Bioorganic & Medicinal Chemistry Letters 23 (2013) 6706–6710

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Bioorganic & Medicinal Chemistry Letters

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

Anion inhibition studies of a b-carbonic anhydrase from Clostridiumperfringens

0960-894X/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.bmcl.2013.10.037

⇑ Corresponding author. Tel.: +39 0554573005; fax: +39 0554573385.E-mail address: claudiu.supuran@unifi.it (C.T. Supuran).

Daniela Vullo a, R. Siva Sai Kumar b, Andrea Scozzafava a, Clemente Capasso c, James G. Ferry b,Claudiu T. Supuran a,d,⇑a Università degli Studi di Firenze, Chemistry Department, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italyb Department of Biochemistry and Molecular Biology, Eberly College of Science, The Pennsylvania State University, University Park, PA 16802-4500, USAc Istituto di Biochimica delle Proteine—CNR, Via P. Castellino 111, 80131 Napoli, Italyd Università degli Studi di Firenze, NEUROFARBA Department, Sezione di Scienze Farmaceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino (Florence), Italy

a r t i c l e i n f o a b s t r a c t

Article history:Received 28 September 2013Accepted 18 October 2013Available online 29 October 2013

Keywords:Carbonic anhydraseBeta class enzymeClostridium perfringensAnionSulfonamide

A b-carbonic anhydrases (CAs, EC 4.2.1.1) was recently cloned, purified and characterized kinetically inthe pathogen Clostridium perfringens. We report here the first inhibition study of this enzyme (CpeCA).CpeCA was poorly inhibited by iodide and bromide, and was inhibited with KIs in the range of 1–10 mM by a range of anions such as (thio)cyanate, azide, bicarbonate, nitrate, nitrite, hydrogensulfite,hydrogensulfide, stannate, tellurate, pyrophosphate, divanadate, tetraborate, peroxydisulfate, sulfate,iminodisulfonate and fluorosulfonate. Better inhibitory power, with KIs of 0.36–1.0 mM, was observedfor cyanide, carbonate, selenate, selenocyanide, trithiocarbonate and diethyldithiocarbamate, whereasthe best CpeCA inhibitors were sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, whichhad KIs in the range of 7–75 lM. This study thus provides the basis for developing better clostridialenzyme inhibitors with potential as antiinfectives with a new mechanism of action.

� 2013 Elsevier Ltd. All rights reserved.

Species belonging to the genus Clostridium, such as Clostridiumtetani, Clostridium botulinum, Clostridium barati, Clostridium butiry-cum, Clostridium hystolyticum and Clostridium perfringens amongothers, are strictly anaerobic pathogens that provoke serioushuman disease, such as tetanus, botulism, gas gangrene, bacterialcorneal keratitis and other infections.1–3 Some progress has beenachieved ultimately for designing pharmacological agents effectiveagainst these diseases, such as for example protease inhibitors tar-geting various metalloproteases essential for the life cycle of thesepathogens.3 However there is a constant search for novel drug tar-gets which may lead to new classes of such agents, considering theserious antibiotic drug resistance problems emerging worldwidewith the clinically used drugs.3

Recently, one of our groups reported4 a b-carbonic anhydrase(CA, EC 4.2.1.1) in C. perfringens. This was the first CA ever reportedin a bacterium belonging to the Clostridia, and it has been alsoshown that this enzyme is involved in crucial processes connectedwith the survival of the pathogen, such as the biosynthesis of lip-ids.4 Indeed, CAs belonging to the a-,5 b-6 and c-CA7 families havebeen described from many organisms, including pathogenic bacte-ria (e.g., Brucella spp., Mycobacterium tuberculosis, Streptococcusspp., Helicobacter pylori, Salmonella enterica, Sulfurihydrogenibium

spp., Vibrio cholera, Porphyromonas gingivalis), fungi (such as Can-dida albicans, Candida glabrata, Cryptococcus neoformans or Malas-sezia globosa) and protozoa (Plasmodium falciparum, Trypanosomacruzi or Leishmania spp.).8–10 Some of these proteins, which gener-ally possess significant catalytic activity for the physiologic, CO2

hydration reaction, are involved in important physiologic pro-cesses of these organisms and interference with them was shownto lead to an impairment of the pathogen growth in vitro andin vivo.8–11 Such CAs were thus considered as new drug targetsfor designing antiinfectives with a new mechanism of action, thatis, CA inhibition.11,12

The b-CA class is ubiquitous, being present in the domainsArchaea,6,11,12 Bacteria,6,9,11,12 and Eukarya, including plants,13 fungiand some arthropods,14 among others. These enzymes are charac-terized by a diverse coordination of the active site Zn(II) ion com-pared to the highly investigated a-CA class (in which the metal ionis tightly coordinated by three His residues and a water molecule/hydroxide ion).5 In fact in the b-CAs there are two patterns of Zn(II)coordination, one involving two Cys and one His residues from theprotein, and a water molecule/hydroxide ion, leading to the nucle-ophilic zinc hydroxide species with the metal ion in a tetrahedralgeometry.6,8,10 Alternatively, in the so-called Type II b-CAs theZn(II) has an additional protein ligand, an Asp residue, and theenzyme is catalytically ineffective at pH values lower than 8.3.15

However at pH of 8.3 or higher, an Arg residue close to the Aspcoordinated to the Zn(II) interacts with the Asp, leading to a

D. Vullo et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6706–6710 6707

decomplexation of the COO� moiety and liberation of the fourthcoordination position of Zn(II), so that an incoming water moleculeis coordinated to the metal, leading thus to a catalytically efficientenzyme.15 The inhibition mechanism of the b-CAs is on the otherhand similar of the inhibition mechanism of a-CAs (with anionor sulfonamides, the most investigated classes of such com-pounds).5,16 In such enzyme-inhibitor adducts, the water moleculecoordinated to the Zn(II) is substituted by the inhibitor, usually inanionic state.5,16 However a different binding mode of the inhibitorwas also observed for both a- and b-CAs, for which some anions,phenols, polyamines, sulfocoumarins and some esters were shownto be anchored to the water coordinated to the Zn(II) ion.5,6,16 It isthus of great interest to investigate inhibition of new such enzymeswith various classes of inhibitors, especially in the search of poten-tial pharmacologic agents with a new mechanism of action.Continuing our interest in investigating pathogenic CAs, we reporthere the first inhibition study of the C. perfringens enzyme (denom-inated here CpeCA) with anions and other small molecules knownto interact with metal ions in metalloproteins, such as sulfamicacid, sulfamide, boronic and arsenic acids.5,16

Table 1 shows the kinetic parameters for the CO2 hydrationreaction catalyzed by several a- and b-CAs, such as the twoa-CAs of human (h) origin, hCA I and II,5,6 as well as several b-CAs from the fungal pathogens Cryptococcus neoformans (Can2),7b

Candida albicans (CalCA),19 the yeast Saccharomyces cerevisiae (Sce-CA),20 the plant Flaveria bidentis (FbiCA 1) and CpeCA, the enzymerecently discovered by Ferry’s group.4 It may be observed that therange of kcat is between 1.5 � 104 and 1.4 � 106 s�1 whereas thekcat/Km ranges between 4.8 � 106 and 1.5 � 108 M�1 � s�1. Mostof these enzymes are also highly sensitive to inhibition by the clin-ically used sulfonamide acetazolamide (5-acetamido-1,3,4-thiadia-zole-2-sulfonamide), with inhibition constants in the range of10.5–250 nM (Table 1). Among the investigated CAs shown in Ta-ble 1, FbiCA 1 and CpeCA are the most inefficient catalyst. Never-theless the value measured for them prove that even of not soexcellent catalysts as hCA II (one of the most rapid enzymes knownin nature), the C. perfringens enzyme is an acceptable catalyst forthe conversion of CO2 to bicarbonate and protons.

To rationalize the kinetic data in Table 1, an alignment of theamino acid sequence of several b-CAs from different organismshas been performed (Fig. 1). We have included cab from Methano-bacterium thermoautotrophicum,7 one of the best characterizedsuch enzymes, as well as the Helicobacter pylori b-CA as represen-tative from an aerobic bacterium, together with the fungal andplant CAs from Table 1. It may be observed that as all theseb-CAs, CpeCA has the conserved Zn(II) ligands Cys160, His220and Cys223 (Pisum sativum CA numbering system),13d as well asthe catalytic dyad, constituted by residues Asp162 and Arg164,which contribute to the activation of the Zn(II) coordinated watermolecule for catalysis. However, from these data it is impossibleto unbderstand why CpeCA has a slightly lower catalytic activity

Table 1Kinetic parameters for the CO2 hydration reaction catalysed by the human cytosolic isozymNa2SO4, and the b-CAs cab (from M. thermoautotrophicum), the fungal Can2, CalCA (from C.bidentis (FbiCA 1) and the newly investigated enzyme from C. perfringens, CpeCA, measuredclinically used sulfonamide acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide)

Isozyme Activity level Class Organism

hCA I Moderate a MammalhCA II Very high a Mammalcab Low b BacteriumCan2 Moderate b FungusCalCA High b FungusSceCA High b YeastFbiCA 1 Low b PlantCpeCA Low b Bacterium

compared to the other b-CAs from Table 1. In fact the kcat valuesfor the enzymes from the two anaerobic organisms from Table 1,cab and CpeCA seem to be rather similar, but cab is 3.75 times abetter catalyst for the hydration reaction compared to CpeCA, con-sidering the kcat/Km values. Probably these differences reflect thedifferences in the CO2 binding pockets in these enzymes, but forthe moment the binding of the substrate to the b-CAs is a poorlyunderstood phenomenon as no X-ray crystal structures with CO2

or bicarbonate were reported for this class of enzymes.Figure 2 presents a phylogenetic analysis of selected b-CAs for

which predicted amino acid sequence is available in data bases.We included sequences from plants, yeasts, fungi, and bacteria,as the corresponding enzymes were characterized completely byour groups,4,7–9 in order to get an overview on the phylogeneticrelationship of this family of CAs. It may be observed that CpeCAclustered on the same branch with cab, the enzyme from theanaerobic bacterium M. thermoautotrophicum,7 whereas the otherb-CAs from plants, fungi and aerobic bacteria were less related toit, being on different branches of the tree (Fig. 2). In fact, alreadyfrom the data of Figure 1 it has been observed that CpeCA andcab seem to be more similar due to an almost identical numberof amino acid residues composing the two proteins, whereas thefungal and plant b-CAs contain a higher number of residues andhave much lower sequence homology (Fig. 1).

Table 2 shows the inhibition data for CpeCA with respect to arather wide range of simple and complex inorganic anions as wellas several small molecules observed in previous studies to act asCA inhibitors (CAIs).5,6,16,18,19 The analogous data for the inhibitionof hCA II and the bacterial enzyme SspCA (from the extremophileSulfurihydrogenibium yellostonense)5c—belonging to the a-CAclass—as well as HpyCA and FbiCA1 (b-CAs, from a bacteriumand a plant, respectively) are provided for comparison. The inhibi-tion profile of CpeCA is distinct from that of other a- or b-CAs asfollows:

(i) A first group of anions, including perrhenate, tetrafluorobo-rate and perchlorate, showed ineffective inhibition of CpeCA, withno inhibitory activity observed up to concentrations as high as200 mM (Table 2). This is not so much unexpected for tetrafluoro-borate and perchlorate, as these anions show lower affinity for me-tal ions in solutions or metalloenzyme (see also the data for hCA I,II and FbiCA 1 from Table 3), but it is rather unexpected for perrh-enate, as this heavy metal anion is a rather efective CA inhibitor(CAI) of many enzymes belonging to the a- or b-classes.18,19

(ii) Unexpectedly, the heavy halogenides bromide and iodidewere quite modest CpeCA inhibitors, with KIs in the range of21.5–63.4 mM (Table 2). In fact for the hCAs the inhibitory powerof the halogenide increased with the increase of the halogen atom-ic weight (see Table 2), but this is not the case for CpeCA. Interest-ingly, for this enzyme the light halogenides, fluoride and chloride,were quite similar as inhibitors, with KIs in the range of4.3–4.8 mM.

es hCA I and II (a-class CAs) at 20 �C and pH 7.5 in 10 mM HEPES buffer and 20 mMneoformans and C. albicans, respectively), SceCA (from S. cerevisiae), the plant Flaveriaat 20 �C, pH 8.3 in 20 mM TRIS buffer and 20 mM NaClO4.17 Inhibition data with the

are also provided.

kcat kcat/Km KI (acetazolamide)(s�1) (M�1 � s�1) (nM)

2.0 � 105 5.0 � 107 2501.4 � 106 1.5 � 108 123.1 � 104 1.8 � 107 12,1003.9 � 105 4.3 � 107 10.58.0 � 105 9.7 � 107 1329.4 � 105 9.8 � 107 821.2 � 105 7.5 � 106 271.5 � 104 4.8 � 106 49

Figure 1. Alignment of the predicted amino acid sequences of selected b-CAs from bacterial, fungal and plant organisms. Multiple alignment was performed with theprogram Clustal W, version 2.1. Pisum sativum numbering system was used. Zinc ligands are indicated in red. The asterisk (⁄) indicates identity at a position; the symbol (:)designates conserved substitutions, while (.) indicates semi-conserved substitutions. Legend: CpeCa_bacterium, Clostridium perfringens F262 (Accession number:EIA18376.1); cab_bacterium, Methanobacterium Thermoautotrophicum (Accession number: gi:13786684); HpyCA_bacterium, Helicobacter pylori (Accession number:BAF34127.1); Can2_fungus; Cryptococcus neoformans (Accession number: GI: 219109194); CalCA_fungus, Candida albicans (Accession number: XP_721672.1); ScCA_yeast,Saccharomyces cerevisiae (Accession number: GAA26059); FbiCA_plant, Flaveria bidentis, isoform I (Accession number: AAA86939.2).

Figure 2. Phylogenetic analysis of the b-CAs from selected prokaryotic andeukaryotic species represented through a radial tree constructed using the programPhyML 3.0. See Figure 1 for organisms, accession numbers and cryptonyms of thesequences used to construct the tree.

6708 D. Vullo et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6706–6710

(iii) Alarge series of other simple or complex anions had a sim-ilar behavior with fluoride and chloride mentioned above. Indeed,(thio)cyanate, azide, bicabonate, nitrate, nitrite, hydrogensulfite,hydrogensulfide, stannate, tellurate, pyrophosphate, divanadate,tetraborate, peroxydisulfate, sulfate, iminodisulfonate and fluoro-sulfonate showed KIs in the range of 1.2–9.2 mM. It may beobserved that both so-called ‘metal pisons’ such as thio)cyanate,azide and hydrogensulfide, as well as anions known for their loweraffinity for metal ions (e.g., nitrate, sulfate), belong to this category

of CpeCA inhibitors, although for other enzymes shown in Table 2,the affinity of these anions for the enzyme is very much different(compare for example HS� and nitrate for hCA I and II, etc.).

(iv) A group of anions, among which cyanide, carbonate, sele-nate, selenocyanide, trithiocarbonate and diethyldithiocarbamatewere efficient, submillimolar or millimolar CpeCA inhibitors, withKIs in the range of 0.36–1.0 mM (Table 1). Except carbonate (whichinhibits CpeCA almost 3-fold stronger compared to bicarbonate) allthese anions possess potent metal ion complexing properties andthis enhanced affinity for the Zn(II) ion from the enzyme active siteis to be expected. In fact many of these anions also effectively in-hibit other a- or b-CAs shown in Table 2.

(v) The most effective CpeCA inhibitors were sulfamate, sulfam-ide, phenylboronic acid and phenylarsonic acid, which had KIs inthe range of 7–75 lM (Table 2). The first two of these small mole-cules incorporate the sulfamoyl moiety connected with the mainclass of CAIs, the sulfonamides and their bioisosteres (sulfamates,sulfamides, etc.).5,6 Many of the clinically used CAIs incorporatesuch moieties together with an organic scaffold to which that moi-ety is appended. Boronic and arsonic acids were also shown by usto strongly inhibit both a-, b-, c- and f-CAs,20 although their mech-anism of inhibition is not entirely understood, as no X-ray crystalstructures with such adduct are available at this moment. It isinteresting to note that apart acetazolamide (Table 1), phenylar-sonic acid is the most potent CpeCA inhibitor detected so far, witha KI of 7 lM (Table 2).

Table 2Inhibition constants of anionic inhibitors against a-CA isozymes derived from human(hCA II), and bacterial (SspCA) sources as well as the b-CA from a bacterium (H. pylori)HpyCA, the plant Flaveria bidentis isoform 1 (FbiCA 1), and the clostridial C. perfringens(CpeCA) enzyme at 20 �C and pH 7.5 (for the a-CAs) and pH 8.3 (for the b-CAs), by astopped flow CO2 hydrase assay17

Inhibitora KIb (mM)

hCA IIc SspCAd HpyCAe FbiCA 1f CpeCAf

F� >300 41.7 0.67 0.71 4.8Cl� 200 8.30 0.56 0.74 4.3Br� 63 49.0 0.38 0.67 21.5I� 26 0.86 0.63 0.71 63.4CNO� 0.03 0.80 0.37 0.93 2.7SCN� 1.60 0.71 0.68 0.83CN� 0.02 0.79 0.54 0.62 1.0N3� 1.51 0.49 0.80 0.46 4.2

HCO3� 85 33.2 0.50 0.66 2.1

CO32� 73 39.3 0.42 0.84 0.65

NO3� 35 0.86 0.78 0.78 4.6

NO2� 63 0.48 0.67 0.57 4.1

HS� 0.04 0.58 0.58 0.86 4.0HSO3

� 89 21.1 0.63 55.3 4.9SnO3

2� 0.83 0.52 0.48 0.53 1.2SeO4

2� 112 0.57 0.65 24.5 0.47TeO4

2� 0.92 0.53 0.45 0.90 3.3P2O7

4� 48.50 0.69 0.75 0.83 2.7V2O7

4� 0.57 0.66 0.18 0.66 2.1B4O7

2� 0.95 0.67 0.68 0.86 2.5ReO4

� 0.75 0.80 0.82 0.52 >100RuO4

� 0.69 0.69 1.10 26.1 4.1S2O8

2� 0.084 84.6 0.93 0.87 9.2CS3

2� 0.0088 0.06 0.21 0.06 1.0Et2NCS2

� 3.1 0.004 0.0074 0.008 0.36SO4

2� >200 0.82 0.57 0.62 7.9ClO4

� >200 >200 6.50 >200 >200BF4

� >200 >200 >200 >200 >200FSO3

� 0.46 0.73 0.75 0.69 3.6NH(SO3)2

2� 0.76 0.75 0.70 50.9 4.9H2NSO2NH2 1.13 0.009 0.072 0.004 0.009H2NSO3H 0.39 0.042 0.094 0.005 0.009Ph�B(OH)2 23.1 0.041 0.073 0.008 0.075Ph�AsO3H2 49.2 0.005 0.092 0.006 0.007

a As sodium salt; nt = not tested.b Errors were in the range of 3–5% of the reported values, from three different

assays.c From Ref. 5c.d From Ref. 5g.e From Ref. 19a.f From Ref. 13f.g This work.

D. Vullo et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6706–6710 6709

In conclusion, we present here the first inhibition study of a CAdiscovered in Clostridia, the CpeCA from C. perfringens. The enzymewas poorly inhibited by iodide and bromide, and was inhibitedwith KIs in the range of 1–10 mM by a range of anions such as(thio)cyanate, azide, bicabonate, nitrate, nitrite, hydrogensulfite,hydrogensulfide, stannate, tellurate, pyrophosphate, divanadate,tetraborate, peroxydisulfate, sulfate, iminodisulfonate and fluoro-sulfonate. Better inhibitory power, with KIs of 0.36–1.0 mM wasobserved for cyanide, carbonate, selenate, selenocyanide, trithio-carbonate and diethyldithiocarbamate, whereas the best CpeCAinhibitors were sulfamate, sulfamide, phenylboronic acid andphenylarsonic acid, which had KIs in the range of 7–75 lM. Thisstudy thus provides the basis for developing better clostridialenzyme inhibitors with potential as antiinfectives with a newmechanism of action.

Acknowledgment

This work was financed in part by two FP7 EU project (Metoxiaand Dynano, to C.T.S. and A.S.), and by the Pennsylvania State Uni-versity Person Endowment (to J.G.F.).

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17. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561. An applied photophysics stopped-flow instrument has been used for assaying the CA catalysed CO2 hydration

6710 D. Vullo et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6706–6710

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 (pH 7.5,for a-CAs) or 20 mM TRIS (pH 8.3 for the b-CAs) as buffers, and 20 mM NaClO4

(for maintaining constant the ionic strength), following the initial rates of theCA-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 thekinetic parameters and inhibition constants. For each inhibitor at least sixtraces of the initial 5–10% of the reaction have been used for determining theinitial velocity. The uncatalyzed rates were determined in the same mannerand subtracted from the total observed rates. Stock solutions of inhibitor(10 mM) were prepared in distilled-deionized water and dilutions up to0.01 lM were done thereafter with the assay buffer. Inhibitor and enzymesolutions were preincubated together for 15 min at room temperature prior toassay, in order to allow for the formation of the E-I complex or for the eventualactive site mediated hydrolysis of the inhibitor. The inhibition constants wereobtained by non-linear least-squares methods using PRISM 3 and the Cheng-

Prusoff equation, as reported earlier,18,19 and represent the mean from at leastthree different determinations. All CA isoforms were recombinant onesobtained in-house as reported earlier.4,5,16.

18. Isik, S.; Kockar, F.; Arslan, O.; Ozensoy Guler, O.; Innocenti, A.; Supuran, C. T.Bioorg. Med. Chem. Lett. 2008, 18, 6327; Isik, S.; Kockar, F.; Aydin, M.; Arslan, O.;Ozensoy Guler, O.; Innocenti, A.; Supuran, C. T. Bioorg. Med. Chem. 2009, 17,1158; (a) Innocenti, A.; Mühlschlegel, F. A.; Hall, R. A.; Steegborn, C.;Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett 2008, 18, 5066.

19. (a) Nishimori, I.; Minakuchi, T.; Kohsaki, T.; Onishi, S.; Takeuchi, H.; Vullo, D.;Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2007, 17, 3585; (b)Maresca, A.; Carta, F.; Vullo, D.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013,28, 407.

20. (a) Winum, J. Y.; Innocenti, A.; Scozzafava, A.; Montero, J. L.; Supuran, C. T.Bioorg. Med. Chem. 2009, 17, 3649; (b) Innocenti, A.; Winum, J. Y.; Hall, R. A.;Mühlschlegel, F. A.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2009,19, 2642.