Anion inhibition studies of the dandruff-producing fungus Malassezia globosa β-carbonic anhydrase MgCA

Bioorganic & Medicinal Chemistry Letters 25 (2015) 5194–5198

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl

Anion inhibition studies of the dandruff-producing fungus Malassezia globosa b-carbonic anhydrase MgCA Sonia Del Prete a,b, Daniela Vullo c, Sameh M. Osman d, Zeid AlOthman d, Clemente Capasso b,⇑, Claudiu T. Supuran a,c,⇑ a

Università degli Studi di Firenze, Dipartimento Neurofarba, Sezione di Scienze Farmaceutiche, Polo Scientifico, Sesto Fiorentino, Firenze, Italy Istituto di Biochimica delle Proteine—CNR, Via P. Castellino 111, 80131 Napoli, Italy Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy d Department of Chemistry, College of Science, King Saud University, PO Box 2455, Riyadh 11451, Saudi Arabia b c

a r t i c l e

i n f o

Article history: Received 3 September 2015 Revised 27 September 2015 Accepted 28 September 2015 Available online 3 October 2015 Keywords: Carbonic anhydrase b-CA-class enzyme Anion Inhibitor Malassezia globosa

a b s t r a c t The genome of the fungal parasite Malassezia globosa, the causative agent of dandruff, contains a single gene annotated as encoding a carbonic anhydrase (CAs, EC 4.2.1.1) belonging to the b-class (MgCA). In an earlier work (J. Med. Chem. 2012, 55, 3513) we have validated this enzyme as an anti-dandruff drug target, reporting that sulfonamide inhibitors show in vitro and in vivo effects, in an animal model of Malassezia infection. However, few classes of compounds apart the sulfonamides, were investigated for their activity against MgCA. Here we present an anion inhibition study of this enzyme, reporting that metal complexing anions such as cyanate, thiocyanate, cyanide, azide are weak MgCA inhibitors (KIs ranging between 6.81 and 45.2 mM) whereas bicarbonate (KI of 0.59 mM) and diethyldithiocarbamate (KI of 0.30 mM) together with sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid were the most effective inhibitors detected so far, with KIs ranging between 83 and 94 lM. This study may help a better understanding of the inhibition profile of this enzyme and may offer the possibility to design new such modulators of activity belonging to different chemical classes. Ó 2015 Elsevier Ltd. All rights reserved.

Dandruff is found in more than 50% of the worldwide population being characterized by an excessive shedding of dead skin cells from the scalp.1 In individuals without this condition, skin cells mature and shed in approximately one month, whereas for those with dandruff, the shedding rate is accelerated and may take only 2–7 days.1 Dandruff can be triggered by several factors, including an increase in sebum production, irritation by pathogenic organisms (particularly fungi belonging to the genus Malassezia) as well as individual susceptibility (hereditary) factors.1 Malassezia (previously known as Pityrosporum) are yeasts, which are naturally found to occur on the skin and scalp of most individuals. There are several recognised species including Malassezia globosa, Malassezia furfur and Malassezia restricta.2 Until recently, M. furfur was thought to be responsible for the onset of dandruff, but instead, the scalp specific species M. globosa/restricta were recently found to be the most probable causative agents.2–4 Malassezia are dependent on external lipids for growth and hence secrete

⇑ Corresponding authors. Tel.: +39 081 6132559; fax: +39 081 6132249 (C.C.); tel.: +39 055 4573005; fax: +39 055 4573385 (C.T.S.). E-mail addresses: [email protected] (C. Capasso), claudiu.supuran@ unifi.it (C.T. Supuran). http://dx.doi.org/10.1016/j.bmcl.2015.09.068 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

lipases to breakdown triglycerides that occur in the sebum of human skin. By-products of this breakdown include unsaturated fatty acids, such as oleic acid, which penetrate the stratum corneum (the top layer of the epidermis), resulting in an inflammatory responses. In susceptible individuals, this results in rapid shedding of the stratum corneum which leads to dandruff.11,12 Several strategies and treatments are available for dandruff, the majority of which aim to target growth of the fungi. A common active ingredient in anti-dandruff shampoos is zinc pyrithione, also known as zinc pyridinethione A,5 its effect being most likely mediated through disruption of fungal membrane activities (Fig. 1).5 Ketoconazole B, an azole antifungal agent interfering with the biosynthesis of fungal sterols is also used in many shampoos.6 Other azoles have been used in anti-dandruff treatments, as they interfere with the synthesis of ergosterol, a key component of fungal cell walls.6 However the effectiveness of A and B for preventing/treating dandruff is not very high, and additional therapeutic strategies are being explored, such as, for example, inhibition of the lipase present in Malassezia spp.,7 or inhibition of the b-carbonic anhydrase (CA, EC 4.2.1.1), which has been proposed as a novel anti-dandruff target recently by this group (in M. globosa).8

S. Del Prete et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5194–5198 N S N

Zn

2+

O S

O A

N O

N Ac

N

N

O

O H Cl

Cl

B

Figure 1. Compounds used in anti-dandruff shampoos, zinc pyrithione (A) and ketoconazole (B).

Indeed, carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes present in all life kingdoms, with six genetically distinct families described to date in various organisms.9–14 Most of them are zinc-containing enzymes, but Fe(II) may be present at the active site of the c-CAs (described so far in Bacteria, Archaea and plants), whereas Cd(II) or Zn(II) ions seem to be equally effective for promoting catalysis in the f-CAs (diatoms encode for this class of CAs).10–12 The metal ion is coordinated by three His residues (in the a-, c- and d-class enzymes), by one His, and two Cys residues (in the b- and f-CAs), and by two His and one Gln residue in the g-CA class,10d,e with the fourth ligand being a water molecule/hydroxide ion.9,12,15,16 The inhibition of many such enzymes, present in mammals (in which there are 16 different isoforms)17–19 or in various pathogens (fungi,8,15 bacteria,10,20,21 or protozoa)10 may be exploited pharmacologically.8–10 Sulfonamides are the main class of CAIs, but they show many side effects, and for this reason a lot of efforts for developing alternative classes of inhibitors were made in the last decade.9,16,21,22 Among them, the inorganic anions,22a phenols,22b, polyamines,22c and dithiocarbamates22d represent interesting cases, which have been investigated in detail by kinetic and crystallographic studies. They allowed a deep understanding of inhibition mechanisms with these classes of compounds and led to interesting drug design campaigns and the discovery of CAIs with a good selectivity ratio for inhibiting enzyme classes or isoforms of medicinal interest.9,10,19–22 Inorganic (complexing) anions represent an interesting class of derivatives which bind metal ions in many types of metalloenzymes.22 Their chemical simplicity represents both a disadvantage as well as an advantage when investigating them as CAIs.22a The disadvantage consists in the fact that due to their propensity to coordinate metal ions in solution or in metalloenzyme active sites, usually this class of inhibitors is non-selectively binding to many metals present in such enzymes, and furthermore, their activities are normally those of weak inhibitors (in the millimolar, rarely tens of micromolar ranges).22 However, the advantage of this chemical simplicity (but also diversity, since a large number of inorganic/organic anions can be envisaged) is represented by the fact that many such compounds can be considered as lead molecules and further elaborated for leading to highly effective inhibitors. The most common examples investigated so far are represented by sulfamide (H2NSO2NH2), sulfamic acid (H2NSO3H), or trithiocarbonate (CS23 ) which were reported as millimolar anionic CAIs by this group,22e,f but led thereafter to the discovery of many classes of organic sulfamides (Ar-NHSO2NH2), sulfamates (Ar-OSO2NH2), and dithiocarbamates (R-NHCS2 ) with low nanomolar activity against many CAs (Ar, R may be aromatic, aliphatic, heterocyclic or sugar moieties).9,18,19,22 Thus considering our interest in anion inhibitors of various CA classes,22a we report here an anion inhibition study of MgCA,8,23–27 the enzyme investigated several years ago for its inhibition profile with sulfonamides, and for which we showed interesting pharmacological properties.8 It should be mentioned that in the earlier study8 we worked with a GST-MgCA fusion protein for the cloning and purification

5195

of the enzyme (the GST part was cleaved from the chimeric protein for investigating its kinetic properties and sulfonamide inhibition). The main problem with that system was that the yield in MgCA was rather low and the protein was often found in inclusion bodies from which it was difficult to separate in a properly folded manner.8 This is the reason why we have recloned MgCA using the synthetic gene to which a His tag (comprising 6 His residues at the amino terminal part) has been attached for allowing the purification with Ni(II)-based affinity columns.28 Indeed, the yield and purity of the protein obtained in this way are better compared to the previously reported one.8 As the preparation of MgCA is diverse from the one reported earlier,8 we re-performed a kinetic investigation of the newly purified MG-CA, comparing its kinetic parameters (kcat and kcat/Km) with those of MgCA prepared as GST-fusion protein, as well as other thoroughly investigated CAs, such as the cytosolic, ubiquitous human isozymes hCA I and II, as well as b-CAs from the fungal pathogen C. neoformans (Can2) or Candida glabrata (CgNce103) characterized4,5,18 (Table 1). Data from Table 1 shows that the new preparation has kinetic data quite similar with those reported earlier for the GST-fusion protein, but the activity of the His-tagged MgCA is better. Similar to other CAs belonging to the a- or bclasses, the enzyme reported here possesses appreciable CO2 hydrase activity, with a kcat of 9.2  105 s 1, and kcat/Km of 8.3  107 M 1 s 1. Data of Table 1 also shows that MgCA (both the old and the new preparations) was inhibited by the clinically used sulfonamide acetazolamide AAZ (5-acetamido-1,3,4-thiadiazole-2-sulfonamide), with an inhibition constant of 76 lM (for the GST-fusion enzyme) and of 74 lM (for the new preparation). However, as shown earlier, some different aromatic sulfonamides show much better inhibitory properties against MgCA.8 We have investigated a range of inorganic/organic anions and other small molecules for their interaction with MgCA (Table 2). They include halides, pseudohalides, nitrite/nitrate, sulfite/sulfate and anions isoelectronic with them, but also complex anions incorporating heavy metals, as well as the simple small molecules known to have affinity for Zn(II) in the CAs, such as sulfamide, sulfamic acid, phenylboronic and phenylarsonic acid (Table 2).22 Data of Table 2 show a very interesting inhibition profile of MgCA with anions and small molecules. The following salient features were observed: (i) Anions known to possess weak affinity for Zn(II) in other CAs,22a such as perchlorate and tetrafluoroborate were also not inhibitory against MgCA (as for hCA I and II, the two major red blood cell isoforms, which may be considered as the main off targets when investigating parasite CAs,18 like the enzyme considered here). Surprisingly, carbonate, bisulfite and peroxydisulfate showed the same activity, with no inhibition of MgCA up to 100 mM concentrations of inhibitor. What is indeed quite interesting is the lack of inhibition by carbonate, also considering the high affinity shown by bicarbonate for MgCA (see later in the text). (ii) Some other anions with poor inhibitory properties against MgCA were bromide, azide, hydrogen sulfide, perrhenate, and sulfate, which had KIs in the range of 11.9–45.2 mM (Table 2). The sulfate data is interesting, as this anion usually has a low affinity for Zn(II) in many CAs (e.g., hCA II and I), whereas azide, which is a very weak MgCA inhibitor acted as a highly potent one for many a-CAs (e.g., it has a KIs in the micromolar rage against hCA I). (iii) The largest majority of anions investigated here showed a medium potency inhibitory power against MgCA with KIs in the low millimolar range (1.73–8.82 mM). Among them are the halides (except bromide), cyanate, thiocyanate, cyanide, nitrate, nitrite, stannate, selenate, tellurate, perosmate,

5196

S. Del Prete et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5194–5198

Table 1 Kinetic parameters for the CO2 hydration reaction catalyzed by various CAs belonging to different enzyme families9,10 Isozyme hCA I hCA II Can2 PfCA MG-CA* MgCA**

Class

Organism

a a

kcat (s

b b b

)

kcat/Km (M

5

2.0  10 1.4  106 3.9  105 1.4  105 (8.6 ± 0.2)  105 (9.2 ± 0.1)  105

Human Human Fungus Protozoa Fungus Fungus

g

1

7

1


s

1

5.0  10 1.5  108 4.3  107 5.4  106 (6.9 ± 0.1)  107 (8.3 ± 0.3)  107

)

KI (acetazolamide) (nM)

References

250 12 10.5 170 76,000 ± 1200 74,000 ± 1600

9 9 15a 10d 8 This work

The a-class CAs were the human cytosolic isozymes hCA I and II, the b-class fungal enzymes Can2 from Cryptococcus neoformans,15a and MgCA (initially cloned as a GST-fusion protein,8 and here recloned as a His-tagged protein), as well as the protozoan, g-CA (pfCA) from Plasmodium falciparum.10d,e Inhibition data with the clinically used sulfonamide acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide) are also provided. All data were obtained in the author’s laboratories. * Cloned as a GST-fusion protein, see Ref. 8. ** Cloned as a His-tagged protein.28

Table 2 Inhibition constants of anion inhibitors against a-CAs from mammals (hCA I, and II, human isoforms, and the protozoan enzyme from T. cruzi, TcCA), and the g-CA PfCA from P. falciparum, for the CO2 hydration reaction, at 20 °C and pH 7.528 Inhibitor§

KI# (mM) a

F Cl Br I CNO SCN CN N3 HCO3 CO23 NO3 NO2 HS HSO3 SnO23 SeO24 TeO24 OsO25 P2O47 V2O47 B4O27 ReO4 RuO4 S2O28 SeCN CS23 Et2NCS2 CF3SO3 PF6 SO24 ClO4 BF4 FSO3 NH(SO3)22 H2NSO2NH2 H2NSO3H Ph-B(OH)2 Ph-AsO3H2

hCA I

hCA IIa

MgCAb

>300 6 4 0.3 0.0007 0.2 0.0005 0.0012 12 15 7 8.4 0.0006 18 0.57 118 0.66 0.92 25.77 0.54 0.64 0.11 0.101 0.107 0.085 0.0087 0.00079 nt nt 63 >200 >200 0.79 0.31 0.31 0.021 58.6 31.7

>300 200 63 26 0.03 1.60 0.02 1.51 85 73 35 63 0.04 89 0.83 112 0.92 0.95 48.50 0.57 0.95 0.75 0.69 0.084 0.086 0.0088 0.0031 nt nt >200 >200 >200 0.46 0.76 1.13 0.39 23.1 49.2

7.13 7.98 18.6 8.73 6.81 8.39 7.19 45.2 0.59 >100 8.13 7.56 11.9 >100 5.07 7.41 5.75 6.16 6.03 6.89 8.45 16.7 8.82 >100 1.73 1.77 0.30 2.28 6.47 19.5 >100 >100 4.06 21.4 0.094 0.083 0.089 0.090

§

As sodium salt, except sulfamide, phenylboronic acid and phenylarsonic acid. Errors were in the range of 3–5% of the reported values, from three different assays, by a CO2 hydrase assay method.23 a From Ref. 22. b This work. #

diphosphate, divanadate, tetraborate, selenocyanide, trithiocarbonate, triflate, hexafluorophosphate, and fluorosulfonate. One may see that this is a highly heterogeneous group of anions, incorporating either highly simple ones (halides) as well as complex ones, which differ considerably in their affinity for metal ions in solutions. Obviously, the environment within the active site dramatically interferes

with the binding, favoring some and probably inducing a less favored binding for other of them, which explains the rather small variation in the inhibition constants for this large group of inhibitors. (iv) A very interesting observation is that among the best anion inhibitors detected here were bicarbonate (KI of 0.59 mM) and diethyldithiocarbamate (KI of 0.30 mM). Bicarbonate is obviously also a substrate/reaction product of the CAs, and this behavior is quite unexpected, also considering the carbonate, as mentioned above, did not show inhibitory properties. It should be mentioned that for other enzymes, the difference in behavior between these two anions (bicarbonate and carbonate) are rather modest, as shown in Table 2 for hCA I and II. Thus, it is difficult to understand why in MgCA bicarbonate is a submillimolar inhibitor, whereas carbonate is not inhibitory up to concentrations as high as 100 mM, but we speculate that this may be physiologically important for the enzyme. It should also be noted the inhibition data between carbonate (not an inhibitor) and trithiocarbonate (KI of 1.77 mM) which is one of the efficient inhibitors in the group of medium potency anions. However, what is even more interesting is the fact that when one of the S atoms in trithiocarbonate is replaced by a diethylamino moiety (as in N,N-diethyldithiocarbamate), the inhibitory power continues to increase, this compound having, as stressed above, a KI of 0.30 mM. Thus, probably dithiocarbamates with longer aliphatic/aromatic chains will possess an increased inhibitory power, as we proved for some other a- and b-class CAs.20,22 (v) The best MgCA inhibitors were sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid, which had inhibition constants in the range of 83–90 lM (comparable with acetazolamide, KI of 74 lM, see discussion above). Thus, as for other pathogenic enzymes investigated ultimately, this study allowed us to reveal that small molecule inhibitors such as dithiocarbamates, sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid show an interesting potential for designing CAIs with an improved potency. For this reason, the catalytic and inhibition mechanisms of this enzyme should be briefly considered (Fig. 2). In fact, as many other b-CAs,15 MgCA possesses the conserved amino acids8 involved in the catalytic cycle of this class of enzymes: (i) the Zn(II) binding residues Cys47, His103 and Cys106 (based on the M. globosa numbering);8 and (ii) the AspArg dyad (in this case Asp49 and Arg51), which activates the metal ion for catalysis. A zinc hydroxide species (shown in A, with HO as the fourth zinc ligand) is the catalytically active one, which attacks CO2 bound in a hydrophobic pocket nearby (B in Fig. 2) with formation of the bicarbonate adduct C (which as shown here is rather stable, as the KI for bicarbonate is of 0.59 mM, see Table 2). This

5197

S. Del Prete et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5194–5198

-

OH

Zn Cys47 B - BH

Inh

+ InhH

Zn

2+

Cys106 His103

-H2O

Cys47

2+

Cys106 His103

E

A

+

-

+ CO2

OH2 Zn Cys47

2+

Cys106 His103

Zn

D

H2O

Cys47

- HCO 3

Inh

Zn

Cys47

+ H2O

-H+

InhH

OH

-

2+

O O

2+

Cys106 0His143 B

O

H

O Zn

Cys40

Cys106 His103

O

2+

-

Cys106 His103 C

F Figure 2. Catalytic and inhibition mechanisms of MgCA. In A–D the catalytic cycle is shown whereas in E and F enzyme–inhibitor adducts (tetrahedral geometry of Zn(II) in E, trigonal bipyramidal geometry in F) are schematically presented.

is thereafter liberated into solution and an incoming water molecules leads to the formation of species D, which is probably catalytically ineffective. The zinc-coordinated water is also activated by the Asp-Arg dyad (enhancing its nucleophilicity), and probably favoring the proton transfer step which leads to the regeneration of the basic form A. However there are no structural or mutagenesis data which prove what amino acid residues are involved in the proton transfer processes in b-CAs. Inhibitors, such as the anions/small molecules investigated here, may bind monodentately, either in tetrahedral geometries of the Zn(II) (as in E) or in trigonal–bipyramidal ones (as in F, when a water molecule is also coordinated to the metal ion).9,15 Alternatively, a bidentate behavior, as for bicarbonate (C), has also been proposed, and proved (by X-ray crystallography), at least for the a-CAs.22a Another interesting aspect that we wish to stress here, is that as shown from Table 2, and also from Figure 2, MgCA seems to have rather different properties regarding both the human isoforms hCA I and II, as well as other b-CAs investigated in detail in the last period.10,15–17 Thus, we performed a detailed phylogenetic analysis of MgCA, comparing its sequence with those of other such enzymes from a variety of organisms, such as bacteria, fungi, algae and superior plants (for the organisms and the corresponding accession numbers of the protein sequences employed see the caption to Fig. 3). The dendrogram of Figure 3 shows that basically MgCA seems to be not closely related to the other fungal such enzymes investigated to date (such as the ones from Saccharomyces cerevisiae SceCA or DbrCA), but instead to cluster and thus to be related to higher plants b-CAs (such as the corn one, Zea mays ZmaCA, Flaveria bidentis, FbiCA, etc).29 This is a rather interesting discovery, which we hypothesize that it may be helpful for the design of specific MgCA inhibitors, considering the differences between this enzyme and that of other bacterial/fungal b-CAs investigated to date. In conclusion, we report an anion inhibition study of the M. globosa b-CA enzyme, reporting that metal complexing anions

Figure 3. Phylogenetic tree of b-CAs from various organisms (bacteria, fungi, algae and plants).

such as cyanate, thiocyanate, cyanide, azide are weak MgCA inhibitors (KIs ranging between 6.81 and 45.2 mM) whereas bicarbonate (KI of 0.59 mM) and diethyldithiocarbamate (KI of 0.30 mM) together with sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid were the most effective inhibitors detected so far (KIs ranging between 83 and 94 lM). Carbonate, similar to perchlorate or tetrafluoroborate, did not inhibit MgCA up to 100 mM. This study may help a better understanding of the inhibition profile of this enzyme and may offer the possibility to design new such modulators of activity belonging to different chemical classes. References and notes 1. (a) Dawson, T. L., Jr. J. Investig. Dermatol. Symp. Proc. 2007, 12, 15; (b) Guillot, J.; Hadina, S.; Guého, E. Parassitologia 2008, 50, 77; (c) Galuppi, R.; Tampieri, M. P. Parassitologia 2008, 50, 73.

5198

S. Del Prete et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5194–5198

2. Clavaud, C.; Jourdain, R.; Bar-Hen, A.; Tichit, M.; Bouchier, C.; Pouradier, F.; El Rawadi, C.; Guillot, J.; Ménard-Szczebara, F.; Breton, L.; Latgé, J. P.; Mouyna, I. PLoS One 2013, 8, e58203. 3. Donnarumma, G.; Perfetto, B.; Paoletti, I.; Oliviero, G.; Clavaud, C.; Del Bufalo, A.; Guéniche, A.; Jourdain, R.; Tufano, M. A.; Breton, L. Arch. Dermatol. Res. 2014, 306, 763. 4. Wang, L.; Clavaud, C.; Bar-Hen, A.; Cui, M.; Gao, J.; Liu, Y.; Liu, C.; Shibagaki, N.; Guéniche, A.; Jourdain, R.; Lan, K.; Zhang, C.; Altmeyer, R.; Breton, L. Exp. Dermatol. 2015, 24, 398. 5. Lodén, M.; Wessman, C. Int. J. Cosmet. Sci. 2000, 22, 285. 6. Hay, R. J. Br. J. Dermatol. 2011, 165, 2. 7. De Angelis, Y. M.; Saunders, C. W.; Johnstone, K. R.; Reeder, N. L.; Coleman, C. G., ; Kaczvinsky, J. R., Jr.; Gale, C.; Walter, R.; Mekel, M.; Lacey, M. P.; Keough, T. W.; Fieno, A.; Grant, R. A.; Begley, B.; Sun, Y.; Fuentes, G.; Youngquist, R. S.; Xu, J., ; Dawson, T. L., Jr. J. Invest. Dermatol. 2007, 127, 2138. 8. Hewitson, K.; Vullo, D.; Scozzafava, A.; Mastrolorenzo, A.; Supuran, C. T. J. Med. Chem. 2012, 55, 3513. 9. (a) Alterio, V.; Di Fiore, A.; D’Ambrosio, K.; Supuran, C. T.; De Simone, G. Chem. Rev. 2012, 112, 4421; (b) Supuran, C. T. Nat. Rev. Drug Disc. 2008, 7, 168; (c) Capasso, C.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2015, 30, 325. 10. (a) Supuran, C. T. Front. Pharmacol. 2011, 2, 34; (b) Del Prete, S.; Vullo, D.; Scozzafava, A.; Capasso, C.; Supuran, C. T. Bioorg. Med. Chem. 2014, 22, 531; (c) Vullo, D.; Del Prete, S.; Osman, S. M.; De Luca, V.; Scozzafava, A.; AlOthman, Z.; Supuran, C. T.; Capasso, C. Bioorg. Med. Chem. Lett. 2014, 24, 275; (d) Del Prete, S.; Vullo, D.; Fisher, G. M.; Andrews, K. T.; Poulsen, S. A.; Capasso, C.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2014, 24, 4389; (e) Vullo, D.; Del Prete, S.; Fisher, G. M.; Andrews, K. T.; Poulsen, S. A.; Capasso, C.; Supuran, C. T. Bioorg. Med. Chem. 2015, 23, 526. 11. (a) Ferry, J. F. Biochim. Biophys. Acta 2010, 1804, 374; (b) Smith, K. S.; Jakubzick, C.; Whittam, T. S.; Ferry, J. G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 15184; (c) Zimmerman, S. A.; Tomb, J. F.; Ferry, J. G. J. Bacteriol. 2010, 192, 1353; (d) Zimmerman, S. A.; Ferry, J. G.; Supuran, C. T. Curr. Top. Med. Chem. 2007, 7, 901; (e) Tripp, B. C.; Bell, C. B., 3rd; Cruz, F.; Krebs, C.; Ferry, J. G. J. Biol. Chem. 2004, 279, 6683. 12. (a) Xu, Y.; Feng, L.; Jeffrey, P. D.; Shi, Y.; Morel, F. M. Nature 2008, 452, 56; (b) Alterio, V.; Langella, E.; Viparelli, F.; Vullo, D.; Ascione, G.; Dathan, N. A.; Morel, F. M.; Supuran, C. T.; De Simone, G.; Monti, S. M. Biochimie 2012, 94, 1232; (c) Viparelli, F.; Monti, S. M.; De Simone, G.; Innocenti, A.; Scozzafava, A.; Xu, Y.; Morel, F. M.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2010, 20, 4745. 13. (a) Alterio, V.; Hilvo, M.; Di Fiore, A.; Supuran, C. T.; Pan, P.; Parkkila, S.; Scaloni, A.; Pastorek, J.; Pastorekova, S.; Pedone, C.; Scozzafava, A.; Monti, S. M.; De Simone, G. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 16233; (b) Vullo, D.; De Luca, V.; Scozzafava, A.; Carginale, V.; Rossi, M.; Supuran, C. T.; Capasso, C. Bioorg. Med. Chem. Lett. 2012, 22, 6324; (c) Harju, A. K.; Bootorabi, F.; Kuuslahti, M.; Supuran, C. T.; Parkkila, S. J. Enzyme Inhib. Med. Chem. 2013, 28, 231. 14. (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. 15. (a) 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; (b) Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 229; (c) Capasso, C.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2014, 29, 379. 16. (a) Supuran, C. T. Bioorg. Med. Chem. Lett. 2010, 20, 3467; (b) Fabrizi, F.; Mincione, F.; Somma, T.; Scozzafava, G.; Galassi, F.; Masini, E.; Impagnatiello, F.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 138; (c) Ekinci, D.; Karagoz, L.; Ekinci, D.; Senturk, M.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 283. ˇ , F.; Remko, M. J. Enzyme Inhib. Med. 17. (a) Supuran, C. T.; Maresca, A.; Gregán Chem. 2013, 28, 289; (b) Alp, C.; Maresca, A.; Alp, N. A.; Gültekin, M. S.; Ekinci, D.; Scozzafava, A.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 294; (c) Carta, F.; Scozzafava, A.; Supuran, C. T. Expert Opin. Ther. Pat. 2012, 22, 747; (d) Supuran, C. T. Expert Opin. Ther. Pat. 2013, 23, 677; (e) Wilkinson, B. L.; Bornaghi, L. F.; Houston, T. A.; Innocenti, A.; Vullo, D.; Supuran, C. T.; Poulsen, S. A. J. Med. Chem. 2007, 50, 1651; (f) Chohan, Z. H.; Scozzafava, A.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2002, 17, 261. 18. (a) Supuran, C. T. Curr. Pharm. Des. 2010, 16, 3233; (b) Carta, F.; Aggarwal, M.; Maresca, A.; Scozzafava, A.; McKenna, R.; Masini, E.; Supuran, C. T. J. Med. Chem. 2012, 55, 1721; (c) Bonneau, A.; Maresca, A.; Winum, J. Y.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 397; (d) Scozzafava, A.; Menabuoni, L.; Mincione, F.; Mincione, G.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2001, 11, 575; (e) Supuran, C. T. Expert Rev. Neurother. 2015, 15, 851. 19. (a) Supuran, C. T.; Scozzafava, A.; Casini, A. Med. Res. Rev. 2003, 23, 146; (b) 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; (c) Capasso, C.; Supuran, C. T. Expert Opin. Ther. Pat. 2013, 23, 693; (d) Supuran, C. T.; Casini, A.; Mastrolorenzo, A.; Scozzafava, A. Mini-Rev. Med. Chem. 2004, 4, 625; (e) Supuran, C. T.; Scozzafava, A.; Mastrolorenzo, A. Expert Opin. Ther. Pat. 2001, 11, 221. 20. (a) Vullo, D.; Nishimori, I.; Scozzafava, A.; Kohler, S.; Winum, J. Y.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2010, 20, 2178; (b) Maresca, A.; Vullo, D.; Scozzafava,

21.

22.

23.

24.

25.

26.

27.

28.

29.

A.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 388; (c) Alp, C.; Özsoy, _ Sßentürk, M.; S.; Alp, N. A.; Erdem, D.; Gültekin, M. S.; Küfreviog˘lu, Ö. I.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 818; (d) Nishimori, I.; Onishi, S.; Takeuchi, H.; Supuran, C. T. Curr. Pharm. Des. 2008, 14, 622. (a) Kazancıog˘lu, E. A.; Güney, M.; S ß entürk, M.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 880; (b) Liu, F.; Martin-Mingot, A.; Lecornué, F.; Maresca, A.; Thibaudeau, S.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 886; (c) Maresca, A.; Scozzafava, A.; Vullo, D.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 384; (d) Koz, O.; Ekinci, D.; Perrone, A.; Piacente, S.; Alankus-Caliskan, O.; Bedir, E.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 412; (e) Winum, J. Y.; Kohler, S.; Supuran, C. T. Curr. Pharm. Des. 2010, 16, 3310. (a) De Simone, G.; Supuran, C. T. J. Inorg. Biochem. 2012, 111, 117; (b) S ß entürk, M.; Gülçin, I.; Dasßtan, A.; Küfreviog˘lu, Ö. I.; Supuran, C. T. Bioorg. Med. Chem. 2009, 17, 3207; (c) Carta, F.; Temperini, C.; Innocenti, A.; Scozzafava, A.; Kaila, K.; Supuran, C. T. J. Med. Chem. 2010, 53, 5511; (d) Carta, F.; Aggarwal, M.; Maresca, A.; Scozzafava, A.; McKenna, R.; Supuran, C. T. Chem. Commun. 2012, 1868; (e) Abbate, F.; Supuran, C. T.; Scozzafava, A.; Orioli, P.; Stubbs, M.; Klebe, G. J. Med. Chem. 2002, 45, 3583; (f) Temperini, C.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2010, 20, 474. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561. An Applied Photophysics stoppedflow instrument has been used for assaying the CA catalyzed CO2 hydration activity. Bromothymol blue (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 10–20 mM TRIS (pH 8.3) as buffer, and 20 mM NaBF4 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 have been 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 inhibitor (10 mM) were prepared in distilled-deionized water and dilutions up to 0.01 lM were done thereafter with distilled-deionized water. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay, in order to allow for the formation of the E–I complex. The inhibition constants were obtained by non-linear least-squares methods using the Cheng–Prusoff equation whereas the kinetic parameters for the uninhibited enzymes from Lineweaver–Burk plots, as reported earlier,24–27 and represent the mean from at least three different determinations. MgCA was a recombinant protein, obtained and purified by a diverse procedure as the one reported earlier.28 (a) Innocenti, A.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 1855, 2009, 19; (b) Innocenti, A.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2010, 20, 1548. (a) Kolayli, S.; Karahalil, F.; Sahin, H.; Dincer, B.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2011, 26, 895; (b) Maresca, A.; Scozzafava, A.; Kohler, S.; Winum, J. Y.; Supuran, C. T. J. Inorg. Biochem. 2012, 110, 36; (c) Ozensoy, O.; Arslan, M.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2011, 26, 749; (d) Migliardini, F.; De Luca, V.; Carginale, V.; Rossi, M.; Corbo, P.; Supuran, C. T.; Capasso, C. J. Enzyme Inhib. Med. Chem. 2014, 29, 146. (a) Demirdag˘, R.; Yerlikaya, E.; Sßentürk, M.; Küfreviog˘lu, Ö. I.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 278; (b) Maresca, A.; Carta, F.; Vullo, D.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 407. (a) Çavdar, H.; Ekinci, D.; Talaz, O.; Saraçog˘lu, N.; Sß entürk, M.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 148; (b) Chohan, Z. H.; Scozzafava, A.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2003, 18, 259; (c) Sharma, A.; Tiwari, M.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2014, 29, 292. The identification of the gene encoding for M. globosa b-CA (MgCA) was performed at the link http://www.ncbi.nlm.nih.gov/genome/. The b-CA gene of M. globosa (accession number:XP_001730815.1) was identified running the ‘BLAST’ program. The GeneArt Company, specialized in gene synthesis, furnished us the synthetic M. globosa gene encoding for MgCA. The fragment was cloned into the expression vector pET100/D-TOPO (Invitrogen), creating the plasmid pET100D-Topo/MgCA. Competent E. coli BL21 (DE3) codon plus cells (Agilent) were transformed with pET100D-Topo/MgCA, grown at 37 °C, induced with 1 mM IPTG, added 0.5 M ZnSO4 and grown for 3 h. After growth, cells were harvested and disrupted by sonication at 4 °C in 20 mM buffer phosphate, pH 8.0. Following sonication, the sample was centrifuged at 1200g at 4 °C for 30 min. The supernatant was loaded onto a His-select HF Nickel affinity column. The MgCA was eluted with 0.02 M phosphate buffer (pH 8.0) containing 250 mM imidazole and 0.5 M NaCl at a flow rate of 1.0 ml/ min. Fractions were collected and dialyzed. At this stage of purification the enzyme was at least 85% pure and the obtained recovery was of 0.3 mg of the recombinant MgCA per liter of culture. (a) Monti, S. M.; Ludwig, M.; Vullo, D.; Scozzafava, A.; Capasso, C.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2013, 23, 1626; (b) Dathan, N. A.; Alterio, V.; Troiano, E.; Vullo, D.; Ludwig, M.; De Simone, G.; Supuran, C. T.; Monti, S. M. J. Enzyme Inhib. Med. Chem. 2014, 29, 500.