Bioorganic & Medicinal Chemistry Letters 23 (2013) 2647–2652

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Bioorganic & Medicinal Chemistry Letters

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Carbonic anhydrase inhibitors: Inhibition of the b-class enzyme fromthe pathogenic yeast Candida glabrata with sulfonamides, sulfamatesand sulfamides

0960-894X/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.bmcl.2013.02.092

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

Daniela Vullo a, Worraanong Leewattanapasuk b, Fritz A. Mühlschlegel b, Antonio Mastrolorenzo c,Clemente Capasso d, Claudiu T. Supuran a,e,⇑a Università degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino, Florence, Italyb School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, United Kingdomc Università degli Studi di Firenze, Centro MTS, Dipartimento di Dermatologia, Firenze, Italyd Istituto di Biochimica delle Proteine—CNR, Via P. Castellino 111, 80131 Napoli, Italye Università degli Studi di Firenze, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche e Nutraceutiche, Via Ugo Schiff 6, I-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 6 February 2013Revised 18 February 2013Accepted 20 February 2013Available online 1 March 2013

Keywords:Carbonic anhydraseb-Class enzymeInhibitorSulfonamideSulfamateSulfamideAntifungal

The fungal pathogen Candida glabrata encodes for a b-carbonic anhydrase (CA, EC 4.2.1.1), CgNce103,recently discovered. Only anions have been investigated as CgNce103 inhibitors up until now. Here wereport the first sulfonamides inhibition study of this enzyme. Simple sulfonamides showed weak or mod-erate CgNce103 inhibitory properties, whereas acetazolamide, and a series of 4-substituted ureido-ben-zene-sulfonamides, sulfamates and sulfamides showed effective CgNce103 inhibitory properties, with KIsin the range of 4.1–115 nM, being also ineffective as human CA II inhibitors. As there is significant resis-tance of C. glabrata clinical isolates to many classical antifungal agents, inhibition of the b-CA from thisorganism may allow an interesting means of controlling the pathogen growth, eventually leading to anti-fungals with a novel mechanism of action.

� 2013 Elsevier Ltd. All rights reserved.

Carbonic anhydrases (CAs, EC 4.2.1.1) are abundant in fungi andyeasts, although these enzymes have only recently started to becharacterized and studied in detail.1–9 Saccharomyces cerevisiae,Candida albicans and Candida glabrata each have one b-CA,1–4 butmultiple copies of b- and/or -CA-encoding genes were reportedin other pathogenic fungi.5 Recent work demonstrated that theseenzymes which catalyze the interconversion between CO2, bicar-bonate and protons,10 play an important role in the CO2-sensingof the fungal pathogens and in their regulation of sexual develop-ment.1–3 In fact physiological concentrations of CO2=HCO�3 induceprominent virulence attributes in C. albicans (filamentation) andC. glabrata or Cryptococcus neoformans (capsule biosynthesis)through direct activation of the fungal adenylyl cyclase.1–3

CO2=HCO�3 equilibration by fungal b-CAs equally plays a criticalrole in fungal CO2 sensing and pathogenesis. For example, the C.albicans enzyme CaNce103 or the analogous C. glabrata one(CgNce103) are essential for pathogenesis of these fungi in nicheswhere the available CO2 is limited (e.g., the skin), or essential for

the growth of C. neoformans in its natural environment (the en-zyme from this pathogen is denominated Can2).7 The newly dis-covered bZIP transcription factor CgRca1p is also required inthese processes in C. albicans, C. glabrata and S. cerevisiae.1–3,8,9a

Thus, the link between cAMP signaling and CO2=HCO�3 sensing isconserved in fungi and revealed CO2 sensing to be an importantmediator of fungal metabolism and pathogenesis.1–3,8

In the model organism S. cerevisiae it has been shown that the b-CA is required for providing sufficient bicarbonate to essential met-abolic carboxylation reactions of the yeast metabolism, such asthose catalyzed by pyruvate carboxylase (PC), acetyl-CoA carboxyl-ase (ACC), carbamoyl phosphate synthase (CPSase) and phosphori-bosylaminoimidazole (AIR) carboxylase.9 This enzyme (calledScCA) has also been investigated in detail for its inhibition and acti-vation with a range of modulators of activity.9b Finally, anotheryeast which has been investigated ultimately for the presence ofCAs is Malassezia globosa, the organism causing dandruff.11 As theabove mentioned fungi/yeasts, M. globosa also contains only oneb-CA, denominated MgCA.11

The main interest in the CAs encoding by these organisms isthat of finding modulators of their activity (inhibitors and/or

Table 1CA inhibition data with sulfonamides 1–22 and clinically used derivatives AAZ-VLXby a stopped-flow CO2 hydrase assay against hCA II and the fungal enzymes Can2,CaNce103 and CgNce10319

Inhibitor KIa (nM)

hCA II b Can2b CaNce103c CgNce103d

1 295 379 1086 91702 240 765 7627 80113 495 440 1277 494 320 1150 1204 8465 170 18,490 1109 8656 160 1394 3195 5507 60 809 1240 988 110 605 1345 979 40 977 408 10910 70 711 719 12811 63 968 6115 9712 75 300 874 8613 60 791 832 9514 19 815 1368 78615 2 42 1092 10616 46 971 1310 10817 50 624 727 9818 33 3887 1108 8319 12 379 1293 42020 80 623 383 6121 125 878 1209 4822 133 484 861 53AAZ 12 10.5 132 11MZA 14 63 484 118EZA 8 87 1070 3125DCP 38 1203 909 112DZA 9 8347 887 6635BRZ 3 87 1095 4350BZA 9 23 1510 952TPM 10 367 1108 110SLP 40 812 760 113IND 15 963 1090 94ZNS 35 971 942 114CLX 21 3056 1017 116VLX 43 704 699 97

a Errors in the range of ±10% of the reported values (data not shown) from threedifferent assays.

b From Ref. 7.c From Ref. 13b.d This work.

2648 D. Vullo et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2647–2652

activators) which may have pharmaceutical applications.3 Forexample, several sulfonamides have been investigated as in vitroMgCA inhibitors and thereafter for the in vivo inhibition of growthof M. globosa, Malassezia dermatis, Malassezia pachydermatic andMalassezia furfur.11 A mouse model of dandruff infection thenshowed that treatment with sulfonamide CA inhibitors (CAIs) ledto fragmented fungal hyphae, a situation also observed after treat-ment with ketoconazole, a clinically used antifungal agent.11 Suchdata offered the proof-of-concept that MgCA is a new antidandruffdrug target, and more generally that fungal CAs may be interestingdrug targets.11 Indeed, the enzymes from the above mentionedorganisms were investigated ultimately for their inhibition withmost classes of CAIs, such as inorganic anions, sulfonamides andtheir isosteres (sulfamates), carboxylates and boronic acids.3,4,6–15

A notable exception is however constituted by the C. glabrata en-zyme, CgNce103, for which only anion inhibition data are availablein the literature.6

As drug resistance problems to the clinically used antifungalsrepresents a challenging medical problem for the C. glabrata infec-tion,16–18 it is important to investigate non-classical drug targets,and the b-CA CgNce103 may represent an interesting, still poorlyinvestigated candidate. Indeed, in comparison to other Candidaspecies, C. glabrata infections are more difficult to treat due tothe organism’s innate resistance to azole antifungal agents, includ-ing the widely prescribed drug fluconazole.16–18 Furthermore, C.glabrata can easily develop antifungal drug resistance during pa-tient treatment.18

Here we report a detailed inhibition study of CgNce103 with alarge number sulfonamides, sulfamates and sulfamides, comparingits inhibition profile with those of the orthologs Can2 (from C. neo-formans) and CaNce103 (from C. albicans). Interesting structure–activity relationship (SAR) as well as several potent in vitro inhib-itors of CgNce103 are reported in the present Letter.

Sulfonamides and sulfamates are well-known inhibitors of a-and b-CAs, many of them possessing clinical applications as diuret-ics, antiglaucoma, antiobesity, anticonvulsant or antitumoragents.10d,15 A large number of such derivatives was investigatedfor the inhibition of the fungal enzymes CaNce103 and Can2 in ear-lier communications from our groups.7,13,15 Simple aromatic andheteroaromatic sulfonamides of types 1–22 were among them, aswell as derivatives AAZ–VLX, which are clinically used drugs.10d

Acetazolamide AAZ, methazolamide MZA, ethoxzolamide EZAand dichlorophenamide DCP, are the classical, systemically actingCAIs.10d Dorzolamide DZA and brinzolamide BRZ are topically-act-ing antiglaucoma agents, benzolamide BZA is an orphan drugbelonging to this class of pharmacological agents, whereas topira-mate TPM and zonisamide ZNS are widely used antiepilepticdrugs.10d Sulpiride SLP and indisulam IND were recently shownby this group to belong to this class of pharmacological agents.10d

The COX2 selective inhibitors celecoxib CLX and valdecoxib VLXwere also included in the study, as they were recently shown toact as CAIs.10f,g Sulfonamides 1–22 and the clinically used agentsinvestigated in this study were either commercially available, orwere prepared as reported earlier by our group.10,13–15

Inhibition data with sulfonamides 1–22 and the cliniaclly usedagents AAZ–VLX are shown in Table 1, and they were obtained by astopped-flow CO2 hydrase assay.19–21 For comparison reasons,inhibition data of the same compounds against the mammalianhCA II (offtarget) enzyme as well as the fungal ones Can2 andCaNce103, reported earlier by us,7,13b are also shown. The follow-ing SAR is observed from the data of Table 1, regarding inhibitionof CgNce103 with these compounds:

(i) A first group of derivatives, including 1, 2, 4–6, 14, 19, EZA,DZA, BRZ and BZA showed weak CgNce103 inhibitory activ-ity, with inhibition constants in the range of 420–9170 nM.

It may be observed that they include the simple aminosulf-onamides orthanilamide and sulfanilamide, together with 4-substituted benzenesulfonamides incorporating alkyl(methyl) or aminoalkyl moeities (4–6). The heterocyclicderivatives 14 and 19 incorporating 1,3,4-thiadiazoline- or1,3,4-thiadiazole-2-sulfonamide moieties are also in thisgroup of inactive compounds together with the bulky het-erocyclic derivatives in clinical use (EZA, DZA, BRZ andBZA). It is thus obvious that bulky, bicyclic ring systemsare detrimental for the inhibition of CgNce103, althooughthe a-class enzyme hCA II is potently inhibited by all ofthem, with inhibition constants in the low nanomolar range(Table 1).

(ii) Better CgNce103 inhibitory activity was observed for the fol-lowing sulfonamides 3, 7–13, 15–18, 20–22, MZA, DCP, SLP–VLX, and one sulfamate, TPM. These compounds showed KIsin the range of 49–128 nM (Table 1). It may be observed thata rather large number of scaffolds, both of aromatic, sugar-like as well as heterocyclic derivatives show this behaviorof medium-potency CgNce103 inhibitor. The SAR is thus dif-ficult to delineate due to the heterogeneity of the investi-gated structures. However several trends are observed,such as for example the fact that the presence of halogenatoms in the molecule of sulfanilamide 2 (a very weak

Table 2CA inhibition data with sulfonamides 23, sulfamates 24 and sulfamides 25 by astopped-flow CO2 hydrase assay, against hCA II and the fungal enzymes Can2,CaNce103 and CgNce10319

SO2NH2NH

NH O

R X-SO2NH2NH

NH O

R

23 24: X = O, 25: X = NH

Inhibitor R KIa (nM)

hCA II b Can2c CaNce103b CgNce103c

23a Ph 3730 69 1086 7323b PhCH2 2200 72 547 8123c PhCH2CH2 660c 79 124c 5623d 4-ClC6H4 781 17.3 62 7.023e 4-BrC6H4 1290 18.2 45 5.423f 3-O2NC6H4 15 62 40.1 6.124a Ph 393c 537 141c 9.624b PhCH2 274c 326 137c 8.424c PhCH2CH2 213d 110 133c 7.524d 4-ClC6H4 291d 118 505c 9.424e 3-ClC6H4 280d 562 124c 8924f 2-NCC6H4 541d 1203 156c 8924g 9H-Fluoren-2-yl 750d 1452 76c 11524h 4-O2NC6H4 450d 87 106c 6.424i 2-Br-4,6F2C6H2 641d 125c 84c 5.024j 2,3,4-F3CC6H2 286d 123c 76c 5.824k 1-Naphthyl 298d 1237c 1431c 8925a PhCH2 1462e 134 146c 24.325b PhCH2CH2 152e 121 130c 15.125c 2-PhC6H4 894e 148c 72c 6.725d 2-i-PrC6H42 673e 187c 55c 33.825e 3-O2NC6H4 423e 62c 109c 4.125f 4-ClC6H4 315e 24 102c 5.525g 4-nBuOC6H4 312e 77c 135c 13.925h 3,5-Me2C6H3 674e 51c 139c 8.525i Furan-2-ylCH2 345e 43c 58c 24.125j Cyclohexyl 354e 176c 134c 80.1

a Errors in the range of ±10% of the reported values (data not shown) from threedifferent assays.

b From Ref. 15b.c This work.d From Ref. 20.e From Ref. 21.

D. Vullo et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2647–2652 2649

inhibitor) leads to a drastic increase of the potency, in deriv-atives 7–10, with those incorporating fluorine and chlorinebeing more effective than those incorporating the heavierhalogens (9 and 10). The 1,3-disulfamoyl-benzenes 11, 12and DCP also showed rather comparable inhibitory activityagainst CgNce103, with KIs in the range of 86–112 nM. Sev-eral unexpected findings should also be noticed. For exam-ple, derivatives 13 and 14 differ only by an extramethylene group in the second derivative, which was 8.3-times a less effective CgNce103 inhibitor compared to 13.The same is observed comparing the pair acetazolamideAAZ—methazolamide MZA. The last compound (again withan extra CH2 compared to acetazolamide) was 10.7 times aweaker CgNce103 inhibitor compared to AAZ, which wasthe best inhibitor in this series of derivatives, with a KI of11 nM (very much comparable to the inhibition constantof this compound against hCA II). Thus, many chemotypesamong those investigated here led to interesting inhibitionof the fungal enzyme, but only one compound, AAZ was apotent inhibitor. The main drawback of this compound isthe fact that it strongly inhibits many other a- or b-CAs,which may lead to side effects in case of its use in vivo.

For this reason we decided to investigate as CgNce103 inhibi-tors a series of sulfonamides, sulfamates and sulfamides, of types23–25, which were recently reported20,21 by one of our groups topossess potent inhibitory activity against the fungal enzyme fromC. albicans,15b but also selectivity for inhibiting this enzyme overthe offtarget human ones hCA I and II (Table 2). As seen from dataof Table 2, these compounds incorporating 4-substituted-ureidomoieties in their molecule, generally showed very good inhibitoryactivity against CgNce103, with KIs in the range of 4.1–115 nM.Furthermore, except one of them, 23f, they were also much weakerhCA II inhibitors. Indeed, 23f was a potent hCA II inhibitor (KI of15 nM)22 and these properties were also rationalized by means ofX-ray crystallography of its adduct with hCA II,22 but all other sul-fonamides, sulfamates and sulfamides of Table 2 are much weakerhCA II inhibitors than 23f, with KIs in the range of 152–3730 nM.

From data of Table 2 it may be observed that some of the inves-tigated derivatives, that is, sulfonamides 23a–23c, sulfamates,24e–24g and 24k, and the sulfamide 25j possessed KIs againstCgNce103 in the range of 56–115 nm, being thus the least effectivesuch inhibitors in the subseries. They incorporate phenyl, benzyland phenethyl moieties (for the sulfonamides) and 2- or 3-substi-tuted phenyl, fluoren-2-yl or 1-naphthyl moieties in the case of thesulfamates. One obvious SAR feature is that the rather bulky fluo-ren-2-yl or 1-naphthyl moieties lead to less effective CgNce103inhiibtors compared to more compact phenyl-substituted groups.However in the case of the sulfamates, this trend is more compli-cated as some compounds with this last substitution pattern werein this category of derivatives (e.g., 24e and 24f). The only sulfam-ide which was in this sub-group was 25j which incorporates acyclohexyl moiety in its scaffold.

The remaining sulfonamides 23d–23f, the sulfamates 24a–24d,24h–24j and the sulfamides 25a–25i were more effective, lownanomolar CgNce103 inhibitors (KIs of 4.1–33.8 nM). For thisgroup of derivatives the SAR is indeed very interesting. The sulfa-mates and sulfamides possessing the same tail (e.g., phenethylure-ido, as in 24c and 25b) were more effective CgNce103 inhibitorscompared to the corresponding sulfonamide (23c). The same wasobserved for example for the sulfonamide/sulfamate pair 23b/24b, with the sulfamate being 10 times a better CgNce103 inhibitorcompared to the corresponding sulfonamide, but the sulfamide25a showed in this case a less effective inhibition of CgNce103(KI of 24.3 nM). However for the 4-chlorophenyl-substitutedderivatives, all these inhibitors, that is, the sulfonamide (23d),

the sulfamate (24d) and the sulfamide (25f) showed comparable,and very strong CgNce103 inhibitory activities (KIs of 5.5–9.4 nM). Among the investigated sulfamides, many other substitu-tion patterns (R moieties) led to effective CgNce103 inhibitors,such as among others the 2-biphenyl (25c), 3-nitrophenyl (25e),4-n-butoxyphenyl (25g), or 3,5-dimethylphenyl (25h) whichshowed inhibition constants in the range of 4.1–13.9 nM (Table 2).

It should be noted that the selectivity profile for inhibitingCgNce103 over hCA II with some of these compounds (e.g., 23d,23e, and 25f) was excellent, with selectivity ratios in the range of57–238. This means that a compound of this type would stronglyinhibit the fungal enzyme but not the widespread, offtarget humanisoform hCA II.

In order to try to rationalize these inhibition data (and consid-ering that no X-ray crystal structure of CgNce103 or CaNce103are available), we present an alignment of the amino acid se-quences of several fungal CAs investigated earlier7–9,11–15 withthe CgNce103 sequence (Fig. 1) and a phylogenetic tree for a largenumber of b-class enzymes from bacteria, plants, algae, fungi andone insect (the only b-CA investigated in detail in an arthropod isthe enzyme from Drosophila melanogaster).23

From data of Figure 1. it may be seen that similar with otherfungal b-CAs investigated in some detail earlier,1–12 such as the en-zymes from C. albicans, C. neoformans, S. cerevisiae and M. globosa,

Figure 1. Alignment of the acid sequences of selected b-CAs from five fungal species. Can 2 numbering system was used. Zinc ligands are indicated in red; amino acidsinvolved in the enzyme catalytic cycle are indicated in blue. The asterisk (⁄) indicates identity at a position; the symbol (:) designates conserved substitutions, while (.)indicates semi-conserved substitutions. Multiple alignment was performed with the program Clustal W, version 2.1. Legend: CgNce103, Candida glabrata (Accession number:XP_446428.1); MgCA, Malassezia globosa (Accession number: XP_001730815.1); CaNce, Candida albicans (Accession number: XP_721792.1); ScCA, Saccharomyces cerevisiae(Accession number: GAA26059); Can2, Cryptococcus Neoformans (Accession number: GI:219109194).

2650 D. Vullo et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2647–2652

CgNce103 possesses the conserved amino acid residues involved inthe catalysis and the binding of inhibitors. Indeed, the Zn(II) li-gands are two Cys and one His residues (Cys68, His124 andCys127, respectively), being thus obvious that similar to Can2, anenzyme for which the X-ray crystal structure was reported,7

CgNce103 has an ‘open’ active site, meaning that the fourth zinc li-gand is the water molecule/hydroxide ion acting as nucleophile inthe catalytic cycle.4,7 Furthemore, the catalytic dyad Asp70–Arg72(and the amino acid separating these two residues, Ser71) are alsoconserved in all fungal b-CAs (Fig. 1). These amino acids are in-volved in the activation of the zinc-bound water molecule, as theyform a relay of hydrogen bonds involving the water molecule, theCOOH moiety of Asp70 and the guanidinium moiety of Arg72,which greatly enhances the nucleophilicity of the zinc-boundwater and favor catalysis.4,7 There is also a cluster of hydrophobicamino acids, conserved in all these enzymes, which is involved inthe binding of inhibitors, as demonstrated by the above-mentionedcrystallographic work on Can2,7 and by homology modeling anddocking studies for the interaction of sulfonamides withCaNce103.12 There are however many diffeernces between theseenzymes, as observed from data of Figures 1 and 2. For example,CaNce103 has a long amino terminal sequence which is not pres-ent in other enzymes investigated so far, whereas the M. globosaand C. glabrata CAs are the shortest ones in the group of fungal/yeast b-CAs considered here. These differences of amino acidswithin the active sites of these enzymes also account for the

different inhibition profiles of Can2, CaNce103 and CgNce103 withthe sulfonamides, sulfamates and sulfamides investigated in thisLetter (Tables 1 and 2).

By examining the phylogenetic tree of Figure 2, it may be ob-served that all the fungal b-CAs cluster together on neighboringbranches, whereas the bacterial enzymes and the D. melanogasterone (which is mitochondrial,23 having thus bacterial ‘ancestors’)are positioned in the upper part of the tree. Only two bacterialCAs (the ones from Burkholderia thailandensis and Brucella suis)are found in the originary, ancestral group from which the b-CAsin plants, algae and fungi originated. Returning to the fungal en-zymes, it is rather surprising (at first sight) to observe that themost related enzyme to the CgNce103 is the yeast one from S.cerevesiae (and not the C. albicans one, which is found alone on aseparate branch). However, conidering the long amino terminalextension mentioned earlier present in CaNce103 (and evidencedin Fig. 1), this is afterwards not so surprising. It is also interestingto note that the M. globosa enzyme is the most related one to Can2from C. neoformans. As mentioned earlier, the sister clade to thefungal b-CAs comprises some algal and bacterial enzymes (posi-tioned in the lower branches of the tree from Fig. 2).

In conclusion, we report a detailed study of sulfonamide, sulfa-mate and sulfamide inhibitors of CgNce103, and enzyme belongingto the b-CA class. Many simple sulfonamides showed weak or mod-erate CgNce103 inhibitory properties, whereas acetazolamide, aswell as some 4-substituted ureido-benzene-sulfonamides,

Figure 2. Phylogenetic tree of the b-CAs from selected prokaryotic and eukaryotic species. The tree was constructed using the program PhyML 3.0. Branch support values arereported at branch points. Legend: PgiCA_bacterium, Porphyromonas gingivalis ATCC 33277 (Accession number: YP_001929649.1); AbaCA_bacterium, Acinetobacterbaumannii AB307-0294 (Accession number: YP_002326524); MinCA_bacterium, Myroides injenensis M09-0166 (Accession number: ZP_10784819); DmeCA_insect,Drosophila melanogaster (Accession number: NP_649849); Cab_bacterium, Methanobacterium thermoautotrophicum (Accession number: GI:13786688); HpyCA_bacterium,Helicobacter pylori (Accession number: BAF34127.1) LpnCA_bacterium, Legionella pneumophila 2300/99 (Accession number: YP_003619232); EcoCa_bacterium, Escherichiacoli (Accession number: ACI70660); VraCA_plant, Vigna radiata (Accession number: AAD27876); PsaCA_plant, Pisum sativum (Accession number: AAA33652); FbiCA_plant,Flaveria bidentis, isoform I (Accession number: AAA86939.2); AthCA_plant, Arabidopsis thaliana (Accession number: AAA50156); ZmaCA_plant, Zea mays (Accession number:NP_001147846.1); OsaCA_plant, Oryza sativa (Accession number: AAA86943); MgCA_fungus, Malassezia globosa (Accession number: XP_001730815.1); Can2_fungus,Cryptococcus Neoformans (Accession number: GI:219109194); CaNce_fungus, Candida albicans (Accession number: XP_721792.1); ScCA_fungus, Saccharomyces cerevisiae(Accession number: GAA26059); DbrCA_fungus, Dekkera bruxellensis AWRI1499 (Accession number: EIF49256); OpaCA_fungus, Ogataea parapolymorpha DL-1 (Accessionnumber: EFW97556); AfuCA_fungus, Aspergillus fumigatus Af293 (Accession number: XP_751704); SmaCA_fungus, Sordaria macrospora (Accession number: CAT00781);TasCA_fungus, Trichosporon asahii CBS 8904 (Accession number: EKD04029); CspCA_alga, Coccomyxa sp. (Accession number: AAC33484.1); CreCA_alga, Chlamydomonasreinhardtii (Accession number: XP_001699151.1); BthCA_bacterium, Burkholderia thailandensis Bt4 (Accession number: ZP_02386321); SpoCA_fungus, Schizosaccharomycespombe (Accession number: CAA21790); BsuCA_bacterium, Brucella suis 1330 (Accession number: NP_699962.1).

D. Vullo et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2647–2652 2651

sulfamates and sulfamides showed effective inhibitory propertiesagainst CgNce103, with KIs in the range of 4.1–115 nM, being alsoineffective as human CA II inhibitors. As there is significant resis-tance of C. glabrata clinical isolates to the classical antifungalagents, inhibition of the b-CA from this fungus may allow an inter-esting means for controlling the pathogen growth, eventually lead-ing to antifungals with a novel mechanism of action.

Acknowledgments

This research was financed in part by two 7th FP EU projects(METOXIA and DYNANO) to CTS.

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19. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561. An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalysed CO2 hydrationactivity. Phenol red (at a concentration of 0.2 mM) has been used as indicator,working at the absorbance maximum of 557 nm, with 10–20 mM Hepes (pH

7.5) or TRIS (pH 8.3) as buffers, and 20 mM Na2SO4 or 20 mM NaClO4 (formaintaining 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 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(1 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. The inhibitionconstants were obtained by non-linear least-squares methods using PRISM 3,whereas the kinetic parameters for the uninhibited enzymes from Lineweaver–Burk plots, as reported earlier,7,20 and represent the mean from at least threedifferent determinations. Full length CgNCE103 was amplified by PCR fromgenomic DNA and ligated into the pGex-6P-2 vector and expressed in BL21E. coli as previously described.6–8 Recombinant protein was purified using FastFlow glutathione S-Sepharose (Amersham) as per the manufacturer’srecommendations. To prevent protein degradation 1 mM PMSF and 25 llproteinase cocktail (Roche complete mini EDTA-free, 1 tablet dissolved in 2 mlPBS) were added to samples prior to lysis. Elution fractions were combined anddialysed against 10 mM HEPES pH 7.5. Protein was size-fractionated ondenaturing 12% SDS Tris–HCl gels (BioRad) with 1� MOPS buffer (BioRad) at150 V.

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21. Tarko, L.; Supuran, C. T. Bioorg. Med. Chem. 2013, 21, 1404.22. Pacchiano, F.; Aggarwal, M.; Avvaru, B. S.; Robbins, A. H.; Scozzafava, A.;

McKenna, R.; Supuran, C. T. Chem. Commun. 2010, 46, 8371.23. (a) Syrjänen, L.; Tolvanen, M.; Hilvo, M.; Olatubosun, A.; Innocenti, A.;

Scozzafava, A.; Leppiniemi, J.; Niederhauser, B.; Hytönen, V. P.; Gorr, T. A.;Parkkila, S.; Supuran, C. T. BMC Biochem. 2010, 11, 28; (b) Maresca, A.; Carta, F.;Vullo, D.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 407.