Bioorganic & Medicinal Chemistry Letters 24 (2014) 1127–1132
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Anion inhibition studies of two new b-carbonic anhydrases from the bacterial pathogen Legionella pneumophila 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, Japan Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy Department of Chemistry, College of Science, King Saud University, PO Box 2455, Riyadh 11451, Saudi Arabia d Istituto di Biochimica delle Proteine—CNR, Via P. Castellino 111, 80131 Napoli, Italy e 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 27 November 2013 Revised 30 December 2013 Accepted 31 December 2013 Available online 8 January 2014 Keywords: Carbonic anhydrase b-Class enzyme Legionella pneumophila Anion Dithiocarbamate Sulfamide
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 and lpCA2, were found in the genome of this pathogen. These enzymes were determined to be efficient catalysts for CO2 hydration, with kcat values in the range of (3.4–8.3) 105 s1 and kcat/KM values of (4.7–8.5) 107 M1 s1. A set of inorganic anions and small molecules was investigated to identify inhibitors 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). The most 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 most potent lpCA2 inhibitors were diethyldithiocarbamate, sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid, with KI values ranging from 2 to 13 lM. As these enzymes seem to be involved in regulation of phagosome pH during Legionella infection, inhibition of these targets may lead to antibacterial agents with a novel mechanism of action. Ó 2014 Elsevier Ltd. All rights reserved.
Legionella pneumophila is a Gram-negative environmental bacterium that normally infects amoebae.1,2 It was discovered in 1976 when it provoked a life-threatening pneumonia-like disease in many participants at the 58th Annual Convention of the American Legion in Philadelphia. This condition was subsequently dubbed Legionnaires’ disease, or legionellosis.1,2 When the bacterial pathogen was characterized in detail, it was shown that a large number of its subspecies and serovars are widespread in nature.2,3 It is now known that L. pneumophila and the related species Legionella longbeachae are responsible for legionellosis in humans. These pathogens are increasingly spread all over the world through the development of artificial water systems for air conditioning, cooling towers, and aerosolizing devices, among other applications.2,3 When infecting Amoeba or human macrophages, L. pneumophila utilizes a sophisticated biochemical machinery to invade its hosts. For example, L. pneumophila can induce the formation of acidic vacuoles within the host cytoplasm and hijack vesicles and organelles to enable effective infection.2 It has been demonstrated that L. pneumophila is able to maintain a neutral pH in its phagosome for at least 6 h, whereas vacuoles that did not contain the ⇑ Corresponding author. Tel.: +39 055 4573005; fax: +39 055 4573385. E-mail address: claudiu.supuran@unifi.it (C.T. Supuran). 0960-894X/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.12.124
bacterium became highly acidic within 15 min of their formation.3b Carbonic anhydrase (CA, EC 4.2.1.1), which hydrates carbon dioxide to bicarbonate and protons (reaction 1),6 is involved in pH control in organisms all over the phylogenetic tree, including bacterial4 and fungal pathogens.5
CO2 þ H2 O $ HCO3 þ Hþ
ðreaction 1Þ
It has been shown that enzymes in this family are closely connected with pH regulation in many pathogenic (as well as nonpathogenic) organisms, as well as with secretion of acidic or alkaline electrolytes.4–10 For example, the extracellular environment around hypoxic tumors becomes more acidic (pH 6.0–6.5)7 through the action of several proteins and enzymes, including the tumor-associated isoforms CA IX and XII.7 Several CAs have been shown to play a significant role in the pathogenicity, invasion and survival of many fungal (e.g., Cryptococcus neoformans,5a Candida albicans,5b Candida glabrata8) and bacterial (e.g., Helicobacter pylori,9a Brucella suis,9b Vibrio cholerae9c) pathogens. These enzymes act by signaling information on its local niche (e.g., low or high availability of CO2 or bicarbonate), or by allowing it to adapt to particular pH conditions (such as high acidity in the stomach for H. pylori,10 or alkalinity and high concentrations of bicarbonate for V. cholerae).11
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The genome of L. pneumophila was recently cloned,3a but no CAs from this bacterium have been reported to date. We previously explored its genome and detected two putative CAs belonging to the b-CA family (accession numbers NC_002942, which we designated as lpCA1, locus tag lpg2500, NCBI reference sequence WP_014844650.1; and lpCA2, locus tag lpg2194, NCBI reference sequence WP_014842179.1). Here, we report that these new b-CAs, which we designated as lpCA1 and lpCA2, are active enzymes for the physiologic hydration of CO2 to form bicarbonate and protons. We also investigated the inhibition profiles of these enzymes in the presence of inorganic anions and other small molecules 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 other bacterial a- and b-CAs.8–10 After removal of the GST tag from the chimeric protein, we obtained the pure enzymes that were investigated for catalytic activity in a stopped-flow CO2 hydrase assay.12 The activity of lpCA1 and lpCA2 can be compared to that of other a- and b-CAs from human (h),6 fungal (C. neoformans,5a Saccharomyces cerevisiae),13 plant (Flaveria bidentis),14 or bacterial (B. suis,9b and H. pylori9c) sources from the data shown in Table 1. Both of these new proteins, lpCA1 and lpCA2, possess enzymatic activity for the hydration of carbon dioxide to yield protons and bicarbonate that is typically catalyzed by CAs. 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),6 Can2 from the fungal pathogen C. neoformans,5b or the bacterial pathogenic enzymes HpyCA and BsuCA219, from H. pylori and B. suis, respectively.9 The second isoform, lpCA2, was more active than lpCA1, with the following kinetic parameters: kcat of 8.3 105 s1 and kcat/KM of 8.5 107 M1 s1 (Table 1). This level of activity is similar to that of the S. cerevisiae enzyme SceCA or to that of BsuCA213 (both enzymes belong to the b-CA family) and is only slightly lower than the activity of hCA II, a highly effective catalyst for the CO2 hydration reaction (Table 1).14,15 It was also observed that acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide), a clinically used drug,6 was an efficient inhibitor of the two Legionella enzymes, with KI values in a range from 36 to 95 nM. The moderately active enzyme (lpCA1) was less sensitive to this compound than was lpCA2, which showed greater catalytic activity and also higher affinity for the sulfonamide inhibitor (Table 1). To better understand the efficient catalytic properties of the two new enzymes, we aligned the amino acid sequences of lpCA1 and lpCA2 with those of other such enzymes that have been characterized previously, such as HpyCA, BsuCA213 and
BsuCA219 (Fig. 1). The data shown in Figure 1 indicate that as has been observed for all other bacterial b-CAs investigated to date, the two Legionella proteins lpCA1 and lpCA2 possess the 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, 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 and leads to the formation of the nucleophilic zinc-hydroxide species in the enzyme.5b,9–11 The proposed catalytic mechanism of the b-CAs is shown schematically in Figure 2. It has been in fact shown by means of kinetic and X-ray crystallographic studies that the catalytic mechanism of the b-CAs is rather similar to that of the a-class enzymes investigated in much greater detail.6,7,11 As mentioned above, the catalytically active species of the enzyme (A in Fig. 2) has a hydroxide ion coordinated to the Zn(II), in addition to the protein ligands (which in Fig. 2 are those from lpCA1, i.e., Cys90, His143 and Cys146). In the case of the a-CAs, the zinc hydroxide species is generated through a proton transfer process from the zinc coordinated water to the environment, assisted by the active site residue His64, acting as a proton shuttle.6,7 In the b-CAs the activation of the zinc-coordinated water molecule (shown in D in Fig. 2) is more complex than for the a-CAs, as the catalytic dyad mentioned above takes part in the process. Furthermore, the nature of the amino acid(s) acting as proton shuttle in the b-class enzymes is not completely elucidated (see discussion later in the text). Unknown is also the CO2 binding site within the b-CAs (B in Fig. 2) but as for the aCAs, probably this is a hydrophobic pocket not far away from the catalytic zinc ion. After the nucleophilic attack of the zinc hydroxide species of the enzyme on the bound CO2, bicarbonate is formed (C in Fig. 2) which presumably is bidentately coordinated to the zinc ion (as in the a-CAs).6,7,11 Inorganic anions are usually weak ligands of the zinc ion in metalloenzymes, so that species C is probably easily converted to species D, by a substitution reaction of the bicarbonate by means of a water molecule. This leads to liberation of the reaction product (bicarbonate) into solution, with generation of the acidic form of the enzyme, D, which is catalytically inactive. To generate the zinc hydroxide, catalytically effective species A, a proton transfer reaction must occur (Fig. 2), probably assisted by an active site residue. In the b-CA from the alga Coccomyxa, it has been hypothesized that there are two residues participating in this process, Tyr88 and His92. However, neither lpCA1 nor lpCA2 possess a sequence of Tyr and His residues close to each other as the algal enzyme, which leads the nature of the proton shuttling residue in these novel b-CAs unknown at this moment. The inhibition
Table 1 Kinetic parameters for the CO2 hydration12 reaction catalyzed by several carbonic anhydrases Isozyme hCA I hCA II Can2 SceCA FbiCA 1 HpyCA BsuCA219 BsuCA213 lpCA1 lpCA2
Activity level Moderate Very high Moderate High Low Moderate Moderate High Moderate High
Class
a a b b b b b b b b
kcat (s1) 5
2.0 10 1.4 106 3.9 105 9.4 105 1.2 105 7.1 105 6.4 105 1.1 106 3.4 105 8.3 105
kcat/KM (M1 s1)
KI (acetazolamide) (nM)
Refs.
5.0 107 1.5 108 4.3 107 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 82 27 40 63 303 95 36
6 6 5b 13 14 9c 9b 9b a a
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. Parameters are also reported for the b-CAs Can2 (from C. neoformans),5b SceCA (from S. cerevisiae),13 the Flaveria bidentis CA (FbiCA 1),14 the Helicobacter pylori b-CA HpyCA,9c the Brucella suis b-CAs BsuCA213 and BsuCA219,9b and the two new enzymes lpCA1 and lpCA2, measured at 20 °C, pH 8.3 in 20 mM TRIS buffer and 20 mM NaClO4. Inhibition data for the clinically used sulfonamide acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide) are also provided. a This work.
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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 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 as follows: Legionella pneumophila, 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.
-
OH
Zn Cys90 B
Inh
+ Inh( BH+)
2+
Cys146 His143
Zn Cys90
-H2O
2+
Cys146
His143 E
A
- BH +
-
+ CO2
OH2 Zn Cys90
2+
Cys146
His143
Zn
D
- HCO 3
O
H
Zn Cys90
Cys146 His143
O
2+
B H
Inh-
2+
O
Cys146 Cys90 His143
+ H2O
Inh-
H
OH
O O Zn
Cys90
O
2+
-
Cys146 His143 C
F Figure 2. Catalytic and inhibition mechanisms of lpCAs (exemplified by using lpCA1, and its amino acid residues numbering system). See text for details.
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mechanism of lpCA1 and lpCA2 (Fig. 2E and F) will be discussed later in the paper. We also examined the phylogenetic relationships of the two new bacterial b-CAs, lpCA1 and lpCA2, to other such enzymes characterized earlier. We compared these enzymes to those from various 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 tree in Figure 3, the two Legionella enzymes are highly similar, clustering on their own branch of the tree. The next most similar enzyme is isoform stCA2 from S. typhimurium. The remaining b-CAs all clustered together on the lower branch of the tree, with HpyCA, stCA1 and BsuCA219 residing on the same branch. The remaining Brucella isoform BsuCA213 was on a separate branch, thus indicating that it is 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 investigated as CA inhibitors (CAIs), as they bind to the metal ion from the enzyme active site and impair catalysis.6,8–11 Herein, we report the first inhibition study of the two Legionella enzymes with a range of such inhibitors, including simple and complex inorganic anions as well as sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid (Table 2). This set of anions and small molecules had been investigated previously for inhibition of a large number 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 lpCA1 and lpCA2 by the anions and small molecules shown in Table 2: (i) Perchlorate and tetrafluoroborate, anions known for their low affinity for metal ions in metalloenzymes (and sometimes also in solution),13–17 did not inhibit the two new b-CAs reported here (KI >200 mM). Similar results have been observed in most of the CAs examined to date: only HpyCA was effectively inhibited by perchlorate, with a KI of 6.5 mM.9c Sulfate was also an ineffective lpCA1 and lpCA2 inhibitor, with KI values between 77.9 and 96.5 mM (Table 2). Iodide and nitrate were also quite weak lpCA2 inhibitors, with inhibition constants of 59.1 and 30.1 mM, respectively. These anions were more effective inhibitors of lpCA1, vide infra. (ii) Another group of anions inhibited lpCA1 and lpCA2 weakly, with inhibition constants in the range of 3.5–9.1 mM. The weak inhibitors 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 submillimolar inhibitors against both lpCA1 and lpCA2. All of the halides, as well as cyanate, thiocyanate, stannate, tellurate, pyrophosphate, divanadate, tetraborate, perrhenate, perruthenate, peroxydisulfate, selenocyanate, and trithiocarbonate inhibited lpCA1 with KI values from 0.24 to 0.98 mM. Iminodisulfonate was slightly less effective
Table 2 Inhibition constants of anionic inhibitors against a-CA isozymes derived from human (hCA II) and bacterial (SspCA)15 sources. Inhibition constants are also reported for bCA from a bacterium (H. pylori)9c HpyCA, the plant Flaveria bidentis isoform 1 (FbiCA 1),14 and the new enzymes lpCA1 and lpCA2 from L. pneumophila. Values were determined at 20 °C in a stopped flow CO2 hydrase assay12 Inhibitor§
KI (mM)# a
hCA II
SspCA
a
a
HpyCAc b
FbiCA 1d b
lpCA1e b
lpCA2 b
>300 200 63 26 0.03 1.60 0.02 1.51 85 73
41.7 8.30 49.0 0.86 0.80 0.71 0.79 0.49 33.2 39.3
0.67 0.56 0.38 0.63 0.37 0.68 0.54 0.80 0.50 0.42
0.71 0.74 0.67 0.71 0.93 0.83 0.62 0.46 0.66 0.84
0.91 0.79 0.65 0.32 0.66 0.52 0.064 0.077 3.5 4.7
0.77 0.81 8.0 59.1 0.96 0.88 0.61 0.45 6.6 4.8
SnO2 3
35 63 0.04 89 0.83
0.86 0.48 0.58 21.1 0.52
0.78 0.67 0.58 0.63 0.48
0.78 0.57 0.86 55.3 0.53
7.6 7.9 0.076 6.6 0.57
30.1 5.8 0.51 7.2 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
P2 O4 7
48.50
0.69
0.75
0.83
0.94
0.83
V2 O4 7
0.57
0.66
0.18
0.66
0.39
0.47
B4 O2 7 ReO 4 RuO 4
0.95
0.67
0.68
0.86
0.60
0.55
0.75 0.69 0.084
0.80 0.69 84.6
0.82 1.10 0.93
0.52 26.1 0.87
0.89 0.82 0.85
0.77 0.86 0.57
0.086 0.0088
0.07 0.06
0.97 0.21
0.88 0.06
0.98 0.53
0.66 0.62
3.1 >200
0.004 0.82
0.0074 0.57
0.008 0.62
0.006 77.9
0.002 96.5
>200 >200 0.46 0.76
>200 >200 0.73 0.75
6.50 >200 0.75 0.70
>200 >200 0.69 50.9
>200 >200 9.1 1.17
>200 >200 0.46 0.59
1.13 0.39 23.1 49.2
0.009 0.042 0.041 0.005
0.072 0.094 0.073 0.092
0.004 0.005 0.008 0.006
0.094 0.076 0.065 0.084
0.009 0.013 0.006 0.008
F Cl Br I CNO SCN CN N 3 HCO 3 CO2 3 NO 3 NO 2 HS HSO 3
S2 O2 8 SeCN CS2 3 Et2 NCS 2 SO2 4 ClO 4 BF4 FSO 3 NHðSO3 Þ2 2 H2 NSO2 NH2 H2 NSO3 H Ph-BðOHÞ2 Ph-AsO3 H2
b
e
§
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. #
as an lpCA1 inhibitor (KI of 1.17 mM). The effective, submillimolar inhibitors of lpCA2 were fluoride, chloride, cyanate, thiocyanate, cyanide, azide, hydrogen sulfide, stannate, tellurate, pyrophosphate, divanadate, tetraborate, perrhenate, perruthenate, peroxydisulfate, selenocyanate, trithiocarbonate, fluorosulfonate and
Figure 3. Phylogenetic tree produced using the b-CA amino acid sequences aligned in Figure 1. The tree was constructed using the program PhyML 3.0, which is phylogeny software based on the maximum-likelihood principle. Branch support values are reported at branch points. Organisms, NCBI sequence numbers and cryptonyms are indicated in Figure 1, except for Salmonella typhimurium,16 stCA1 and stCA2 (NCBI sequence numbers: NP_459176.1 for stCA1, and YP_007905873.1, for stCA2).
I. Nishimori et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1127–1132
iminodisulfonate. These ions showed KI values ranging from 0.29 to 0.96 mM. The best inhibitor in this subseries was tellurate, with KI values of 0.24 and 0.29 mM against lpCA1 and lpCA2, respectively. This is a very potent inhibitor compared to the salts of elements from the same group. Specifically, the Se(VI) and S(VI) derivatives selenate and sulfate were much weaker inhibitors of the two enzymes (Table 2). (iv) The best anionic inhibitors of lpCA1 detected in this study were 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 from these data. The metal ‘poisons’ cyanide, azide, and hydrogen sulfide had very similar inhibitory effects against lpCA1. This similarity can be extended further to the four small molecules from Table 2, that is, sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid. In fact all these compounds inhibited the enzymes over a narrow range of potencies, with inhibition constants from 64 to 94 lM. The compound N,N-diethyldithiocarbamate had a much higher affinity for this enzyme, with a low micromolar value for KI of 6 M. In fact, the dithiocarbamates were recently reported as a potent new class of CAIs targeting both the a- and b-classes of such enzymes.18 However, all of the small molecules were low micromolar inhibitors of lpCA2, with KI values from 2 to 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 Legionella enzymes towards the anionic inhibitors investigated here. We also observed significant differences between these two enzymes, lpCA1 and lpCA2, and other a- and b-class CAs for which such data are available. These include the enzymes shown in Table 2, which are of human, bacterial or plant origin. Thus, lpCA1 seems to have higher affinity for some poisonous metal anions such as cyanide, azide, and hydrogen sulfide, which are around one order of magnitude more potent against lpCA1 than lpCA2. However, lpCA2 showed higher affinity for N,N-diethyldithiocarbamate, sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid compared to lpCA1. The heavy halides, bromide and iodide, also behaved quite differently against the two enzymes. These two anions are submillimolar inhibitors of lpCA1 and much weaker inhibitors of lpCA2 (KI values ranging from 8 to 59 mM). However, there were also anions that showed comparable affinities for these two enzymes (e.g., chloride, bicarbonate, carbonate, tellurate—see Table 2). The inhibitory potencies of these anions against lpCA1 and lpCA2 were also quite different when compared to those observed in other bacterial (SspCA or HpyCA), plant (FbiCA 1) or human (hCA II) enzymes. For example, while chloride was a submillimolar inhibitor of lpCA1, lpCA2, FbiCA 1 and HpyCA, it showed KI values from 8.3 to 200 mM against the extremophilic bacterial enzyme SspCA or human hCA II (Table 2). Trithiocarbonate was an effective inhibitor of hCA II, SspCA and FbiCA 1, but was less effective against HpyCA, lpCA1 and lpCA2. The inhibition mechanism of these anions against lpCA1 and lpCA2 is probably similar to that of the inorganic anions against a-CAs, investigated in greater detail, by means of kinetic, spectroscopic and X-ray crystallographic techniques.6,7,11,16 As shown in Figure 2, the inhibitor may substitute the fourth, non-protein zinc ligand, 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 shown to possess this inhibition mechanism by means of high resolution X-ray crystallography. An alternative inhibition mechanism has been also reported again for the Coccomyxa b-CA11c for iodide, which was found anchored to the zinc-coordinated water
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molecule/hydroxide ion, as shown schematically in Figure 2F. This mechanism was also observed for phenols,19 polyamines 20 and sulfocoumarins21 for the inhibition of a-CAs. In conclusion, we investigated the cloning, catalytic activity and anion inhibition of two b-CAs from the bacterial pathogen L. pneumophila, lpCA1 and lpCA2. The two new enzymes were efficient catalysts for CO2 hydration, with kcat values ranging from (3.4– 8.3) 105 s1 and kcat/KM of (4.7–8.5) 107 M1 s1. A set of inorganic anions and small molecules was investigated for inhibition of these enzymes. Perchlorate and tetrafluoroborate were not active inhibitors (KIs >200 mM), whereas sulfate was a very weak inhibitor of both lpCA1 and lpCA2 (KIs of 77.9–96.5 mM). The most potent lpCA1 inhibitors were cyanide, azide, hydrogen sulfide, N,N-diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with KI values in a range from 6 to 94 lM. The most active lpCA2 inhibitors were N,N-diethyldithiocarbamate, sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid, with KI values between 2 and 13 lM. As these enzymes seem to be involved in regulation of phagosome pH during Legionella infection, inhibition of these targets may lead to antibacterial 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 stoppedflow 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
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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 the initial rates of the CA-catalyzed CO2 hydration reaction for a period of 10– 100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor, at least six traces of the initial 5–10% of the reaction were used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitors (10 mM) were prepared in distilled–deionized water, and dilutions up to 0.01 M were performed thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay 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 Cheng–Prusoff equation, whereas the kinetic parameters for the uninhibited enzymes 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.; De Luca, 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.
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Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Anion inhibition studies of two new b-carbonic anhydrases from the bacterial pathogen Legionella pneumophila 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, Japan Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy Department of Chemistry, College of Science, King Saud University, PO Box 2455, Riyadh 11451, Saudi Arabia d Istituto di Biochimica delle Proteine—CNR, Via P. Castellino 111, 80131 Napoli, Italy e 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 27 November 2013 Revised 30 December 2013 Accepted 31 December 2013 Available online 8 January 2014 Keywords: Carbonic anhydrase b-Class enzyme Legionella pneumophila Anion Dithiocarbamate Sulfamide
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 and lpCA2, were found in the genome of this pathogen. These enzymes were determined to be efficient catalysts for CO2 hydration, with kcat values in the range of (3.4–8.3) 105 s1 and kcat/KM values of (4.7–8.5) 107 M1 s1. A set of inorganic anions and small molecules was investigated to identify inhibitors 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). The most 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 most potent lpCA2 inhibitors were diethyldithiocarbamate, sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid, with KI values ranging from 2 to 13 lM. As these enzymes seem to be involved in regulation of phagosome pH during Legionella infection, inhibition of these targets may lead to antibacterial agents with a novel mechanism of action. Ó 2014 Elsevier Ltd. All rights reserved.
Legionella pneumophila is a Gram-negative environmental bacterium that normally infects amoebae.1,2 It was discovered in 1976 when it provoked a life-threatening pneumonia-like disease in many participants at the 58th Annual Convention of the American Legion in Philadelphia. This condition was subsequently dubbed Legionnaires’ disease, or legionellosis.1,2 When the bacterial pathogen was characterized in detail, it was shown that a large number of its subspecies and serovars are widespread in nature.2,3 It is now known that L. pneumophila and the related species Legionella longbeachae are responsible for legionellosis in humans. These pathogens are increasingly spread all over the world through the development of artificial water systems for air conditioning, cooling towers, and aerosolizing devices, among other applications.2,3 When infecting Amoeba or human macrophages, L. pneumophila utilizes a sophisticated biochemical machinery to invade its hosts. For example, L. pneumophila can induce the formation of acidic vacuoles within the host cytoplasm and hijack vesicles and organelles to enable effective infection.2 It has been demonstrated that L. pneumophila is able to maintain a neutral pH in its phagosome for at least 6 h, whereas vacuoles that did not contain the ⇑ Corresponding author. Tel.: +39 055 4573005; fax: +39 055 4573385. E-mail address: claudiu.supuran@unifi.it (C.T. Supuran). 0960-894X/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.12.124
bacterium became highly acidic within 15 min of their formation.3b Carbonic anhydrase (CA, EC 4.2.1.1), which hydrates carbon dioxide to bicarbonate and protons (reaction 1),6 is involved in pH control in organisms all over the phylogenetic tree, including bacterial4 and fungal pathogens.5
CO2 þ H2 O $ HCO3 þ Hþ
ðreaction 1Þ
It has been shown that enzymes in this family are closely connected with pH regulation in many pathogenic (as well as nonpathogenic) organisms, as well as with secretion of acidic or alkaline electrolytes.4–10 For example, the extracellular environment around hypoxic tumors becomes more acidic (pH 6.0–6.5)7 through the action of several proteins and enzymes, including the tumor-associated isoforms CA IX and XII.7 Several CAs have been shown to play a significant role in the pathogenicity, invasion and survival of many fungal (e.g., Cryptococcus neoformans,5a Candida albicans,5b Candida glabrata8) and bacterial (e.g., Helicobacter pylori,9a Brucella suis,9b Vibrio cholerae9c) pathogens. These enzymes act by signaling information on its local niche (e.g., low or high availability of CO2 or bicarbonate), or by allowing it to adapt to particular pH conditions (such as high acidity in the stomach for H. pylori,10 or alkalinity and high concentrations of bicarbonate for V. cholerae).11
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The genome of L. pneumophila was recently cloned,3a but no CAs from this bacterium have been reported to date. We previously explored its genome and detected two putative CAs belonging to the b-CA family (accession numbers NC_002942, which we designated as lpCA1, locus tag lpg2500, NCBI reference sequence WP_014844650.1; and lpCA2, locus tag lpg2194, NCBI reference sequence WP_014842179.1). Here, we report that these new b-CAs, which we designated as lpCA1 and lpCA2, are active enzymes for the physiologic hydration of CO2 to form bicarbonate and protons. We also investigated the inhibition profiles of these enzymes in the presence of inorganic anions and other small molecules 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 other bacterial a- and b-CAs.8–10 After removal of the GST tag from the chimeric protein, we obtained the pure enzymes that were investigated for catalytic activity in a stopped-flow CO2 hydrase assay.12 The activity of lpCA1 and lpCA2 can be compared to that of other a- and b-CAs from human (h),6 fungal (C. neoformans,5a Saccharomyces cerevisiae),13 plant (Flaveria bidentis),14 or bacterial (B. suis,9b and H. pylori9c) sources from the data shown in Table 1. Both of these new proteins, lpCA1 and lpCA2, possess enzymatic activity for the hydration of carbon dioxide to yield protons and bicarbonate that is typically catalyzed by CAs. 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),6 Can2 from the fungal pathogen C. neoformans,5b or the bacterial pathogenic enzymes HpyCA and BsuCA219, from H. pylori and B. suis, respectively.9 The second isoform, lpCA2, was more active than lpCA1, with the following kinetic parameters: kcat of 8.3 105 s1 and kcat/KM of 8.5 107 M1 s1 (Table 1). This level of activity is similar to that of the S. cerevisiae enzyme SceCA or to that of BsuCA213 (both enzymes belong to the b-CA family) and is only slightly lower than the activity of hCA II, a highly effective catalyst for the CO2 hydration reaction (Table 1).14,15 It was also observed that acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide), a clinically used drug,6 was an efficient inhibitor of the two Legionella enzymes, with KI values in a range from 36 to 95 nM. The moderately active enzyme (lpCA1) was less sensitive to this compound than was lpCA2, which showed greater catalytic activity and also higher affinity for the sulfonamide inhibitor (Table 1). To better understand the efficient catalytic properties of the two new enzymes, we aligned the amino acid sequences of lpCA1 and lpCA2 with those of other such enzymes that have been characterized previously, such as HpyCA, BsuCA213 and
BsuCA219 (Fig. 1). The data shown in Figure 1 indicate that as has been observed for all other bacterial b-CAs investigated to date, the two Legionella proteins lpCA1 and lpCA2 possess the 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, 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 and leads to the formation of the nucleophilic zinc-hydroxide species in the enzyme.5b,9–11 The proposed catalytic mechanism of the b-CAs is shown schematically in Figure 2. It has been in fact shown by means of kinetic and X-ray crystallographic studies that the catalytic mechanism of the b-CAs is rather similar to that of the a-class enzymes investigated in much greater detail.6,7,11 As mentioned above, the catalytically active species of the enzyme (A in Fig. 2) has a hydroxide ion coordinated to the Zn(II), in addition to the protein ligands (which in Fig. 2 are those from lpCA1, i.e., Cys90, His143 and Cys146). In the case of the a-CAs, the zinc hydroxide species is generated through a proton transfer process from the zinc coordinated water to the environment, assisted by the active site residue His64, acting as a proton shuttle.6,7 In the b-CAs the activation of the zinc-coordinated water molecule (shown in D in Fig. 2) is more complex than for the a-CAs, as the catalytic dyad mentioned above takes part in the process. Furthermore, the nature of the amino acid(s) acting as proton shuttle in the b-class enzymes is not completely elucidated (see discussion later in the text). Unknown is also the CO2 binding site within the b-CAs (B in Fig. 2) but as for the aCAs, probably this is a hydrophobic pocket not far away from the catalytic zinc ion. After the nucleophilic attack of the zinc hydroxide species of the enzyme on the bound CO2, bicarbonate is formed (C in Fig. 2) which presumably is bidentately coordinated to the zinc ion (as in the a-CAs).6,7,11 Inorganic anions are usually weak ligands of the zinc ion in metalloenzymes, so that species C is probably easily converted to species D, by a substitution reaction of the bicarbonate by means of a water molecule. This leads to liberation of the reaction product (bicarbonate) into solution, with generation of the acidic form of the enzyme, D, which is catalytically inactive. To generate the zinc hydroxide, catalytically effective species A, a proton transfer reaction must occur (Fig. 2), probably assisted by an active site residue. In the b-CA from the alga Coccomyxa, it has been hypothesized that there are two residues participating in this process, Tyr88 and His92. However, neither lpCA1 nor lpCA2 possess a sequence of Tyr and His residues close to each other as the algal enzyme, which leads the nature of the proton shuttling residue in these novel b-CAs unknown at this moment. The inhibition
Table 1 Kinetic parameters for the CO2 hydration12 reaction catalyzed by several carbonic anhydrases Isozyme hCA I hCA II Can2 SceCA FbiCA 1 HpyCA BsuCA219 BsuCA213 lpCA1 lpCA2
Activity level Moderate Very high Moderate High Low Moderate Moderate High Moderate High
Class
a a b b b b b b b b
kcat (s1) 5
2.0 10 1.4 106 3.9 105 9.4 105 1.2 105 7.1 105 6.4 105 1.1 106 3.4 105 8.3 105
kcat/KM (M1 s1)
KI (acetazolamide) (nM)
Refs.
5.0 107 1.5 108 4.3 107 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 82 27 40 63 303 95 36
6 6 5b 13 14 9c 9b 9b a a
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. Parameters are also reported for the b-CAs Can2 (from C. neoformans),5b SceCA (from S. cerevisiae),13 the Flaveria bidentis CA (FbiCA 1),14 the Helicobacter pylori b-CA HpyCA,9c the Brucella suis b-CAs BsuCA213 and BsuCA219,9b and the two new enzymes lpCA1 and lpCA2, measured at 20 °C, pH 8.3 in 20 mM TRIS buffer and 20 mM NaClO4. Inhibition data for the clinically used sulfonamide acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide) are also provided. a This work.
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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 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 as follows: Legionella pneumophila, 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.
-
OH
Zn Cys90 B
Inh
+ Inh( BH+)
2+
Cys146 His143
Zn Cys90
-H2O
2+
Cys146
His143 E
A
- BH +
-
+ CO2
OH2 Zn Cys90
2+
Cys146
His143
Zn
D
- HCO 3
O
H
Zn Cys90
Cys146 His143
O
2+
B H
Inh-
2+
O
Cys146 Cys90 His143
+ H2O
Inh-
H
OH
O O Zn
Cys90
O
2+
-
Cys146 His143 C
F Figure 2. Catalytic and inhibition mechanisms of lpCAs (exemplified by using lpCA1, and its amino acid residues numbering system). See text for details.
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mechanism of lpCA1 and lpCA2 (Fig. 2E and F) will be discussed later in the paper. We also examined the phylogenetic relationships of the two new bacterial b-CAs, lpCA1 and lpCA2, to other such enzymes characterized earlier. We compared these enzymes to those from various 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 tree in Figure 3, the two Legionella enzymes are highly similar, clustering on their own branch of the tree. The next most similar enzyme is isoform stCA2 from S. typhimurium. The remaining b-CAs all clustered together on the lower branch of the tree, with HpyCA, stCA1 and BsuCA219 residing on the same branch. The remaining Brucella isoform BsuCA213 was on a separate branch, thus indicating that it is 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 investigated as CA inhibitors (CAIs), as they bind to the metal ion from the enzyme active site and impair catalysis.6,8–11 Herein, we report the first inhibition study of the two Legionella enzymes with a range of such inhibitors, including simple and complex inorganic anions as well as sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid (Table 2). This set of anions and small molecules had been investigated previously for inhibition of a large number 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 lpCA1 and lpCA2 by the anions and small molecules shown in Table 2: (i) Perchlorate and tetrafluoroborate, anions known for their low affinity for metal ions in metalloenzymes (and sometimes also in solution),13–17 did not inhibit the two new b-CAs reported here (KI >200 mM). Similar results have been observed in most of the CAs examined to date: only HpyCA was effectively inhibited by perchlorate, with a KI of 6.5 mM.9c Sulfate was also an ineffective lpCA1 and lpCA2 inhibitor, with KI values between 77.9 and 96.5 mM (Table 2). Iodide and nitrate were also quite weak lpCA2 inhibitors, with inhibition constants of 59.1 and 30.1 mM, respectively. These anions were more effective inhibitors of lpCA1, vide infra. (ii) Another group of anions inhibited lpCA1 and lpCA2 weakly, with inhibition constants in the range of 3.5–9.1 mM. The weak inhibitors 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 submillimolar inhibitors against both lpCA1 and lpCA2. All of the halides, as well as cyanate, thiocyanate, stannate, tellurate, pyrophosphate, divanadate, tetraborate, perrhenate, perruthenate, peroxydisulfate, selenocyanate, and trithiocarbonate inhibited lpCA1 with KI values from 0.24 to 0.98 mM. Iminodisulfonate was slightly less effective
Table 2 Inhibition constants of anionic inhibitors against a-CA isozymes derived from human (hCA II) and bacterial (SspCA)15 sources. Inhibition constants are also reported for bCA from a bacterium (H. pylori)9c HpyCA, the plant Flaveria bidentis isoform 1 (FbiCA 1),14 and the new enzymes lpCA1 and lpCA2 from L. pneumophila. Values were determined at 20 °C in a stopped flow CO2 hydrase assay12 Inhibitor§
KI (mM)# a
hCA II
SspCA
a
a
HpyCAc b
FbiCA 1d b
lpCA1e b
lpCA2 b
>300 200 63 26 0.03 1.60 0.02 1.51 85 73
41.7 8.30 49.0 0.86 0.80 0.71 0.79 0.49 33.2 39.3
0.67 0.56 0.38 0.63 0.37 0.68 0.54 0.80 0.50 0.42
0.71 0.74 0.67 0.71 0.93 0.83 0.62 0.46 0.66 0.84
0.91 0.79 0.65 0.32 0.66 0.52 0.064 0.077 3.5 4.7
0.77 0.81 8.0 59.1 0.96 0.88 0.61 0.45 6.6 4.8
SnO2 3
35 63 0.04 89 0.83
0.86 0.48 0.58 21.1 0.52
0.78 0.67 0.58 0.63 0.48
0.78 0.57 0.86 55.3 0.53
7.6 7.9 0.076 6.6 0.57
30.1 5.8 0.51 7.2 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
P2 O4 7
48.50
0.69
0.75
0.83
0.94
0.83
V2 O4 7
0.57
0.66
0.18
0.66
0.39
0.47
B4 O2 7 ReO 4 RuO 4
0.95
0.67
0.68
0.86
0.60
0.55
0.75 0.69 0.084
0.80 0.69 84.6
0.82 1.10 0.93
0.52 26.1 0.87
0.89 0.82 0.85
0.77 0.86 0.57
0.086 0.0088
0.07 0.06
0.97 0.21
0.88 0.06
0.98 0.53
0.66 0.62
3.1 >200
0.004 0.82
0.0074 0.57
0.008 0.62
0.006 77.9
0.002 96.5
>200 >200 0.46 0.76
>200 >200 0.73 0.75
6.50 >200 0.75 0.70
>200 >200 0.69 50.9
>200 >200 9.1 1.17
>200 >200 0.46 0.59
1.13 0.39 23.1 49.2
0.009 0.042 0.041 0.005
0.072 0.094 0.073 0.092
0.004 0.005 0.008 0.006
0.094 0.076 0.065 0.084
0.009 0.013 0.006 0.008
F Cl Br I CNO SCN CN N 3 HCO 3 CO2 3 NO 3 NO 2 HS HSO 3
S2 O2 8 SeCN CS2 3 Et2 NCS 2 SO2 4 ClO 4 BF4 FSO 3 NHðSO3 Þ2 2 H2 NSO2 NH2 H2 NSO3 H Ph-BðOHÞ2 Ph-AsO3 H2
b
e
§
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. #
as an lpCA1 inhibitor (KI of 1.17 mM). The effective, submillimolar inhibitors of lpCA2 were fluoride, chloride, cyanate, thiocyanate, cyanide, azide, hydrogen sulfide, stannate, tellurate, pyrophosphate, divanadate, tetraborate, perrhenate, perruthenate, peroxydisulfate, selenocyanate, trithiocarbonate, fluorosulfonate and
Figure 3. Phylogenetic tree produced using the b-CA amino acid sequences aligned in Figure 1. The tree was constructed using the program PhyML 3.0, which is phylogeny software based on the maximum-likelihood principle. Branch support values are reported at branch points. Organisms, NCBI sequence numbers and cryptonyms are indicated in Figure 1, except for Salmonella typhimurium,16 stCA1 and stCA2 (NCBI sequence numbers: NP_459176.1 for stCA1, and YP_007905873.1, for stCA2).
I. Nishimori et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1127–1132
iminodisulfonate. These ions showed KI values ranging from 0.29 to 0.96 mM. The best inhibitor in this subseries was tellurate, with KI values of 0.24 and 0.29 mM against lpCA1 and lpCA2, respectively. This is a very potent inhibitor compared to the salts of elements from the same group. Specifically, the Se(VI) and S(VI) derivatives selenate and sulfate were much weaker inhibitors of the two enzymes (Table 2). (iv) The best anionic inhibitors of lpCA1 detected in this study were 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 from these data. The metal ‘poisons’ cyanide, azide, and hydrogen sulfide had very similar inhibitory effects against lpCA1. This similarity can be extended further to the four small molecules from Table 2, that is, sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid. In fact all these compounds inhibited the enzymes over a narrow range of potencies, with inhibition constants from 64 to 94 lM. The compound N,N-diethyldithiocarbamate had a much higher affinity for this enzyme, with a low micromolar value for KI of 6 M. In fact, the dithiocarbamates were recently reported as a potent new class of CAIs targeting both the a- and b-classes of such enzymes.18 However, all of the small molecules were low micromolar inhibitors of lpCA2, with KI values from 2 to 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 Legionella enzymes towards the anionic inhibitors investigated here. We also observed significant differences between these two enzymes, lpCA1 and lpCA2, and other a- and b-class CAs for which such data are available. These include the enzymes shown in Table 2, which are of human, bacterial or plant origin. Thus, lpCA1 seems to have higher affinity for some poisonous metal anions such as cyanide, azide, and hydrogen sulfide, which are around one order of magnitude more potent against lpCA1 than lpCA2. However, lpCA2 showed higher affinity for N,N-diethyldithiocarbamate, sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid compared to lpCA1. The heavy halides, bromide and iodide, also behaved quite differently against the two enzymes. These two anions are submillimolar inhibitors of lpCA1 and much weaker inhibitors of lpCA2 (KI values ranging from 8 to 59 mM). However, there were also anions that showed comparable affinities for these two enzymes (e.g., chloride, bicarbonate, carbonate, tellurate—see Table 2). The inhibitory potencies of these anions against lpCA1 and lpCA2 were also quite different when compared to those observed in other bacterial (SspCA or HpyCA), plant (FbiCA 1) or human (hCA II) enzymes. For example, while chloride was a submillimolar inhibitor of lpCA1, lpCA2, FbiCA 1 and HpyCA, it showed KI values from 8.3 to 200 mM against the extremophilic bacterial enzyme SspCA or human hCA II (Table 2). Trithiocarbonate was an effective inhibitor of hCA II, SspCA and FbiCA 1, but was less effective against HpyCA, lpCA1 and lpCA2. The inhibition mechanism of these anions against lpCA1 and lpCA2 is probably similar to that of the inorganic anions against a-CAs, investigated in greater detail, by means of kinetic, spectroscopic and X-ray crystallographic techniques.6,7,11,16 As shown in Figure 2, the inhibitor may substitute the fourth, non-protein zinc ligand, 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 shown to possess this inhibition mechanism by means of high resolution X-ray crystallography. An alternative inhibition mechanism has been also reported again for the Coccomyxa b-CA11c for iodide, which was found anchored to the zinc-coordinated water
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molecule/hydroxide ion, as shown schematically in Figure 2F. This mechanism was also observed for phenols,19 polyamines 20 and sulfocoumarins21 for the inhibition of a-CAs. In conclusion, we investigated the cloning, catalytic activity and anion inhibition of two b-CAs from the bacterial pathogen L. pneumophila, lpCA1 and lpCA2. The two new enzymes were efficient catalysts for CO2 hydration, with kcat values ranging from (3.4– 8.3) 105 s1 and kcat/KM of (4.7–8.5) 107 M1 s1. A set of inorganic anions and small molecules was investigated for inhibition of these enzymes. Perchlorate and tetrafluoroborate were not active inhibitors (KIs >200 mM), whereas sulfate was a very weak inhibitor of both lpCA1 and lpCA2 (KIs of 77.9–96.5 mM). The most potent lpCA1 inhibitors were cyanide, azide, hydrogen sulfide, N,N-diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with KI values in a range from 6 to 94 lM. The most active lpCA2 inhibitors were N,N-diethyldithiocarbamate, sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid, with KI values between 2 and 13 lM. As these enzymes seem to be involved in regulation of phagosome pH during Legionella infection, inhibition of these targets may lead to antibacterial 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 stoppedflow 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
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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 the initial rates of the CA-catalyzed CO2 hydration reaction for a period of 10– 100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor, at least six traces of the initial 5–10% of the reaction were used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitors (10 mM) were prepared in distilled–deionized water, and dilutions up to 0.01 M were performed thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay 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 Cheng–Prusoff equation, whereas the kinetic parameters for the uninhibited enzymes 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.; De Luca, 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.
