Anion and sulfonamide inhibition studies of an α-carbonic anhydrase from the Antarctic hemoglobinless fish Chionodraco hamatus

Bioorganic & Medicinal Chemistry Letters 25 (2015) 5485–5489

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Anion and sulfonamide inhibition studies of an a-carbonic anhydrase from the Antarctic hemoglobinless fish Chionodraco hamatus Alessandra Cincinelli a, Tania Martellini a, Daniela Vullo a, Claudiu T. Supuran a,b,⇑ a

Università degli Studi di Firenze, Dipartimento di Chimica Ugo Schiff, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino (Firenze), Italy Università degli Studi di Firenze, Polo Scientifico, Dipartimento NEUROFARBA;Sezione di Scienze Farmaceutiche e Nutraceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino (Firenze), Italy b

a r t i c l e

i n f o

Article history: Received 4 October 2015 Revised 22 October 2015 Accepted 23 October 2015 Available online 26 October 2015 Keywords: Carbonic anhydrase Sulfonamide Anion Chionodraco hamatus

a b s t r a c t An a-carbonic anhydrase (CA, EC 4.2.1.1) has been purified from the Antarctic hemoglobinless fish Chionodraco hamatus (icefish). The new enzyme, denominated ChaCA, has a good catalytic activity for the physiologic CO2 hydration to bicarbonate reaction, similar to that of the low activity human isoform hCA I, with a kcat of 5.3
105 s1, and a kcat/Km of 3.7
107 M1 s1. The enzyme was inhibited in the submillimolar range by most inorganic anions (cyanate, thiocyanate, cyanide, bicarbonate, halides), whereas sulfamide, sulfamate, phenylboronic/phenylarsonic acids were micromolar inhibitors, with KIs in the range of 9–77 lM. Many clinically used drugs, such as acetazolamide, methazolamide, dorzolamide, brinzolamide, topiramate and benzolamide were low nanomolar inhibitors, with KIs in the range of 39.1–77.6 nM. As the physiology of CO2/bicarbonate transport or the Root effect in this Antarctic fish are poorly understood at this moment, such inhibition data may give a more detailed insight in the role that CAs play in these phenomena, by the use of inhibitors described here as physiologic tools. Ó 2015 Elsevier Ltd. All rights reserved.

Chionodraco hamatus, also known as icefish, is one of the few vertebrates which is devoid of red blood cells, and thus hemoglobin, being found in the Antarctic seas at depth of 400–600 m.1 Lacking the oxygen carried by haemoglobin and myglobin, C. hamatus is probably able to survive because the cold Antarctic water is well oxygenated, but many aspects regarding the physiology of this fish are in fact poorly understood.1,2 The ‘white’ blood and pale anaemic flesh of this fish is probably a consequence of its adaptation to live within the cold, highly oxygenated waters of the Antarctic, where metabolic requirements are lower than for warm-blooded animals.1,2 However, as for all vertebrates, the metabolism produces CO2, a gas which is not highly water soluble (being thus able to damage membranes or other cell substructures), which by hydration leads to a base (bicarbonate) and H+ ions, which may possibly lead to acid-base imbalance. The spontaneous process is rather slow and is normally catalyzed by the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1), which is thus involved in pH buffering of extra- and intracellular spaces.3–6 The ⇑ Corresponding author. Tel.: +39 055 4573005; fax: +39 055 4573385. E-mail address: [email protected] (C.T. Supuran). http://dx.doi.org/10.1016/j.bmcl.2015.10.074 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

equilibration between CO2, bicarbonate and protons is steadfastly assured by the catalytic activity of these enzymes, present in organisms all over the phylogenetic tree, in prokaryotes as well as eukaryotes.5–9 CAs are in fact encoded by six distinct, evolutionarily unrelated gene families, the a-, b-, c-, d-, f- and g-CAs, but vertebrates seem to possess only members of the first family, although in a rather large number of isoforms.7–11 These different isoforms are involved in physiological processes connected with respiration and transport of CO2/bicarbonate between metabolizing tissues and lungs, pH and CO2 homeostasis, electrolyte secretion in a variety of tissues/organs, biosynthetic reactions (e.g., gluconeogenesis, lipogenesis and ureagenesis), bone resorption, calcification, tumorigenicity, epileptogenesis, tumorigenesis, etc.3–11 As mentioned above, vertebrate genomes encode for a variable number of a-CA isoforms but their precise number is not known for most orders.1–3,8 Except humans and rodents (for which 15 and respectively 16 CAs were described and characterized in detail),4,5 few other vertebrates were thoroughly investigated regarding the number of isoforms, their catalytic activity, and inhibition susceptibility with various classes of inhibitors.4,10,11

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Indeed, literature data are available for several fish species10 (such as the model animal zebra fish, Danio rerio,11 the rainbow trout Oncorhyncus mykiss;12a the sea bass Dicentrarchus labrax12b and to a lesser extent the icefish Chionodraco hamatus).13 Among birds, the red blood cell enzyme from the ratid Struthio camelus (ostrich)14 has been investigated in some detail, but as outlined above, these data for enzymes which are of non human or non rodent origin, are rather scarce. The gas exchange processes are very much dependent on the CA activity (in the blood, kidneys, lungs, etc.), both through the BohrHaldane effect (O2 loading and unloading of hemoglobin, a process dependent on pH, which is regulated by the CAs and in the teleost fish this is known as the Root effect),15 and through their direct involvement in the CO2 excretion pathways (in gills and kidneys, as bicarbonate ion, or in the lungs as gaseous CO2).10,15,16 It appears thus of interest to investigate these enzymes in organisms adapted to live in extreme condition, such as the ice fish C. hamatus, which as mentioned above, do not have hemoglobin/myoglobin in its tissues and is adapted to live in the drastic conditions of the Antarctic ocean. It should be noted that although there are literature data reporting the biochemical characterization and molecular modeling of the gill CA from this fish, from Maffia’s group,13 no detailed kinetic and inhibition studies of this enzyme were reported until now. In this paper we report the purification, characterization and inhibition studies of a high activity a-CA isoform from C. hamatus and its inhibitory properties towards inorganic anions and sulfonamides. Samples of C. hamatus were collected in the Ross Sea, Antarctica, south the Italian ‘Mario Zucchelli’ scientific station (74°420 0000 S, 164°080 4000 E)17 in the framework of the Italian National Programme of Research in Antarctica (PNRA). Samples of C. hamatus (n = 5) have been used for the isolation and purification of the enzyme, as described earlier for similar purification procedures of CAs from other vertebrates.18 Only one ChaCA isoform, with a molecular weight of around 29 kDa, has been isolated and purified by sulfonamide affinity chromatography.19,21 The catalytic activity of the enzyme preparation has been measured by using a stopped flow spectrophotometric method,21–24 monitoring the physiologic reaction catalyzed by these enzymes, that is, CO2 hydration to bicarbonate and protons. Data of Table 1 show the fish enzyme ChaCA to possess a significant catalytic activity as CO2 hydrase, with a kcat/Km of 3.7
107 M1 s1, similar to that of the cytosolic human (h) isoform hCA I (highly abundant in the blood and gastrointestinal tract).3,4 The ice fish has a first order rate constant of 5.3
105 s1, which is more than two times higher than that of hCA I (kcat of 2.0
105 s1), whereas the Michaelis–Menten constants (Km) of the two enzymes are quite different, of 14.2 mM for the fish enzyme and of 4.0 mM for hCA I (Table 1). The higher value of Km for the fish enzyme explains why it has a slightly lower catalytic efficacy compared to hCA I. It should be observed that ChaCA is catalytically much more active than another vertebrate

enzyme investigated earlier,3 hCA III, which is more than two orders of magnitude a weaker catalyst compared to ChaCA (Table 1). However, ChaCA is less catalytically active compared to the physiologically dominant human isoform hCA II or the Weddel seal enzyme lwCA investigated earlier by our group.18 The fish enzyme ChaCA, similar to all other CAs explored to date apart CA III,3a was also significantly inhibited by the clinically used sulfonamide CAI acetazolamide (1,3,4-thiadiazole-2-sulfonamide) with an inhibition constant of 65.4 nM (Table 1). Considering the methodology used for the enzyme purification (see Experimental) and the similar activities of ChaCA and hCA I (a cytosolic isoform),3a we speculate that ChaCA is also a cytosolic enzyme. Metal-complexing anions are well-known inhibitors of many metalloenzymes, including the CAs, and such compounds usually bind to the metal ion from the enzyme cavity, coordinating to it or adding to the metal coordination sphere (which in all a-CAs is constituted by three His residues and a water molecule/hydroxide ion coordinated to a catalytically crucial Zn(II) ion).3,18 We thus investigated the inhibition of ChaCA with a range of such anions and small molecules reported earlier to act as CAIs,3 such as sulfamide, sulfamic acid, phenyl-boronic- and phenyl-arsonic acid, Table 2). Inhibition data of the human isoforms hCA I and II and the fish enzyme ChaCA with these anions is also provided in Table 2, for comparison reasons, as they were reported earlier by this group.3,18 Data of Table 2 show that the only anion not inhibiting significantly these enzymes (KI > 200 mM) was perchlorate, known for its low propensity to bind metal ions in solution or in other metalloenzymes, such as many CAs of various origin, including those from Antarctic organisms. 27 Most of the other anions investigated here, such as the ‘‘metal poisons” cyanate, thiocyanate, cyanide, the halides/pseudohalides, nitrate, nitrite, sulfate, bisulfite as well as bicarbonate/carbonate, showed a very similar behaviour of submillimollar-millimolar inhibitors, with KIs varying in a small range of 0.58–1.21 mM (Table 2). The most effective inhibitor was cyanate and the least effective one bromide, but the difference of affinity is minimal, as outlined above. This is in a strong contrast with other enzymes investigated earlier and shown in Table 2, for which a much higher variation in the range of KIs was always observed.3,18,25 Other small molecules such as sulfamic acid (as sulfamate), sulfamide, phenylboronic and phenylarsonic acids, on the other hand acted as much more potent ChaCA inhibitors, with inhibition constants of 9–77 lM. Except sulfamate which was a better hCA I than ChaCA inhibitor, all the other molecules discussed in this subsection are much more effective as inhibitors of the Antarctic fish enzyme than of hCA I and II, whereas their affinity for the Weddel seal18a enzyme is intermediate between the ChaCA and human isoforms CA inhibitory profile (Table 2). However, the sulfonamides constitute the most important class of CAIs, with many representatives used as drugs (diuretics,

Table 1 Kinetic parameters and inhibition with acetazolamide of CAs from various vertebrates (hCA = human isoforms; lwCA = Weddel seal enzyme; ChaCA = ice fish CA), by a CO2 hydrase assay, at 20 °C and pH 7.521 Enzyme* hCA I hCA II hCA III lwCA ChaCA *

Species Human Human Human Seal Ice fish

kcat (s1) 5

2.0
10 1.4
106 1.3
104 1.1
106 5.3
105

Km (mM) 4.0 9.3 52.0 7.5 14.2

kcat/Km (M1 s1) 7

5.0
10 1.5
108 2.5
105 1.4
108 3.7
107

Ki (AAZ) (nM)

Localization

Ref.

250 12 240,000 63.0 65.4

Cytosol Cytosol Cytosol Cytosol Cytosol?

3 3 3 17a This work

Errors of the kinetic/thermodynamic parameters were in the range of ±5–10% of the reported value, from at least 3 different assays.

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antiglaucoma, antiobesity, antiepileptics, etc).3,26 It appeared thus of interest to investigate inhibition of the fish enzyme ChaCA with some of these compounds.

H3C

N N CH3CONH

SO2NH2

S

N

CH3CON

the heterocyclic as well as aromatic sulphonamide class, such as AAZ, MZA, EZA, DZA, BRZ, BZA, ZNS, SLT and the sulfamate TPM were potent ChaCA inhibitors, with KIs in the range of

N

N SO2NH2

S

AAZ

EtO

MZA

S

O

NHEt

SO2NH2

S

MeO(CH2)3

O

N O

DZA

S

O

H N NH

S

O O ZNS

HN

O

O

SAC

OMe O N H

O

O

O

N

SO2NH2

O NH2 O S O O

SO2NH2

S

SO2NH2

BZA

O

S

BRZ

N N PhSO2NH

SO2NH2

EZA

NHEt

Me

S

S

SO2NH2

O HCT

H N

N

Cl

O O S N H

Cl

SO2NH2

SO2NH2

O

TPM

SLP

O

IND

O

O S NH2

O S NH2

O N

N

CH3

S

H3C O N

F F F

CLX

N

S O NH2

O O

SLT

VLX

We have included 15 clinically used compounds (sulfonamides and one sulfamate): acetazolamide AAZ, methazolamide MZA, dorzolamide DZA, brinzolamide BRZ, benzolamide BZA, zonisamide ZNS, saccharin SAC, hydrochlorothiazide HCT, topiramate TPM, sulpiride SLP, indisulam IND, celecoxib CLX, valdecoxib VLX and sulthiame SLT.26 They include both aromatic and heteroaromatic derivatives. Inhibition data of the two human enzymes hCA I and II3,26 are also provided in Table 3, for comparison reasons. Data of Table 3 show that similar to other a-CAs, ChaCA is sensitive to inhibition with sulphonamides/sulfamates, as the 15 derivatives investigated here showed KIs varying between 39.1 and 877 nM. A number of these derivatives, belonging both to

39.1–77.6 nM. The secondary, acylated sulphonamide SAC, as well as the compounds possessing a bulkier (HCT) or more complex (SLP, IND, CLX and VLX) scaffolds, were on the other hand less effective as ChaCA inhibitors, with KIs in the range of 385–877 nM. It should be also noted that the inhibition profile of the ice fish enzyme is different both from the ones of the human red blood cell enzymes hCA I and II, as well as the Weddel seal enzyme lwCA, which shares a similar habitat as C. hamatus.18a In conclusion, an a-CA has been purified from the Antarctic hemoglobinless fish C. hamatus (icefish). The new enzyme, ChaCA, has a good catalytic activity for the physiologic CO2 hydration to bicarbonate reaction, similar to that of the low activity human

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Table 2 Inhibition constants of anionic inhibitors against the cytosolic human isozymes hCA I, II, lwCA and ChaCA, for the CO2 hydration reaction, at 20 °C and pH 7.521 KI# [mM]

Inhibitor



F Cl Br I CNO SCN CN N 3 HCO 3 CO2 3 NO 3 NO 2  HS HSO 3 SO2 4  ClO4 H2NSO3H* H2NSO2NH2 PhB(OH)2 PhAsO3H2*

hCA Ia

hCA IIa

lwCAb

ChaCAc

>300 6 4 0.3 0.0007 0.2 0.0005 0.0012 12 15 7 8.4 0.0006 18 63 >200 0.021 0.31 58.6 31.7

>300 200 63 26 0.03 1.6 0.02 1.5 85 73 35 63 0.04 89 >200 >200 0.39 1.13 23.1 49.2

0.74 0.89 1.01 20.0 0.087 0.097 0.070 0.83 0.10 0.060 55.6 0.90 1.00 0.59 0.82 >200 0.055 0.070 0.046 0.057

0.91 0.93 1.21 0.70 0.58 0.95 0.79 0.85 0.73 1.02 0.80 0.82 0.87 0.74 0.67 >200 0.077 0.009 0.010 0.009

a

Human recombinant isozyme, data from Ref. 3a. Seal enzyme, Ref. 18a. c This work. # Errors were in the range of 3–5% of the reported values, from three different assays. * As sodium salt. b

Table 3 Human (h) hCA I, II, seal and fish CA inhibition data with sulfonamides AAZ–HCT, by a stopped flow CO2 hydrase assay method at 20 °C and pH 7.521 KI* (nM)

Inhibitor a

AAZ MZA EZA DZA BRZ BZA ZNS SAC HCT TPM SLP IND CLX VLX SLT

a

hCA I

hCA II

lwCA

ChaCA

250 50 25 50,000 45,000 15 56 18,540 328 250 12,000 31 50,000 54,000 374

12 14 8 9 3 9 35 5950 290 10 40 15 21 43 9

63 61 51 5.7 6.4 67 517 5390 630 nt nt nt nt nt nt

65.4 65.0 49.9 39.1 88.1 65.3 52.9 877 866 77.6 511 385 866 726 49.5

a

From Ref. 3,25. Errors were in the range of 5–10% of the reported values, from three different assays. *

isoform hCA I, with a kcat of 5.3
105 s1, and a kcat/Km of 3.7
107 M1 s1. ChaCA was inhibited in the submillimolar range by most inorganic anions (cyanate, thiocyanate, cyanide, bicarbonate, halides), whereas sulfamide, sulfamate, phenylboronic- and phenylarsonic acids were micromolar inhibitors, with KIs in the range of 9–77 lM. Many clinically used drugs, such as acetazolamide, methazolamide, dorzolamide, brinzolamide, topiramate and benzolamide were low nanomolar inhibitors, with KIs in the range of 39.1–77.6 nM. As the physiology of CO2/bicarbonate transport or the Root effect in this Antarctic fish are poorly understood at this moment, such inhibition data may give a more detailed insight in the role that CAs play in these phenomena by the use of inhibitors described here as physiologic tools.

Acknowledgments The research was funded in part by the PNRA projects PdR 2009/A2.10 ‘Environmental contamination in Antarctica: levels and trends of legacy persistent organic pollutants’, PdR 2009/ A1.04 ‘Flows of persistent organic pollutants between polar abiotic and biotic compartments (POP-LAB)’ and PdR 2013/AZ2.05 ‘Evaluation and modelling of environmental impact related to chemical substances and pollutants released from massive snow/ice melting in Antarctica’. References and notes 1. (a) Ruud, J. T. Nature 1954, 173, 848; (b) Near, T. J. Antarct. Sci. 2004, 16, 37. 2. (a) Bargelloni, L.; Ritchie, P. A.; Patarnello, T.; Battaglia, B.; Lambert, D. M.; Meyer, A. Mol. Biol. Evol. 1994, 11, 854; (b) Scapigliati, G.; Chausson, F.; Cooper, E. L.; Scalia, D.; Mazzini, M. Polar Biol. 1997, 18, 209. 3. (a) Supuran, C. T. Nat. Rev. Drug Disc. 2008, 7, 168; (b) Supuran, C. T. Bioorg. Med. Chem. Lett. 2010, 20, 3467; (c) Supuran, C. T.; Scozzafava, A. Bioorg. Med. 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Enzyme Inhib. Med. Chem. 2013, 28, 231; (d) Aggarwal, M.; Kondeti, B.; McKenna, R. Expert Opin. Ther. Pat. 2013, 23, 717. 6. (a) Xu, Y.; Feng, L.; Jeffrey, P. D.; Shi, Y.; Morel, F. M. Nature 2008, 452, 56; (b) Capasso, C.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2014, 29, 379; (c) Supuran, C. T.; Capasso, C. Expert Opin. Ther. Targets 2015, 19, 551; (d) Capasso, C.; Supuran, C. T. Curr. Med. Chem. 2015, 22, 2130. 7. (a) Tripp, B. C.; Smith, K. S.; Ferry, J. G. J. Biol. Chem. 2001, 276, 48615; (b) Smith, K. S.; Ferry, J. G. FEMS Microbiol. Rev. 2000, 24, 335; (c) Ferry, J. G. Biochim. Biophys. Acta 2010, 1804, 374; (d) Pettersen, E. O.; Ebbesen, P.; Gieling, R. G.; Williams, K. J.; Dubois, L.; Lambin, P.; Ward, C.; Meehan, J.; Kunkler, I. H.; Langdon, S. P.; Ree, A. H.; Flatmark, K.; Lyng, H.; Calzada, M. J.; del Peso, L.; Landazuri, M. O.; Görlach, A.; Flamm, H.; Kieninger, J.; Urban, G.; Weltin, A.; Singleton, D.; Buffa, F. M.; Harris, A.; Scozzafava, A.; Supuran, C. T.; Moser, I.; Jobst, G.; Pouyssegur, J.; Chiche, J.; Mazure, N.; Marchiq, I.; Parks, S.; Ahmed, A.; Ashcroft, M.; Pastorekova, S.; Cao, Y.; Rouschop, K. M.; Wouters, B.; Koritzinsky, M.; Mujcic, H.; Cojocari, D. J. Enzyme Inhib. Med. Chem. 2015, 30, 689; (e) Zimmerman, S. A.; Ferry, J. G.; Supuran, C. T. Curr. Top. Med. Chem. 2007, 7, 901. 8. (a) Syrjänen, L.; Tolvanen, M.; Hilvo, M.; Olatubosun, A.; Innocenti, A.; Scozzafava, A.; Leppiniemi, J.; Niederhauser, B.; Hytönen, V. P.; Gorr, T. A.; Parkkila, S.; Supuran, C. T. BMC Biochem. 2010, 11, 28; (b) Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 759; (c) De Simone, G.; Supuran, C. T. J. Inorg. Biochem. 2012, 111, 117; (d) Capasso, C.; Supuran, C. T. Expert Opin. Ther. Pat. 2013, 23, 693; (e) Supuran, C. T. Front. Pharmacol. 2011, 2, 34. 9. (a) Rowlett, R. S. Biochim. Biophys. Acta 2010, 1804, 362. 10. Gilmour, K. M. Comp. Biochem. Physiol. A 2010, 157, 193. 11. Lin, T. Y.; Liao, B. K.; Horng, J. L.; Hsiao, C. D.; Hwang, O. P. Am. J. Physiol. 2008, 294, C1250. _ Küfrevioglu, Ö. I.; _ Supuran, C. T. J. J. 12. (a) Hisar, O.; Beydemir, S ß .; Gülcin, I.; Enzyme Inhib. Med. Chem. 2005, 20, 35; (b) Ekinci, D.; Bug˘rahan Ceyhun, S.; S ß entürk, M.; Erdem, D.; Küfreviog˘lu, Ö. I.; Supuran, C. T. Bioorg. Med. Chem. 2011, 19, 744. 13. (a) Rizzello, A.; Ciardiello, M. A.; Acierno, R.; Carratore, V.; Verri, T.; di Prisco, G.; Storelli, C.; Maffia, M. Protein J. 2007, 26, 335; (b) Maffia, M.; Rizzello, A.; Acierno, R.; Rollo, M.; Chiloiro, R.; Storelli, C. J. Exp. Biol. 2001, 204, 3983. 14. (a) Ozensoy, O.; Isik, S.; Arslan, O.; Arslan, M.; Scozzafava, A.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2005, 20, 383; (b) Casini, A.; Scozzafava, A.; Mincione, F.; Menabuoni, L.; Ilies, M. A.; Supuran, C. T. J. Med. Chem. 2000, 43, 4884; (c) Supuran, C. T.; Ilies, M. A.; Scozzafava, A. Eur. J. Med. Chem. 1998, 33, 739. 15. Rummer, J. L.; McKenzie, D. J.; Innocenti, A.; Supuran, C. T.; Brauner, C. J. Science 2013, 340, 1327. 16. Endevard, V.; Musa-Aziz, R.; Cooper, G. J.; Chen, L. M.; Plletier, M. F.; Virkki, L. K.; Supuran, C. T.; King, L. S.; Boron, W. F.; Gros, G. FASEB J. 1974, 2006, 20. 17. (a) Stortini, A. M.; Martellini, T.; Del Bubba, M.; Lepri, L.; Capodaglio, G.; Cincinelli, A. Microchem. J. 2009, 92, 37; (b) Cincinelli, A.; Martellini, T.; Del Bubba, M.; Lepri, L.; Corsolini, S.; Borghesi, N.; King, M. D.; Dickhut, R. M. Environ. Pollut. 2009, 157, 2153; (c) Dickhut, R. M.; Cincinelli, A.; Cochran, M.; Kylin, H. Environ. Sci. Technol. 2012, 46, 3135.

A. Cincinelli et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5485–5489 18. (a) Cincinelli, A.; Martellini, T.; Innocenti, A.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. 1847, 2011, 19; (b) Ozensoy, O.; Arslan, M.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2011, 26, 749. 19. Samples of C. hamatus were taken from five adult fishes weighing 300-400 g, captured during the XXVI Expedition of the Italian Antarctic Research Program (PNRA) in Terra Nova Bay, Antarctica, during the Austral summer months in 2010-2011. Samples were stored at -20 °C until analysis. Fish samples were washed twice with NaCl (0.9%) and homogenized. Cell were lysed by immersion in liquid nitrogen at -163 °C. The lysed samples underwent three freeze–thaw cycles in dry ice with the addition of five times their volume of ice-cold distilled water, and then sonicated, in order to solubilize all cytosolic enzymes. The pH of the homogenate was adjusted to 8.7 using solid Tris, and the supernatant was applied to an activated CH Sepharose 4B-4-(2aminoethyl)-benzensulfonamide affinity column (1.36
30 cm) in order to separate the CA. The enzyme was purified by using this affinity gel chromatography according to the published method.19 All procedures were performed at 4 °C. Protein concentrations in the column effluents were determined at 280 nm spectrophotometrically and its activity by a stopped flow assay.20 20. Khalifah, R. G.; Strader, D. J.; Bryant, S. H.; Gibson, S. M. Biochemistry 1977, 16, 2241. 21. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561 An Applied Photophysics stopped-flow instrument has been used for assaying the CA-catalyzed CO2 hydration activity. Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes buffer (pH 7.5) and 20 mM NaClO4 for maintaining constant ionic strength, following the initial rates of the CA-catalyzed CO2 hydration reaction for a period of 10-100 s, at 20 °C. 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 to determine the initial velocity. The uncatalyzed rates were measured in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (1 mM) were prepared in distilleddeionized water and dilutions down to 0.01 nM were made thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for

22.

23.

24.

25.

26.

27.

5489

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 leastsquares 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,21–23 and represent the mean from at least three different determinations. (a) Maresca, A.; Carta, F.; Vullo, D.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 407; (b) Ekinci, D.; Kurbanoglu, N. I.; Salamci, E.; Senturk, M.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 845; (c) Ekinci, D.; Karagoz, L.; Ekinci, D.; Senturk, M.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 283; (d) 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. (a) Liu, F.; Martin-Mingot, A.; Lecornué, F.; Maresca, A.; Thibaudeau, S.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 886; (b) Demirdag˘, R.; Yerlikaya, E.; S ß entürk, M.; Küfreviog˘lu, Ö. I.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 278; (c) Moya, A.; Tambutté, S.; Bertucci, A.; Tambutté, E.; Lotto, S.; Vullo, D.; Supuran, C. T.; Allemand, D.; Zoccola, D. J. Biol. Chem. 2008, 283, 25475; (d) Scozzafava, A.; Menabuoni, L.; Mincione, F.; Mincione, G.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2001, 11, 575. (a) Maresca, A.; Vullo, D.; Scozzafava, A.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 388; (b) Koz, O.; Ekinci, D.; Perrone, A.; Piacente, S.; AlankusCaliskan, O.; Bedir, E.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 412; (c) Supuran, C. T.; Maresca, A.; Gregánˇ, F.; Remko, M. J. Enzyme Inhib. Med. Chem. 2013, 28, 289; (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) DeLuca, V.; DelPrete, S.; Carginale, V.; Vullo, D.; Supuran, C. T.; Capasso, C. Bioorg. Med. Chem. Lett. 2015, 15, 15; (b) De Luca, V.; Vullo, D.; Del Prete, S.; Carginale, V.; Scozzafava, A.; Osman, S. M.; AlOthman, Z.; Supuran, C. T.; Capasso, C. Bioorg. Med. Chem. 2015, 23, 4405. (a) Supuran, C. T. Expert Opin. Ther. Pat. 2013, 23, 677; (b) Supuran, C. T. Bioorg. Med. Chem. 2013, 21, 1377; (c) De Simone, G.; Alterio, V.; Supuran, C. T. Expert Opin. Drug Discov. 2013, 8, 793. Vullo, D.; De Luca, V.; Del Prete, S.; Carginale, V.; Scozzafava, A.; Capasso, C.; Supuran, C. T. Bioorg. Med. Chem. 2015, 23, 1728.