Poly(amidoamine) dendrimers show carbonic anhydrase inhibitory activity against α-, β-, γ- and η-class enzymes

Bioorganic & Medicinal Chemistry 23 (2015) 6794–6798

Contents lists available at ScienceDirect

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

Poly(amidoamine) dendrimers show carbonic anhydrase inhibitory activity against a-, b-, c- and g-class enzymes Fabrizio Carta a, Sameh M. Osman b, Daniela Vullo a, Zeid AlOthman b, Sonia Del Prete c,d, Clemente Capasso c, Claudiu T. Supuran b,d,⇑ a

Università degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino (Firenze), Italy Department of Chemistry, College of Science, King Saud University, PO Box 2455, Riyadh 11451, Saudi Arabia Istituto di Bioscienze e Biorisorse, CNR, Via Pietro Castellino 111, 80131 Napoli, Italy d 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 c

a r t i c l e

i n f o

Article history: Received 24 August 2015 Revised 3 October 2015 Accepted 5 October 2015 Available online 8 October 2015 Keywords: PAMAM dendrimer Sulfonamide Carbonic anhydrase Pathogens Bacteria Fungi Protozoa

a b s t r a c t Four generations of poly(amidoamine) (PAMAM) dendrimers incorporating benzenesulfonamide moieties were investigated as inhibitors of carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the a-, b-, cand g-classes which are present in pathogenic bacteria, fungi or protozoa. The following bacterial, fungal and protozoan organisms were included in the study: Vibrio cholerae, Trypanosoma cruzi, Leishmania donovani chagasi, Porphyromonas gingivalis, Cryptococcus neoformans, Candida glabrata, and Plasmodium falciparum. The eight pathozymes present in these organisms were efficiently inhibited by the four generations PAMAM–sulfonamide dendrimers, but multivalency effects were highly variable among the different enzyme classes. The Vibrio enzyme VchCA was best inhibited by the G3 dendrimer incorporating 32 sulfamoyl moieties. The Trypanosoma enzyme TcCA on the other hand was best inhibited by the first generation dendrimer G0 (with 4 sulfamoyl groups), whereas for other enzymes the optimal inhibitory power was observed for the G1 or G2 dendrimers, with 8 and 16 sulfonamide functionalities. This study thus proves that the multivalency may be highly relevant for enzyme inhibition for some but not all CAs from pathogenic organisms. On the other hand, some dendrimers investigated here showed a better inhibitory power compared to acetazolamide for enzymes from widespread pathogens, such as the g-CA from Plasmodium falciparum. Overall, the main conclusion is that this class of molecules may lead to important developments in the field of anti-infective CA inhibitors. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Dendrimers, a class of molecules incorporating repetitively branched units around a symmetric central core, are highly attractive for a large number of biomedical and biotechnological applications, such as among others, drug delivery, preparation of synthetic enzymes, gene transfection systems and catalysis, as well as for developing alternative contrast agents for magnetic resonance imaging or for optical sensors.1–5 Poly(amidoamine) (PAMAM) dendrimers were the first family of such derivatives to be prepared,1,5 they are well characterized, commercially available, and consist of repetitively branched subunits of amide and amine functionalities. Due to their interesting physico-chemical properties, versatility and ease of derivatization, they are widely used for such ⇑ 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.bmc.2015.10.006 0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

biomedical applications as those mentioned above.1,5,6 The branched, tree-like concentric layers of the dendrimers, referred to as ’generations’, allow for a precise number of various functional groups to be incorporated in the macromolecules, which thereafter may act as a platform for controlling the interactions with the receptor, enzyme or tissue of interest. In addition, the particular three-dimensional conformation that functionalized dendrimer generations adopt may be exploited for targeting nano-drugs to different tissues or cell compartments as well as for enhancing bioavailability of some drugs.1–6 Recently we reported PAMAM dendrimers functionalized with aromatic sulfonamide functionalities which showed excellent inhibitory activity against several human (h) isoforms of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1), as well as excellent antiglaucoma action in vivo, in an animal model of the disease.6 CAs are widely spread enzymes in all life kingdoms, being involved in many crucial physiologic processes, as they catalyze a

F. Carta et al. / Bioorg. Med. Chem. 23 (2015) 6794–6798

simple but fundamental reaction, the reversible hydration of carbon dioxide to bicarbonate and protons.7–13 CAs are encoded by six different genetic families, the a-, b-, c-, d-, f- and g-CA classes, an interesting example of convergent evolution at molecular level.10–16 In vertebrates, including humans, at least 15 different a-CA isoforms are known,7,8 which play various physiologic functions, such as pH and CO2 homeostasis, respiration and transport of CO2/bicarbonate, electrolyte secretion in many tissues/organs, biosynthetic reactions (e.g., gluconeogenesis, lipogenesis and ureagenesis), bone resorption, calcification, tumorigenicity, etc.6–8,17 In algae, plants and cyanobacteria CAs play an important role in photosynthesis, by concentrating CO2/bicarbonate nearby the RUBISCO enzyme complex, as well as in several other biosynthetic reactions.11 In diatoms d- and f-CAs also play a crucial role in CO2 fixation but probably also in the SiO2 cycle.11,12b Inhibition of the mammalian CAs (mainly with primary sulfonamides and their isosteres such as the sulfamates) has been exploited therapeutically for decades for obtaining diuretics, antiglaucoma agents, antiepileptics, antiobesity drugs and more recently for antitumor therapies.6–8,17 However these enzymes are also widespread (and play crucial roles) in many pathogens belonging to the bacteria,13,16 fungi,14 and protozoa12d,e,15 domains, which were less investigated for the moment as drug targets. Only recently our and other groups explored the possibility of inhibiting enzymes form microorganisms with the goal of obtaining anti-infectives with a mechanism of action different from the clinically used drugs, to which significant drug-resistance problems emerged worldwide.13–16 Although the classical inhibitors of the sulfonamide type17 or the newer classes of such agents reported ultimately (coumarins, polyamines, dithiocarbamates, etc.)18–20 show potent inhibitory action in vitro, in many cases the in vivo efficacy of these agents is diminished due to lack of penetration through membranes or association with biomolecules which impair their action. This is the reason why we have explored alternative delivery systems for the CA inhibitors (CAIs) such as nanoparticles,21,22 fullerenes derivatized with various classes of CAIs,23 and more recently, dendrimers.6 These alternative classes of CAIs were however only investigated for their interaction with human CAs. Here we report the first inhibition study of pathogenic a-, b-, c- and g-class CAs with dendrimers, in the search of novel applications of the CAIs in the anti-infective field. 2. Results and discussion 2.1. Chemistry Four generations of commercially available PAMAM dendrimers, which incorporate free aminoethyl moieties, have been used for preparing the corresponding sulfonamide–dendrimers G0–G3, as reported earlier.6 These dendrimers incorporate 4, 8, 16 and 32 sulfonamide moieties, respectively (Fig. 1). Our main interest was to investigate structure–activity relationship related to the inhibition of pathogenic enzymes (pathozymes) from bacteria, fungi and protozoa with these compounds and how the multivalency may influence inhibition of various classes of CAs from such organisms. 2.2. Carbonic anhydrase inhibition The four generation of dendrimers G0–G3 (as well as the underivatized corresponding commercially available dendrimers) were assayed in vitro for the inhibition of several physiologically relevant CAs from various pathogens, as well as the cytosolic hCA

6795

I and II which are the physiologically dominant human isoforms (Table 1). We included in our study bacterial, fungal and protozoan CAs belonging to four enzyme classes, the a-, b-, c- and g-CAs, from the following pathogenic organisms: the bacterium Vibrio cholerae (the a-CA VchCA reported earlier16 by our groups was used as an example of bacterial enzymes); the protozoan Trypanosoma cruzi (TcCA, again an a-CA);15b,c as well as another protozoan provoking serious disease in humans and animals, Leishmania donovani chagasi (LdcCA, belonging to the b-CA class).15a In the oral cavity bacterium Porphyromonas gingivalis (responsible of periodontitis), we recently cloned and characterized two CAs, one belonging to the b-class (PgiCAb)12c and the other one to the c-CA class (PgiCA).12a We also investigated the inhibition of these two bacterial CAs with dendrimers G0–G3 incorporating sulfonamide moieties, since they may constitute new drug targets for developing antibiotics against this pathogen.13 Two fungal b-CAs were also included in our study, Can2 from Cryptococcus neoformans14a and CgCA, from Candida glabrata,14b pathogens which provoke serious infections worldwide, and for which significant drug resistance problems to the common anti-mycotic agents were reported.14c Finally, the recently discovered g-CA from the malaria protozoan Plasmodium falciparum,12d,e PfaCA was also included in this study, in order to assay the effects of these dendrimers against a wide type of different CAs (Table 1). It should be mentioned that the underivatized PAMAM dendrimers were first assayed for inhibitory action against all these enzymes and no significant loss of activity was observed up to 50 lM concentrations. The only exceptions were PgiCAb and PfaCA for which weak inhibitory effects were observed with some of the underivatized dendrimers (inhibition constants in the range of 8–10 lM, data not shown). The sulfonamide-derivatized dendrimers G0–G3 on the other hand showed a substantial inhibitory activity against all these enzymes, as shown in Table 1. The following SAR should be observed: (i) The bacterial enzyme VchCA was highly inhibited by dendrimers G0–G3 with KIs ranging between 8.1 and 47.2 nM (the best performing dendrimers were comparable to acetazolamide (AAZ) a highly efficient CAI against many a-CAs).2 For this particular case, the G0 generation showed a KI of 47.2 nM whereas the higher generations G1–G3, incorporating an increasing number of sulfonamides moieties, of 8, 16 and 32, respectively, showed a quite similar inhibitory power (KIs ranging between 8.1 and 9.2 nM). Thus, the multivalency effect reached to a saturation already for the compound with 8 sulfonamide units, with no further improvement of activity for dendrimers incorporating 16 and 32 such groups. (ii) The situation is quite different for the other a-CA investigated here, from the protozoan T. cruzi, TcCA. In this case G0 was the most effective inhibitor (KI of 17.7 nM), being more effective than AAZ, which has an inhibition constant of 61.6 nM against this enzyme. The inhibitory power constantly diminished thereafter for the next dendrimer generations, from G1 to G3 (KIs ranging between 95.4 and 639 nM), proving that for this protozoan a-CA (unlike the bacterial one VchCA discussed above), no multivalency inhibitory effects are being present. (iii) The other protozoan enzyme investigated here, LdcCA, a bCA from L. donovani chagasi, showed a diverse behavior to inhibition with dendrimers G0–G3, compared to both enzymes discussed above. Thus, for LdcCA the best inhibitor was G2 (KI of 34.8 nM) whereas G0, G1 and G3 had rather similar, less effective inhibitory activities, with KIs ranging between 75.4 and 90.7 nM (Table 1). It should be mentioned

6796

F. Carta et al. / Bioorg. Med. Chem. 23 (2015) 6794–6798 O NH O

H2NO2S

O

NH

O

NH

N

O

NH

N

O

SO2NH2

O

2

H2NO2S

N

O

2

HN

O

HN

O

SO2NH2

NH

HN

O

NH

O

NH

HN

N

O

HN

SO2NH2

HN

SO2NH2

NH O

G0

G1

H 2NO2S

O H2 NO 2S

O

O O

HN

O NH

O

HN NH

N

O

O

N

O

N H

H2NO2 S

O

O

SO 2NH 2

O N

NH N

HN

HN

HN

NH

O

NH

N

SO 2NH 2

2 NH

O

O

SO2 NH 2

NH

N 2

O

NH

O

O

N O

O

O

O

N H

NH O

NH

O

N HN

SO2NH2

SO 2NH2

HN N

HN

O

NH

HN N

O

N H

N

O

O

HN

O

O

H N

H2NO2 S

N H

O

NH

NH O

H N

N H

SO2NH2

N

O

O

O

NH

HN

HN

O

SO2NH2

SO2NH2

HN

H N

NH

H2 NO 2S

N

O

HN

O

O

O

O

N

HN HN

O O

O

NH

HN

NH N

NH

O

O

H N NH

HN

O H2 NO 2S

O

NH

HN

G2

H2NO2 S

N

N

O

HN NH O

O

N H

O

O HN

O

NH

SO2 NH 2

H N O

N

NH N H

NH

HN

SO2NH2

O

O

HN

O

O SO2NH2

O O

H N

N

N

O

O

SO2NH2

HN

O HN

SO2NH2

O

HN

HN

H 2NO2 S

NH

SO2 NH 2

O

O

H N

N

NH

N

O SO 2NH2

N H SO2NH2

G3

Figure 1. Chemical structure of sulfonamide-derivatized PAMAM dendrimers G0–G3.

that G2 is almost 3-fold a better LdcCA inhibitor compared to acetazolamide. (iv) The bacterial b-CA from P. gingivalis, PgiCAb was also inhibited by all dendrimers investigated here, with KIs ranging between 77.0 and 84.9 nM. Thus, all these dendrimers showed a very similar behavior of medium potency inhibitors, and were in fact more effective compared to the simple sulfonamide AAZ (KI of 214 nM).

(v) The fungal b-CAs Can2 and CgCA showed a very different behavior towards dendrimers G0–G3 as inhibitors, although these ortholog enzymes are encoded by similar genes in the two pathogens.14 Indeed, Can2 was well inhibited by G0 (incorporating 4 sulfamoyl moieties) with a KI of 24.0 nM, whereas the upper generations, G1–G3, showed diminished inhibitory potency (KIs in the range of 67.6 and 93.7 nM, Table 1). CgCA on the other hand was poorly inhibited by

F. Carta et al. / Bioorg. Med. Chem. 23 (2015) 6794–6798 Table 1 CA inhibition data against isoforms hCA I and II (a-class enzymes) as well as pathogenic enzymes from bacteria, protozoa and fungi, belonging to the a-, b-, c- and g-CA classes, with sulfonamide functionalized PAMAM dendrimers G0–G3 and acetazolamide (AAZ) as standard drug, by a stopped-flow CO2 hydrase assay24

* **

Enzyme

Class

hCA I** hCA II** VchCA TcCA LdcCA PgiCAb Can2 CgCA PgiCA PfaCA

a a a a b b b b

c g

KI* (nM) G0

G1

G2

G3

AAZ

24.1 10.4 47.2 17.7 75.4 84.9 24.0 427 405 69.7

12.0 3.1 8.1 95.4 85.6 77.0 83.6 342 563 58.6

10.8 0.93 9.2 414 34.8 76.5 93.7 66.4 682 47.8

10.5 0.07 8.5 639 90.7 80.5 67.6 509 283 85.5

250 12 6.8 61.6 91.7 214 10.5 11 324 170

Errors in the range of ±5% of the reported values, from three different assays. From Ref. 6.

G0, G1 and G3 (KIs ranging between 342 and 509 nM) with only G2 showing a better inhibitory effect (KI of 66.4 nM, which is much weaker compared to AAZ, see Table 1). (vi) The only c-class CA included in our study, PgiCA, was poorly inhibited by dendrimers G0–G3 investigated here, with KIs in the range of 283–682 nM. The best inhibitor was G3 but no regular variation of the inhibition effects with the number of sulfonamide moieties was evidenced for this enzyme (Table 1). It should be mentioned that c-CAs are trimeric enzymes with shallow active sites at the interface between monomers,10 and probably these bulky sulfonamides are sterically impaired and unable to easily interact with the Zn(II) ion from their active site. (vii) An interesting behavior was observed for PfaCA, the g-class enzyme from the malaria-provoking protozoa P. falciparum. Acetazolamide is a rather ineffective inhibitor for this enzyme (KI of 170 nM) and many other simple sulfonamides investigated earlier showed the same activity.12e To our surprise, dendrimers G0–G3 investigated here showed a potent inhibition of this enzyme, with KIs in the range of 47.8–85.5 nM. The best inhibitors were G2 and G1, whereas the activity was slightly worse for G0 and G3 (Table 1). However we should mention that the human isoforms hCA I and II were much more sensitive to inhibition by the dendrimers incorporating sulfonamide moieties compared to the pathogenic enzymes included in this study, which may lead to offtarget effects.

3. Conclusions We report here the first dendrimer inhibition study of pathogenic a-, b-, c- and g-CAs from the following bacterial, fungal and protozoan organisms: Vibrio cholerae, Trypanosoma cruzi, Leishmania donovani chagasi, Porphyromonas gingivalis, Cryptococcus neoformans, Candida glabrata, and Plasmodium falciparum. The eight pathozymes were efficiently inhibited by four generations of PAMAM–sulfonamide dendrimers but the multivalency effects were highly variable among the different enzyme classes. VchCA was best inhibited by the older generation (G3) dendrimer, incorporating 32 sulfonamide moieties. TcCA on the other hand was best inhibited by the first generation, G0 dendrimer (with only 4 such groups), whereas for other enzymes the optimal inhibitory power was observed for the G1 or G2 dendrimers. This study thus proves that the multivalency for enzyme inhibition may be highly relevant for some but not all CAs from

6797

pathogenic organisms. On the other hand, some of the dendrimers investigated here showed a better inhibitory power compared to acetazolamide for enzymes from important pathogens, such as the g-CA from Plasmodium falciparum. Overall, the main conclusion is that this class of molecules may lead to important developments in the field of anti-infective CA inhibitors. 4. Experimental 4.1. Chemistry The non-derivatized PAMAM dendrimers were commercially available from Sigma–Aldrich (Milan, Italy) whereas the derivatized dendrimers G0–G3 were prepared as described recently by us.6 4.2. CA enzyme assay An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed CO2 hydration activity.24 Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nM, with 20 mM HEPES (pH 7.5 for a- and g-class enzymes) or 20 mM Tris (pH 8.3, for the b- and c-CAs) as buffer. All buffers contained 20 mM Na2SO4 for maintaining constant the ionic strength. The initial rates of the CA-catalyzed CO2 hydration reaction were followed 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 (0.1 mM) were prepared in distilled-deionized water and dilutions up to 0.01 nM were done thereafter with the assay buffer. 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 PRISM 3, as reported earlier,6,25 and represent the mean from at least three different determinations. All CA isoforms were recombinant ones obtained in-house as reported earlier.6,12–16 Acknowledgements We thank The Distinguished Scientist Fellowship Program (DSFP) at King Saud University, Saudi Arabia for funding this project. This work was also supported by an European Union FP7 ITN Project (Dynano). References and notes 1. (a) Tomalia, D. A.; Naylor, A. M.; Goddard, W. A. Angew. Chem., Int. Ed. Engl. 1990, 29, 138; (b) Newkome, G. R.; Moorefield, C. N.; Vögtle, F. Dendritic Molecules: Concepts, Syntheses, Perspectives; WILEY-VCH, Verlag GmbH, 1997. 2. (a) Cai, X.; Hu, J.; Xiao, J.; Cheng, Y. Expert Opin. Ther. Pat. 2013, 23, 515; (b) Soršak, E.; Valh, J. V.; Urek, Š. K.; Lobnik, A. Analyst 2015, 140, 976; (c) Bagul, R. S.; Jayaraman, N. Inorg. Chim. Acta 2014, 409, 34. 3. (a) Smith, D. K.; Diederich, F. Chem. Eur. J. 1998, 4, 1353; (b) Baussanne, I.; Benito, J. M.; Mellet, C. O.; Fernandez, J. M. G.; Law, H.; Defaye, J. Chem. Commun. 2000, 1489. 4. (a) Wiener, E. C.; Brechbiel, M. W.; Brothers, H.; Magin, R. L.; Gansow, O. A.; Tomalia, D. A.; Lauterbur, P. C. Magn. Reson. Med. 1994, 31, 1; (b) Albertazzi, L.; Storti, B.; Marchetti, L.; Beltram, F. J. Am. Chem. Soc. 2010, 132, 18158; (c) Esfand, R.; Tomalia, D. A. Drug Discovery Today 2001, 6, 427. 5. (a) Svenson, S.; Tomalia, D. A. Adv. Drug Deliv. Rev. 2005, 57, 2106; (b) Tomalia, D. A.; Reyna, L. A.; Svenson, S. Biochem. Soc. Trans. 2007, 35, 61; (c) Kannan, R. M.; Nance, E.; Kannan, S.; Tomalia, D. A. J. Intern. Med. 2014, 276, 579. 6. (a) Carta, F.; Osman, S. M.; Vullo, D.; Gullotto, A.; Winum, J. Y.; AlOthman, Z.; Masini, E.; Supuran, C. T. J. Med. Chem. 2015, 58, 4039; (b) Carta, F.; Osman, S. M.; Vullo, D.; AlOthman, Z.; Supuran, C. T. Org. Biomol. Chem. 2015, 13, 6453.

6798

F. Carta et al. / Bioorg. Med. Chem. 23 (2015) 6794–6798

7. (a) Alterio, V.; Di Fiore, A.; D’Ambrosio, K.; Supuran, C. T.; De Simone, G. Chem. Rev. 2012, 112, 4421; (b) Neri, D.; Supuran, C. T. Nat. Rev. Drug Disc. 2011, 10, 767; (c) Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 229. 8. (a) Supuran, C. T. Nat. Rev. Drug Disc. 2008, 7, 168; (b) Supuran, C. T. Curr. Pharm. Des. 2008, 14, 601; (c) Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 759; (d) De Simone, G.; Supuran, C. T. J. Inorg. Biochem. 2012, 111, 117. 9. (a) Capasso, C.; Supuran, C. T. Expert Opin. Ther. Pat. 2013, 23, 693; (b) Supuran, C. T. Front. Pharmacol. 2011, 2, 34. 10. (a) Smith, K. S.; Jakubzick, C.; Whittam, T. S.; Ferry, J. G. Proc. Natl. Acad. Sci. U.S. A. 1999, 96, 15184; (b) Harju, A. K.; Bootorabi, F.; Kuuslahti, M.; Supuran, C. T.; Parkkila, S. J. Enzyme Inhib. Med. Chem. 2013, 28, 231; (c) Aggarwal, M.; Kondeti, B.; McKenna, R. Expert Opin. Ther. Pat. 2013, 23, 717. 11. (a) 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; (b) Bertucci, A.; Moya, A.; Tambutté, S.; Allemand, D.; Supuran, C. T.; Zoccola, D. Bioorg. Med. Chem. 2013, 21, 1437. 12. (a) Del Prete, S.; De Luca, V.; Vullo, D.; Scozzafava, A.; Carginale, V.; Supuran, C. T.; Capasso, C. J. Enzyme Inhib. Med. Chem. 2014, 29, 532; (b) 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; (c) Del Prete, S.; Vullo, D.; Osman, S. M.; Scozzafava, A.; AlOthman, Z.; Capasso, C.; Supuran, C. T. Bioorg. Med. Chem. 2014, 22, 4537; (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. 13. (a) Capasso, C.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2014, 29, 379; (b) Supuran, C. T.; Capasso, C. Expert Opin. Ther. Targets 2015, 19, 551; (c) Capasso, C.; Supuran, C. T. Curr. Med. Chem. 2015, 22, 2130; d S ß entürk, M.; Gülçin, I.; Dasßtan, A.; Küfreviog˘lu, Ö. I.; Supuran, C. T. Bioorg. Med. Chem. 2009, 17, 3207. 14. (a) Schlicker, C.; Hall, R. A.; Vullo, D.; Middelhaufe, S.; Gertz, M.; Supuran, C. T.; Mühlschlegel, F. A.; Steegborn, C. J. Mol. Biol. 2009, 385, 1207; (b) Cottier, F.; Leewattanapasuk, W.; Kemp, L. R.; Murphy, M.; Supuran, C. T.; Kurzai, O.; Mühlschlegel, F. A. Bioorg. Med. Chem. 2013, 21, 1549; (c) Innocenti, A.; Muhlschlegel, F. A.; Hall, R. A.; Steegborn, C.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2008, 18, 5066. 15. (a) Syrjanen, L.; Vermelho, A. B.; de Almeida Rodrigues, I.; Corte-Real, S.; Salonen, T.; Pan, P.; Vullo, D.; Parkkila, S.; Capasso, C.; Supuran, C. T. J. Med. Chem. 2013, 56, 7372; (b) Pan, P.; Vermelho, A. B.; Capaci Rodrigues, G.; Scozzafava, A.; Tolvanen, M. E.; Parkkila, S.; Capasso, C.; Supuran, C. T. J. Med. Chem. 2013, 56, 1761; (c) Güzel-Akdemir, Ö.; Akdemir, A.; Pan, P.; Vermelho, A. B.; Parkkila, S.; Scozzafava, A.; Capasso, C.; Supuran, C. T. J. Med. Chem. 2013, 56, 5773. 16. (a) Del Prete, S.; De Luca, V.; Scozzafava, A.; Carginale, V.; Supuran, C. T.; Capasso, C. J. Enzyme Inhib. Med. Chem. 2014, 29, 23; (b) Del Prete, S.; Isik, S.; Vullo, D.; De Luca, V.; Carginale, V.; Scozzafava, A.; Supuran, C. T.; Capasso, C. J.

17.

18.

19.

20.

21. 22.

23.

24. 25.

Med. Chem. 2012, 55, 10742; (c) 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. (a) Carta, F.; Supuran, C. T. Expert Opin. Ther. Pat. 2013, 23, 681; (b) Supuran, C. T. Expert Opin. Ther. Pat. 2013, 23, 677; (c) Scozzafava, A.; Supuran, C. T.; Carta, F. Expert Opin. Ther. Pat. 2013, 23, 725; (d) Masini, E.; Carta, F.; Scozzafava, A.; Supuran, C. T. Expert Opin. Ther. Pat. 2013, 23, 705; (e) Supuran, C. T.; Casini, A.; Mastrolorenzo, A.; Scozzafava, A. Mini-Rev. Med. Chem. 2004, 4, 625; (f) Supuran, C. T.; Scozzafava, A.; Mastrolorenzo, A. Expert Opin. Ther. Pat. 2001, 11, 221. (a) Maresca, A.; Temperini, C.; Vu, H.; Pham, N. B.; Poulsen, S. A.; Scozzafava, A.; Quinn, R. J.; Supuran, C. T. J. Am. Chem. Soc. 2009, 131, 3057; (b) Maresca, A.; Temperini, C.; Pochet, L.; Masereel, B.; Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2010, 53, 335. (a) Carta, F.; Temperini, C.; Innocenti, A.; Scozzafava, A.; Kaila, K.; Supuran, C. T. J. Med. Chem. 2010, 53, 5511; (b) Davis, R.; Vullo, D.; Supuran, C. T.; Poulsen, S. A. BioMed. Res. Int. 2014, 2014, 374079. (a) Carta, F.; Aggarwal, M.; Maresca, A.; Scozzafava, A.; McKenna, R.; Supuran, C. T. Chem. Commun. 2012, 1868; (b) Maresca, A.; Carta, F.; Vullo, D.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 407; (c) Carta, F.; Aggarwal, M.; Maresca, A.; Scozzafava, A.; McKenna, R.; Masini, E.; Supuran, C. T. J. Med. Chem. 2012, 55, 1721; (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. Stiti, M.; Cecchi, A.; Rami, M.; Abdaoui, M.; Scozzafava, A.; Guari, Y.; Winum, J. Y.; Supuran, C. T. J. Am. Chem. Soc. 2008, 130, 16130. Ratto, F.; Witort, E.; Tatini, F.; Centi, S.; Lazzeri, L.; Carta, F.; Lulli, M.; Vullo, D.; Fusi, F.; Supuran, C. T.; Scozzafava, A.; Capaccioli, S.; Pini, R. Adv. Funct. Mater. 2015, 25, 316. (a) Abellán-Flos, M.; Tanç, M.; Supuran, C. T.; Vincent, S. P. Org. Biomol. Chem. 2015, 13, 7445; (b) Innocenti, A.; Durdagi, S.; Doostdar, N.; Strom, T. A.; Barron, A. R.; Supuran, C. T. Bioorg. Med. Chem. 2010, 28, 2822. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561. (a) Ekinci, D.; Kurbanoglu, N. I.; Salamci, E.; Senturk, M.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 845; (b) Ekinci, D.; Karagoz, L.; Ekinci, D.; Senturk, M.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 283; (c) 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; (d) Maresca, A.; Vullo, D.; Scozzafava, A.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 388; (e) Koz, O.; Ekinci, D.; Perrone, A.; Piacente, S.; Alankus-Caliskan, O.; Bedir, E.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 412; (f) Supuran, C. T.; Maresca, A.; Gregánˇ, F.; Remko, M. J. Enzyme Inhib. Med. Chem. 2013, 28, 289; (g) Migliardini, F.; De Luca, V.; Carginale, V.; Rossi, M.; Corbo, P.; Supuran, C. T.; Capasso, C. J. Enzyme Inhib. Med. Chem. 2014, 29, 146.