these enzymes seem to be involved in reg-ulation of phagosome pH during Legionella infection, inhibition of these targets may lead to antibacterialagents with a novel mechanism of action.

� 2014 Elsevier Ltd. All rights reserved.

Legionella pneumophila is a Gram-negative environmental bacterium became highly acidic within 15 min of their forma-

bacterium that normally infects amoebae.1,2 It was discovered in1976 when it provoked a life-threatening pneumonia-like diseasein many participants at the 58th Annual Convention of the Amer-ican Legion in Philadelphia. This condition was subsequentlydubbed Legionnaires’ disease, or legionellosis.1,2 When the bacte-rial pathogen was characterized in detail, it was shown that a largenumber of its subspecies and serovars are widespread in nature.2,3

It is now known that L. pneumophila and the related species Legion-ella longbeachae are responsible for legionellosis in humans. Thesepathogens are increasingly spread all over the world through thedevelopment of artificial water systems for air conditioning, cool-ing towers, and aerosolizing devices, among other applications.2,3

When infecting Amoeba or human macrophages, L. pneumophilautilizes a sophisticated biochemical machinery to invade its hosts.For example, L. pneumophila can induce the formation of acidic vac-uoles within the host cytoplasm and hijack vesicles and organellesto enable effective infection.2 It has been demonstrated that L.pneumophila is able to maintain a neutral pH in its phagosomefor at least 6 h, whereas vacuoles that did not contain the

tion.3b Carbonic anhydrase (CA, EC 4.2.1.1), which hydrates carbondioxide to bicarbonate and protons (reaction 1),6 is involved in pHcontrol in organisms all over the phylogenetic tree, includingbacterial4 and fungal pathogens.5

CO2 þH2O$ HCO�3 þHþ ðreaction 1Þ

It has been shown that enzymes in this family are closely con-nected with pH regulation in many pathogenic (as well as non-pathogenic) organisms, as well as with secretion of acidic or alka-line electrolytes.4–10 For example, the extracellular environmentaround hypoxic tumors becomes more acidic (pH 6.0–6.5)7

through the action of several proteins and enzymes, includingthe tumor-associated isoforms CA IX and XII.7 Several CAs havebeen shown to play a significant role in the pathogenicity, invasionand survival of many fungal (e.g., Cryptococcus neoformans,5a Can-dida albicans,5b Candida glabrata8) and bacterial (e.g., Helicobacterpylori,9a Brucella suis,9b Vibrio cholerae9c) pathogens. These enzymesact by signaling information on its local niche (e.g., low or highavailability of CO2 or bicarbonate), or by allowing it to adapt toparticular pH conditions (such as high acidity in the stomach forH. pylori,10 or alkalinity and high concentrations of bicarbonatefor V. cholerae).11

1128 I. Nishimori et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1127–1132

The genome of L. pneumophila was recently cloned,3a but no CAsfrom this bacterium have been reported to date. We previously ex-plored its genome and detected two putative CAs belonging to theb-CA family (accession numbers NC_002942, which we designatedas lpCA1, locus tag lpg2500, NCBI reference sequenceWP_014844650.1; and lpCA2, locus tag lpg2194, NCBI referencesequence WP_014842179.1). Here, we report that these newb-CAs, which we designated as lpCA1 and lpCA2, are activeenzymes for the physiologic hydration of CO2 to form bicarbonateand protons. We also investigated the inhibition profiles of theseenzymes in the presence of inorganic anions and other smallmolecules known to interfere with metalloenzymes.

We cloned the two b-CAs from L. pneumophila, lpCA1 and lpCA2,as GST-fusion proteins using the method reported earlier for otherbacterial a- and b-CAs.8–10 After removal of the GST tag from thechimeric protein, we obtained the pure enzymes that were inves-tigated for catalytic activity in a stopped-flow CO2 hydrase assay.12

The activity of lpCA1 and lpCA2 can be compared to that ofother a- and b-CAs from human (h),6 fungal (C. neoformans,5a Sac-charomyces cerevisiae),13 plant (Flaveria bidentis),14 or bacterial (B.suis,9b and H. pylori9c) sources from the data shown in Table 1. Bothof these new proteins, lpCA1 and lpCA2, possess enzymatic activityfor the hydration of carbon dioxide to yield protons and bicarbon-ate that is typically catalyzed by CAs. The first isoform, lpCA1,showed a moderate degree of activity with a kcat of 3.4 � 105 s�1

and kcat/KM of 4.7 � 107 M�1 � s�1. This activity is in fact compara-ble to that of other a- and b-CAs, such as hCA I (a physiologicallyrelevant human isoform),6 Can2 from the fungal pathogen C.neoformans,5b or the bacterial pathogenic enzymes HpyCA and Bsu-CA219, from H. pylori and B. suis, respectively.9 The second isoform,lpCA2, was more active than lpCA1, with the following kineticparameters: kcat of 8.3 � 105 s�1 and kcat/KM of 8.5 � 107 M�1 � s�1

(Table 1). This level of activity is similar to that of the S. cerevisiaeenzyme SceCA or to that of BsuCA213 (both enzymes belong to theb-CA family) and is only slightly lower than the activity of hCA II, ahighly effective catalyst for the CO2 hydration reaction(Table 1).14,15 It was also observed that acetazolamide (5-acetam-ido-1,3,4-thiadiazole-2-sulfonamide), a clinically used drug,6 wasan efficient inhibitor of the two Legionella enzymes, with KI valuesin a range from 36 to 95 nM. The moderately active enzyme(lpCA1) was less sensitive to this compound than was lpCA2, whichshowed greater catalytic activity and also higher affinity for thesulfonamide inhibitor (Table 1).

To better understand the efficient catalytic properties of thetwo new enzymes, we aligned the amino acid sequences oflpCA1 and lpCA2 with those of other such enzymes that havebeen characterized previously, such as HpyCA, BsuCA213 and

Table 1Kinetic parameters for the CO2 hydration12 reaction catalyzed by several carbonic anhydr

Isozyme Activity level Class kcat (s�1)

hCA I Moderate a 2.0 � 105

hCA II Very high a 1.4 � 106

Can2 Moderate b 3.9 � 105

SceCA High b 9.4 � 105

FbiCA 1 Low b 1.2 � 105

HpyCA Moderate b 7.1 � 105

BsuCA219 Moderate b 6.4 � 105

BsuCA213 High b 1.1 � 106

lpCA1 Moderate b 3.4 � 105

lpCA2 High b 8.3 � 105

The human cytosolic isozymes hCA I and II (a-class CAs) at 20 �C and pH 7.5 in 10 mM HEPC. neoformans),5b SceCA (from S. cerevisiae),13 the Flaveria bidentis CA (FbiCA 1),14 the Helithe two new enzymes lpCA1 and lpCA2, measured at 20 �C, pH 8.3 in 20 mM TRIS buffer a(5-acetamido-1,3,4-thiadiazole-2-sulfonamide) are also provided.

a This work.

BsuCA219 (Fig. 1). The data shown in Figure 1 indicate thatas has been observed for all other bacterial b-CAs investigatedto date, the two Legionella proteins lpCA1 and lpCA2 possessthe amino acids crucial for the catalytic cycle of CO2 hydration.These amino acids include: (i) the metal coordinating residues,constituted by two Cys and one His, specifically Cys90, His143and Cys146 (lpCA1 numbering system) and (ii) the catalyticdyad constituted by residues Asp92 and Arg94, which is in-volved in the activation of the zinc-coordinated water moleculeand leads to the formation of the nucleophilic zinc-hydroxidespecies in the enzyme.5b,9–11

The proposed catalytic mechanism of the b-CAs is shown sche-matically in Figure 2. It has been in fact shown by means of kineticand X-ray crystallographic studies that the catalytic mechanism ofthe b-CAs is rather similar to that of the a-class enzymes investi-gated in much greater detail.6,7,11 As mentioned above, the catalyt-ically active species of the enzyme (A in Fig. 2) has a hydroxide ioncoordinated to the Zn(II), in addition to the protein ligands (whichin Fig. 2 are those from lpCA1, i.e., Cys90, His143 and Cys146). Inthe case of the a-CAs, the zinc hydroxide species is generatedthrough a proton transfer process from the zinc coordinated waterto the environment, assisted by the active site residue His64, actingas a proton shuttle.6,7 In the b-CAs the activation of the zinc-coor-dinated water molecule (shown in D in Fig. 2) is more complexthan for the a-CAs, as the catalytic dyad mentioned above takespart in the process. Furthermore, the nature of the amino acid(s)acting as proton shuttle in the b-class enzymes is not completelyelucidated (see discussion later in the text). Unknown is also theCO2 binding site within the b-CAs (B in Fig. 2) but as for the a-CAs, probably this is a hydrophobic pocket not far away from thecatalytic zinc ion. After the nucleophilic attack of the zinc hydrox-ide species of the enzyme on the bound CO2, bicarbonate is formed(C in Fig. 2) which presumably is bidentately coordinated to thezinc ion (as in the a-CAs).6,7,11 Inorganic anions are usually weakligands of the zinc ion in metalloenzymes, so that species C is prob-ably easily converted to species D, by a substitution reaction of thebicarbonate by means of a water molecule. This leads to liberationof the reaction product (bicarbonate) into solution, with generationof the acidic form of the enzyme, D, which is catalytically inactive.To generate the zinc hydroxide, catalytically effective species A, aproton transfer reaction must occur (Fig. 2), probably assisted byan active site residue. In the b-CA from the alga Coccomyxa, it hasbeen hypothesized that there are two residues participating in thisprocess, Tyr88 and His92. However, neither lpCA1 nor lpCA2 pos-sess a sequence of Tyr and His residues close to each other as thealgal enzyme, which leads the nature of the proton shuttling resi-due in these novel b-CAs unknown at this moment. The inhibition

ases

kcat/KM (M�1 � s�1) KI (acetazolamide) (nM) Refs.

5.0 � 107 250 61.5 � 108 12 64.3 � 107 10.5 5b9.8 � 107 82 137.5 � 106 27 144.8 � 107 40 9c3.9 � 107 63 9b8.9 � 107 303 9b4.7 � 107 95 a

8.5 � 107 36 a

ES buffer and 20 mM Na2SO4. Parameters are also reported for the b-CAs Can2 (fromcobacter pylori b-CA HpyCA,9c the Brucella suis b-CAs BsuCA213 and BsuCA219,9b andnd 20 mM NaClO4. Inhibition data for the clinically used sulfonamide acetazolamide

Figure 1. Amino acid sequences alignment of selected b-CAs from three bacterial species (the lpCA1 numbering system was used). Amino acid residues participating in thecoordination 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 inred. The asterisk (⁄) indicates identity at a position, the symbol (:) designates conserved substitutions, whereas (.) indicates semi-conserved substitutions. The multiplealignment was performed with the program MUSCLE and refined using the program Gblocks. Organisms, NCBI sequence numbers and cryptonyms are as follows: Legionellapneumophila, WP_014844650.1 and WP_014842179.1, lpCA1 and lpCA2; Helicobacter pylori, YP_005769368.1, HpyCA; Brucella suis, WP_012243428.1 and YP_005616633.1,BsuCA219 and BsuCA213.

Zn2+

OH

CO2

Zn2+

OHO

O

Zn2+

OO

HOHCO3

Zn2+

OH2

B

BH

Zn2+

Inh

Zn2+

OHH

Cys146His143

Cys90

--

+

+ H2O

A

B

C

D

-

-

-

+-

Inh-+

-H2O

E

-

F

Inh-

Cys90

Cys90

Cys90

Cys90

Cys90

His143

His143

His143

His143

His143

Cys146

Cys146

Cys146

Cys146

Cys146

Inh-

( BH+)

Figure 2. Catalytic and inhibition mechanisms of lpCAs (exemplified by using lpCA1, and its amino acid residues numbering system). See text for details.

I. Nishimori et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1127–1132 1129

Table 2Inhibition constants of anionic inhibitors against a-CA isozymes derived from human(hCA II) and bacterial (SspCA)15 sources. Inhibition constants are also reported for b-CA from a bacterium (H. pylori)9c HpyCA, the plant Flaveria bidentis isoform 1 (FbiCA1),14 and the new enzymes lpCA1 and lpCA2 from L. pneumophila. Values weredetermined at 20 �C in a stopped flow CO2 hydrase assay12

Inhibitor§ KI (mM)#

hCA IIa SspCAb HpyCAc FbiCA 1d lpCA1e lpCA2 e

a a b b b b

F� >300 41.7 0.67 0.71 0.91 0.77Cl� 200 8.30 0.56 0.74 0.79 0.81Br� 63 49.0 0.38 0.67 0.65 8.0I� 26 0.86 0.63 0.71 0.32 59.1CNO� 0.03 0.80 0.37 0.93 0.66 0.96SCN� 1.60 0.71 0.68 0.83 0.52 0.88CN� 0.02 0.79 0.54 0.62 0.064 0.61N�3 1.51 0.49 0.80 0.46 0.077 0.45HCO�3 85 33.2 0.50 0.66 3.5 6.6

CO2�3

73 39.3 0.42 0.84 4.7 4.8

NO�3 35 0.86 0.78 0.78 7.6 30.1NO�2 63 0.48 0.67 0.57 7.9 5.8HS� 0.04 0.58 0.58 0.86 0.076 0.51HSO�3 89 21.1 0.63 55.3 6.6 7.2

SnO2�3

0.83 0.52 0.48 0.53 0.57 0.63

SeO2�4

112 0.57 0.65 24.5 7.3 0.66

TeO2�4

0.92 0.53 0.45 0.90 0.24 0.29

P2O4�7

48.50 0.69 0.75 0.83 0.94 0.83

V2O4�7

0.57 0.66 0.18 0.66 0.39 0.47

B4O2�7

0.95 0.67 0.68 0.86 0.60 0.55

ReO�4 0.75 0.80 0.82 0.52 0.89 0.77RuO�4 0.69 0.69 1.10 26.1 0.82 0.86

S2O2�8

0.084 84.6 0.93 0.87 0.85 0.57

SeCN� 0.086 0.07 0.97 0.88 0.98 0.66

CS2�3

0.0088 0.06 0.21 0.06 0.53 0.62

Et2NCS�2 3.1 0.004 0.0074 0.008 0.006 0.002

SO2�4

>200 0.82 0.57 0.62 77.9 96.5

ClO�4 >200 >200 6.50 >200 >200 >200BF�4 >200 >200 >200 >200 >200 >200FSO�3 0.46 0.73 0.75 0.69 9.1 0.46

NHðSO3Þ2�20.76 0.75 0.70 50.9 1.17 0.59

H2NSO2NH2 1.13 0.009 0.072 0.004 0.094 0.009H2NSO3H 0.39 0.042 0.094 0.005 0.076 0.013Ph-BðOHÞ2 23.1 0.041 0.073 0.008 0.065 0.006Ph-AsO3H2 49.2 0.005 0.092 0.006 0.084 0.008

§ As sodium salt.# Errors were in the range of 3–5% of the reported values, from three different

assays.a From Ref. 6.b From Ref. 15.c From Ref. 9c.d From Ref. 14.e This work.

1130 I. Nishimori et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1127–1132

mechanism of lpCA1 and lpCA2 (Fig. 2E and F) will be discussedlater in the paper.

We also examined the phylogenetic relationships of the twonew bacterial b-CAs, lpCA1 and lpCA2, to other such enzymescharacterized earlier. We compared these enzymes to those fromvarious pathogens, such as H. pylori (one b-CA in its genome),9c B.suis (two b-CAs in the genome)9b and Salmonella typhimurium(again with two such isoforms encoded in the bacterial genome),as shown in Figure 3.16 As observed from the phylogenetic treein Figure 3, the two Legionella enzymes are highly similar, cluster-ing on their own branch of the tree. The next most similar enzymeis isoform stCA2 from S. typhimurium. The remaining b-CAs all clus-tered together on the lower branch of the tree, with HpyCA, stCA1and BsuCA219 residing on the same branch. The remaining Brucellaisoform BsuCA213 was on a separate branch, thus indicating that itis more distantly related to the other bacterial CAs examined here(Fig. 3).

Anions and other small molecules (e.g., sulfamide, sulfamate,phenylboronic acid or phenylarsonic acid) were highly investi-gated as CA inhibitors (CAIs), as they bind to the metal ion fromthe enzyme active site and impair catalysis.6,8–11 Herein, we reportthe first inhibition study of the two Legionella enzymes with arange of such inhibitors, including simple and complex inorganicanions as well as sulfamide, sulfamate, phenylboronic acid andphenylarsonic acid (Table 2). This set of anions and small mole-cules had been investigated previously for inhibition of a largenumber of CAs from all of the known genetic families, a-, b-, c-,d-, and f-CAs.6,8–11,13–17

The following should be noted regarding inhibition of lpCA1and lpCA2 by the anions and small molecules shown in Table 2:

(i) Perchlorate and tetrafluoroborate, anions known for theirlow affinity for metal ions in metalloenzymes (and sometimes alsoin solution),13–17 did not inhibit the two new b-CAs reported here(KI >200 mM). Similar results have been observed in most of theCAs examined to date: only HpyCA was effectively inhibited byperchlorate, with a KI of 6.5 mM.9c Sulfate was also an ineffectivelpCA1 and lpCA2 inhibitor, with KI values between 77.9 and96.5 mM (Table 2). Iodide and nitrate were also quite weak lpCA2inhibitors, with inhibition constants of 59.1 and 30.1 mM, respec-tively. These anions were more effective inhibitors of lpCA1, videinfra.

(ii) Another group of anions inhibited lpCA1 and lpCA2 weakly,with inhibition constants in the range of 3.5–9.1 mM. The weakinhibitors of lpCA1 were bicarbonate, carbonate, nitrate, nitrite,hydrogen sulfite, selenate and fluorosulfonate, whereas for lpCA2,the weak inhibitors included bromide, bicarbonate, carbonate,nitrite and hydrogen sulfite (Table 2).

(iii) A large number of the anions investigated were submillim-olar inhibitors against both lpCA1 and lpCA2. All of the halides, aswell as cyanate, thiocyanate, stannate, tellurate, pyrophosphate,divanadate, tetraborate, perrhenate, perruthenate, peroxydisulfate,selenocyanate, and trithiocarbonate inhibited lpCA1 with KI valuesfrom 0.24 to 0.98 mM. Iminodisulfonate was slightly less effective

Figure 3. Phylogenetic tree produced using the b-CA amino acid sequences aligned in Fisoftware based on the maximum-likelihood principle. Branch support values are reportedin Figure 1, except for Salmonella typhimurium,16 stCA1 and stCA2 (NCBI sequence numb

as an lpCA1 inhibitor (KI of 1.17 mM). The effective, submillimolarinhibitors of lpCA2 were fluoride, chloride, cyanate, thiocyanate,cyanide, azide, hydrogen sulfide, stannate, tellurate, pyrophos-phate, divanadate, tetraborate, perrhenate, perruthenate, peroxy-disulfate, selenocyanate, trithiocarbonate, fluorosulfonate and

gure 1. The tree was constructed using the program PhyML 3.0, which is phylogenyat branch points. Organisms, NCBI sequence numbers and cryptonyms are indicateders: NP_459176.1 for stCA1, and YP_007905873.1, for stCA2).

I. Nishimori et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1127–1132 1131

iminodisulfonate. These ions showed KI values ranging from 0.29to 0.96 mM. The best inhibitor in this subseries was tellurate, withKI values of 0.24 and 0.29 mM against lpCA1 and lpCA2, respec-tively. This is a very potent inhibitor compared to the salts ofelements from the same group. Specifically, the Se(VI) and S(VI)derivatives selenate and sulfate were much weaker inhibitors ofthe two enzymes (Table 2).

(iv) The best anionic inhibitors of lpCA1 detected in this studywere cyanide, azide, hydrogen sulfide, and diethyldithiocarbamate.Taken together with the small molecules sulfamide, sulfamate,phenylboronic acid and phenylarsonic acid, this group showed KI

values from 6 to 94 lM. Several observations can be gleaned fromthese data. The metal ‘poisons’ cyanide, azide, and hydrogen sul-fide had very similar inhibitory effects against lpCA1. This similar-ity can be extended further to the four small molecules fromTable 2, that is, sulfamide, sulfamate, phenylboronic acid andphenylarsonic acid. In fact all these compounds inhibited the en-zymes over a narrow range of potencies, with inhibition constantsfrom 64 to 94 lM. The compound N,N-diethyldithiocarbamate hada much higher affinity for this enzyme, with a low micromolarvalue for KI of 6 M. In fact, the dithiocarbamates were recentlyreported as a potent new class of CAIs targeting both the a- andb-classes of such enzymes.18 However, all of the small moleculeswere low micromolar inhibitors of lpCA2, with KI values from 2to 13 lM. These inhibitors include N,N-diethyldithiocarbamate,sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid.Again, the dithiocarbamate was the most potent lpCA2 inhibitor.

(v) There are net differences in the behavior of the two Legion-ella enzymes towards the anionic inhibitors investigated here. Wealso observed significant differences between these two enzymes,lpCA1 and lpCA2, and other a- and b-class CAs for which such dataare available. These include the enzymes shown in Table 2, whichare of human, bacterial or plant origin.

Thus, lpCA1 seems to have higher affinity for some poisonousmetal anions such as cyanide, azide, and hydrogen sulfide, whichare around one order of magnitude more potent against lpCA1 thanlpCA2. However, lpCA2 showed higher affinity for N,N-diethyldi-thiocarbamate, sulfamide, sulfamate, phenylboronic acid andphenylarsonic acid compared to lpCA1. The heavy halides, bromideand iodide, also behaved quite differently against the two enzymes.These two anions are submillimolar inhibitors of lpCA1 and muchweaker inhibitors of lpCA2 (KI values ranging from 8 to 59 mM).However, there were also anions that showed comparable affinitiesfor these two enzymes (e.g., chloride, bicarbonate, carbonate, tellu-rate—see Table 2).

The inhibitory potencies of these anions against lpCA1 andlpCA2 were also quite different when compared to those observedin other bacterial (SspCA or HpyCA), plant (FbiCA 1) or human (hCAII) enzymes. For example, while chloride was a submillimolarinhibitor of lpCA1, lpCA2, FbiCA 1 and HpyCA, it showed KI valuesfrom 8.3 to 200 mM against the extremophilic bacterial enzymeSspCA or human hCA II (Table 2). Trithiocarbonate was an effectiveinhibitor of hCA II, SspCA and FbiCA 1, but was less effectiveagainst HpyCA, lpCA1 and lpCA2.

The inhibition mechanism of these anions against lpCA1 andlpCA2 is probably similar to that of the inorganic anions againsta-CAs, investigated in greater detail, by means of kinetic, spectro-scopic and X-ray crystallographic techniques.6,7,11,16 As shown inFigure 2, the inhibitor may substitute the fourth, non-protein zincligand, leading to tetrahedral species, as those shown in E (Fig. 2).Anions such as acetate bound to Can2,5b as well as thiocyanate,azide and phosphate bound to the Coccomyxa b-CA,11c were shownto possess this inhibition mechanism by means of high resolutionX-ray crystallography. An alternative inhibition mechanism hasbeen also reported again for the Coccomyxa b-CA11c for iodide,which was found anchored to the zinc-coordinated water

molecule/hydroxide ion, as shown schematically in Figure 2F. Thismechanism was also observed for phenols,19 polyamines 20 andsulfocoumarins21 for the inhibition of a-CAs.

In conclusion, we investigated the cloning, catalytic activity andanion inhibition of two b-CAs from the bacterial pathogen L. pneu-mophila, lpCA1 and lpCA2. The two new enzymes were efficientcatalysts for CO2 hydration, with kcat values ranging from (3.4–8.3) � 105 s�1 and kcat/KM of (4.7–8.5) � 107 M�1 s�1. A set of inor-ganic anions and small molecules was investigated for inhibition ofthese enzymes. Perchlorate and tetrafluoroborate were not activeinhibitors (KIs >200 mM), whereas sulfate was a very weak inhibi-tor of both lpCA1 and lpCA2 (KIs of 77.9–96.5 mM). The mostpotent lpCA1 inhibitors were cyanide, azide, hydrogen sulfide,N,N-diethyldithiocarbamate, sulfamate, sulfamide, phenylboronicacid and phenylarsonic acid, with KI values in a range from 6 to94 lM. The most active lpCA2 inhibitors were N,N-diethyldithio-carbamate, sulfamide, sulfamate, phenylboronic acid and pheny-larsonic acid, with KI values between 2 and 13 lM. As theseenzymes seem to be involved in regulation of phagosome pHduring Legionella infection, inhibition of these targets may lead toantibacterial agents with a novel mechanism of action.

Acknowledgment

We thank The Distinguished Scientist Fellowship Program(DSFP) at KSU for funding this project.

References and notes

1. (a) 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. Eng. J. Med. 1977, 297, 1189; (b) Edelstein, P. H.; Finegold, S.M. J. Clin. Microbiol. 1979, 9, 457.

2. (a) Gomez-Valero, L.; Rusniok, C.; Cazalet, C.; Buchrieser, C. Front. Microbiol.2011, 2, 208; (b) Escoll, P.; Rolando, M.; Gomez-Valero, L.; Buchrieser, C. Curr.Top. Microbiol. Immunol. 2013, 376, 1; (c) Xu, L.; Luo, Z. Q. Microbes Infect. 2013,15, 157.

3. (a) Chien, M.; Morozova, I.; Shi, S.; Sheng, H.; Chen, J.; Gomez, S. M.; Asamani,G.; Hill, K.; Nuara, J.; Feder, M.; Rineer, J.; Greenberg, J. J.; Steshenko, V.; Park, S.H.; Zhao, B.; Teplitskaya, E.; Edwards, J. R.; Pampou, S.; Georghiou, A.; Chou, I.C.; Iannuccilli, W.; Ulz, M. E.; Kim, D. H.; Geringer-Sameth, A.; Goldsberry, C.;Morozov, P.; Fischer, S. G.; Segal, G.; Qu, X.; Rzhetsky, A.; Zhang, P.; Cayanis, E.;De Jong, P. J.; Ju, J.; Kalachikov, S.; Shuman, H. A.; Russo, J. J. Science 2004, 305,1966; (b) Horwitz, M. A.; Maxfield, F. R. J. Cell Biol. 1984, 99, 1936.

4. (a) Supuran, C. T. Front. Pharmacol. 2011, 2, 34; (b) Supuran, C. T. Curr. Med.Chem. 2012, 19, 831.

5. (a) Bahn, Y. S.; Cox, G. M.; Perfect, J. R.; Heitman, J. Curr. Biol. 2005, 15, 2013; (b)Schlicker, C.; Hall, R. A.; Vullo, D.; Middelhaufe, S.; Gertz, M.; Supuran, C. T.;Muhlschlegel, F. A.; Steegborn, C. J. Mol. Biol. 2009, 385, 1207.

6. (a) Supuran, C. T. Nat. Rev. Drug Disc. 2008, 7, 168; (b) Capasso, C.; Supuran, C. T.Expert Opin. Ther. Pat. 2013, 23, 693; (c) Alterio, V.; Di Fiore, A.; D’Ambrosio, K.;Supuran, C. T.; De Simone, G. Chem. Rev. 2012, 112, 4421.

7. (a) Neri, D.; Supuran, C. T. Nat. Rev. Drug Disc. 2011, 10, 767; (b) Supuran, C. T. J.Enzyme Inhib. Med. Chem. 2012, 27, 759; (c) Rummer, J. L.; McKenzie, D. J.;Innocenti, A.; Supuran, C. T.; Brauner, C. J. Science 2013, 340, 1327.

8. (a) Innocenti, A.; Leewattanapasuk, W.; Mühlschlegel, F. A.; Mastrolorenzo, A.;Supuran, C. T. Bioorg. Med. Chem. Lett. 2009, 19, 4802; (b) Cottier, F.;Leewattanapasuk, W.; Kemp, L. R.; Murphy, M.; Supuran, C. T.; Kurzai, O.;Mühlschlegel, F. A. Bioorg. Med. Chem. 2013, 21, 1549.

9. (a) Minakuchi, T.; Nishimori, I.; Vullo, D.; Scozzafava, A.; Supuran, C. T. J. Med.Chem. 2009, 52, 2226; (b) Joseph, P.; Turtaut, F.; Ouahrani-Bettache, S.;Montero, J. L.; Nishimori, I.; Minakuchi, T.; Vullo, D.; Scozzafava, A.; Kohler, S.;Winum, J. Y.; Supuran, C. T. J. Med. Chem. 2010, 53, 2277; (c) Nishimori, I.;Minakuchi, T.; Kohsaki, T.; Onishi, S.; Takeuchi, H.; Vullo, D.; Scozzafava, A.;Supuran, C. T. Bioorg. Med. Chem. Lett. 2007, 17, 3585.

10. (a) Nishimori, I.; Onishi, S.; Takeuchi, H.; Supuran, C. T. Curr. Pharm. Des. 2008,14, 622; (b) Morishita, S.; Nishimori, I.; Minakuchi, T.; Onishi, S.; Takeuchi, H.;Sugiura, T.; Vullo, D.; Scozzafava, A.; Supuran, C. T. J. Gastroenterol. 2008, 43,849.

11. (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) 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; (c) Huang, S.; Hainzl, T.; Grundström,C.; Forsman, C.; Samuelsson, G.; Sauer-Eriksson, A. E. PLoS ONE 2011, 6, e28458.

12. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561. An Applied Photophysics stopped-flow instrument was used for assaying the CA catalyzed CO2 hydration activity.Phenol red (at a concentration of 0.2 mM) was used as indicator, working at the

1132 I. Nishimori et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1127–1132

absorbance maximum of 557 nm, with 10–20 mM Hepes (pH 7.5) as buffer,and 20 mM NaClO4 for maintaining constant the ionic strength, following theinitial 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 determinationof the kinetic parameters and inhibition constants. For each inhibitor, at leastsix traces of the initial 5–10% of the reaction were used for determining theinitial velocity. The uncatalyzed rates were determined in the same mannerand subtracted from the total observed rates. Stock solutions of inhibitors(10 mM) were prepared in distilled–deionized water, and dilutions up to0.01 M were performed thereafter with the assay buffer. Inhibitor and enzymesolutions were preincubated together for 15 min at room temperature prior toassay to allow for the formation of the E–I complex. The inhibition constantswere obtained by non-linear least-squares methods using PRISM 3 and theCheng–Prusoff equation, whereas the kinetic parameters for the uninhibitedenzymes were obtained from Lineweaver–Burk plots, as reported earlier,9,11–15

and represent the mean from at least three different determinations..13. Isik, S.; Kockar, F.; Arslan, O.; Ozensoy Guler, O.; Innocenti, A.; Supuran, C. T.

Bioorg. Med. Chem. Lett. 2008, 18, 6327.14. Monti, S. M.; Ludwig, M.; Vullo, D.; Scozzafava, A.; Capasso, C.; Supuran, C. T.

Bioorg. Med. Chem. Lett. 2013, 23, 1626.15. (a) De Luca, V.; Vullo, D.; Scozzafava, A.; Carginale, V.; Rossi, M.; Supuran,

C. T.; Capasso, C. Bioorg. Med. Chem. 2013, 21, 1465; (b) Capasso, C.; DeLuca, V.; Carginale, V.; Rossi, M. J. Enzyme Inhib. Med. Chem. 2012, 27, 892;(c) De Luca, V.; Vullo, D.; Scozzafava, A.; Rossi, M.; Supuran, C. T.; Capasso,C. Bioorg. Med. Chem. Lett. 2012, 22, 5630; (d) Vullo, D.; De Luca, V.;Scozzafava, A.; Carginale, V.; Rossi, M.; Supuran, C. T.; Capasso, C. Bioorg.Med. Chem. Lett. 2012, 6324.

16. (a) Nishimori, I.; Minakuchi, T.; Vullo, D.; Scozzafava, A.; Supuran, C. T. Bioorg.Med. Chem. 2011, 19, 5023; (b) Vullo, D.; Nishimori, I.; Minakuchi, T.;Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2011, 21, 3591; (c)Briganti, F.; Pierattelli, R.; Scozzafava, A.; Supuran, C. T. Eur. J. Med. Chem. 1996,31, 1001.

17. (a) Innocenti, A.; Zimmerman, S.; Ferry, J. G.; Scozzafava, A.; Supuran, C. T.Bioorg. Med. Chem. Lett. 2004, 14, 3327; (b) Vullo, D.; SaiKumar, R. S.;Scozzafava, A.; Capasso, C.; Ferry, J. G.; Supuran, C. T. Bioorg Med. Chem. Lett.2014, 23, 6706; (c) Del Prete, S.; Vullo, D.; Scozzafava, A.; Capasso, C.; Supuran,C. T. Bioorg. Med. Chem. 2014, 22, 531.

18. (a) Carta, F.; Aggarwal, M.; Maresca, A.; Scozzafava, A.; McKenna, R.; Supuran,C. T. Chem. Commun. 2012, 48, 1868; (b) Maresca, A.; Carta, F.; Vullo, D.;Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 407; (c) Monti, S. M.;Maresca, A.; Carta, F.; De Simone, G.; Mühlschlegel, F. A.; Scozzafava, A.;Supuran, C. T. Bioorg. Med. Chem. Lett. 2012, 22, 859; (d) Carta, F.; Aggarwal, M.;Maresca, A.; Scozzafava, A.; McKenna, R.; Masini, E.; Supuran, C. T. J. Med. Chem.2012, 55, 1721.

19. (a) Innocenti, A.; Vullo, D.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett.2008, 18, 1583; (b) Innocenti, A.; Hilvo, M.; Scozzafava, A.; Parkkila, S.;Supuran, C. T. Bioorg. Med. Chem. Lett. 2008, 18, 3593; (c) Innocenti, A.; Vullo,D.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. 2008, 16, 7424.

20. Carta, F.; Temperini, C.; Innocenti, A.; Scozzafava, A.; Kaila, K.; Supuran, C. T. J.Med. Chem. 2010, 53, 5511.

21. (a) Tars, K.; Vullo, D.; Kazaks, A.; Leitans, J.; Lends, A.; Grandane, A.;Zalubovskis, R.; Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2013, 56, 293; (b)Tanc, M.; Carta, F.; Bozdag, M.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem.2013, 21, 4502.