Carbonic anhydrases: novel ... - Akdeniz Üniversitesiicerik.akdeniz.edu.tr › _dinamik › 1 ›...

Carbonic anhydrases (CAs; also known as carbonate dehydratases EC 4.2.1.1) are ubiquitous metalloenzymes present in prokaryotes and eukaryotes that are encoded by four evolutionarily unrelated gene families. These are the α‑CAs (present in vertebrates, bacteria, algae and cytoplasm of green plants); the β‑CAs (predominantly in bacteria, algae and chloroplasts of monodicotyle‑dons and dicotyledons); the γ‑CAs (mainly in archaea and some bacteria); and the δ‑CAs (present in some marine diatoms)1–7. In mammals, 16 α‑CA isozymes or CA‑related proteins have been described (TABLE 1), with different catalytic activity, subcellular localization and tissue distribution8–18. There are five cytosolic forms (CA I, CA II, CA III, CA VII and CA XIII), five membrane‑bound isozymes (CA IV, CA IX, CA XII, CA XIV and CA XV), two mitochondrial forms (CA VA and CA VB), and a secreted CA isozyme (CA VI)19–25.

CAs catalyse a simple physiological reaction (BOX 1): the conversion of CO2 to the bicarbonate ion and pro‑tons. The active site of most CAs contains a zinc ion (Zn2+), which is essential for catalysis. The CA reaction is involved in many physiological and pathological pro‑cesses, including respiration and transport of CO2 and bicarbonate between metabolizing tissues and lungs; pH and CO2 homeostasis; electrolyte secretion in vari‑ous tissues and organs; biosynthetic reactions (such as gluconeogenesis, lipogenesis and ureagenesis); bone resorption; calcification; and tumorigenicity8–18.

Many of the CA isozymes involved in these processes are important therapeutic targets with the potential to be inhibited to treat a range of disorders including oedema, glaucoma, obesity, cancer, epilepsy and osteoporosis. Two main classes of CA inhibitors (CAIs) are known: the metal‑complexing anions and the unsubstituted sulphonamides and their bioisosteres — for example, sulphamates and sulphamides compounds 1–25 (FIG. 1;

TABLE 1). These inhibitors bind to the Zn2+ ion of the enzyme either by substituting the non‑protein zinc ligand to generate a tetrahedral adduct or by addition to the metal coordination sphere to generate a trigonal‑bipyramidal species1–7 (FIG. 2). At least 25 clinically used drugs have been reported to possess significant CA inhibitory properties (discussed below), in addition to many other derivatives belonging to the sulphonamide, sulphamate or sulphamide families1–3,5,7,8,19–25.

Furthermore, the potential use of CAIs to fight infec‑tions caused by protozoa, fungi and bacteria has recently emerged as a new research direction. CAs belonging to various families were cloned and characterized in many such organisms (such as Plasmodium falciparum, Helicobacter pylori, Mycobacterium tuberculosis, Candida albicans and Cryptococcus neoformans)26–34 and have been shown to be crucial for the virulence, growth or acclimatization of the parasite. In addition, impor‑tant advances have been achieved in the understanding of CA activation by several classes of activators35–41.

Laboratorio di Chimica Bioinorganica, Università degli Studi di Firenze, Rm 188, Via della Lastruccia 3, I‑50019 Sesto Fiorentino (Firenze), Italy. e‑mail: [email protected]:10.1038/nrd2467 Published online 2 January 2008

GlaucomaA chronic, degenerative eye disease, characterized by high intraocular pressure that causes irreversible damage to the optic nerve head, resulting in the progressive loss of visual function and eventually blindness.

Carbonic anhydrases: novel therapeutic applications for inhibitors and activatorsClaudiu T. Supuran

Abstract | Carbonic anhydrases (CAs), a group of ubiquitously expressed metalloenzymes, are involved in numerous physiological and pathological processes, including gluconeogenesis, lipogenesis, ureagenesis, tumorigenicity and the growth and virulence of various pathogens. In addition to the established role of CA inhibitors (CAIs) as diuretics and antiglaucoma drugs, it has recently emerged that CAIs could have potential as novel anti-obesity, anticancer and anti-infective drugs. Furthermore, recent studies suggest that CA activation may provide a novel therapy for Alzheimer’s disease. This article discusses the biological rationale for the novel uses of inhibitors or activators of CA activity in multiple diseases, and highlights progress in the development of specific modulators of the relevant CA isoforms, some of which are now being evaluated in clinical trials.

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168 | FEBruAry 2008 | VOluME 7 www.nature.com/reviews/drugdisc

© 2008 Nature Publishing Group

http://www.expasy.org/uniprot/P00915




http://www.expasy.org/uniprot/Q8N1Q1


http://www.expasy.org/uniprot/Q16790

http://www.expasy.org/uniprot/O43570

http://www.expasy.org/uniprot/Q9ULX7

http://www.expasy.org/uniprot/Q99N23


http://www.expasy.org/uniprot/Q9Y2D0


mailto:[email protected]

Such compounds might lead to pharmacological agents that have the potential to treat Alzheimer’s disease, ageing and other conditions involving memory deficits40.

Carbonic anhydrase inhibitorsCAIs include the classical inhibitors acetazolamide (com‑pound 1), methazolamide (compound 2), ethoxzolamide (compound 3), sulthiame (compound 4) and dichloro‑phenamide (compound 5). CAIs also include more recent drugs/investigational agents such as dorzolamide (compound 6), brinzolamide (compound 7), indisulam (compound 8), topiramate (compound 9), zonisamide (compound 10), sulpiride (compound 11), COuMATE (compound 12), EMATE (compound 13), celecoxib (compound 14), valdecoxib (compound 15) and saccharin (compound 16) (FIG. 1; TABLE 1). Derivatives 17 and 18 are investigational agents for targeting the tumour‑associated isoform CA IX (see later in the text). Many of these compounds were initially developed years ago during the search for diuretics, among which the thiazides,

compounds 19a–e, as well as derivatives 20–25 are still widely clinically used2,3 (FIG. 1). However, some of these enzyme inhibitors could also be used for the systemic treatment of glaucoma (see below), and more recently, newer derivatives have been discovered that have the potential as topical antiglaucoma agents, as well as anti‑tumour, anti‑obesity or anti‑infective drugs1–3,5,7,9–26.

The inhibitory effects of some of these clinically used drugs against the mammalian isoforms CA I–XIV — of human or mouse origin — are shown in TABLE 1. As specific isozymes are responsible for different bio‑logical responses, the diverse inhibition profiles of the various isozymes may explain the different actual and potential clinical applications of the CAIs, which range from diuretics and antiglaucoma agents, to anticancer, anti‑obesity and anti‑epileptic drugs. However, a crucial problem in CAI design is related to the high number of isoforms, their diffuse localization in many tissues and organs (TABLE 2), and the lack of isozyme selectivity of the presently available inhibitors. It can be observed that

Table 1 | Inhibition data with selected sulphonamides/sulphamates/sulphamides 1–25 against isozymes I–XIV*

KI (nm)

Isozyme (h = human, m = mouse)

hCA I‡ hCA II‡ hCA III‡ hCA IV‡ hCA VA‡ hCA VB‡ hCA VI‡ hCA VII‡ hCA IX§ hCA XII§ mCA XIII‡ hCA XIV‡

1 250 12 2 × 105 74 63 54 11 2.5 25 5.7 17 41

2 50 14 7 × 105 6,200 65 62 10 2.1 27 3.4 19 43

3 25 8 1 × 106 93 25 19 43 0.8 34 22 50 2.5

4 374 9 6.3 × 105 95 81 91 134 6 43 56 1,450 1,540

5 1,200 38 6.8 × 105 15,000 630 21 79 26 50 50 23 345

6 50,000 9 7.7 × 105 8,500 42 33 10 3.5 52 3.5 18 27

7 45,000 3 1.1 × 105 3,950 50 30 0.9 2.8 37 3.0 10 24

8 31 15 10,400 65 79 23 47 122 24 3.4 11 106

9 250 10 7.8 × 105 4,900 63 30 45 0.9 58|| 3.8 47 1,460

10 56 35 2.2 × 106 8,590 20 6,033 89 117 5.1 11,000 430 5,250

11 12,000 40 10,600 6.5 × 105 174 18 0.8 3,630 46 3.9 295 110

12 3,450 21 7.0 × 105 24 765 720 653 23 34 12 1,050 755

13 37 10 6.5 × 105 NT NT NT NT NT 30 7.5 NT NT

14 50,000 21 7.4 × 104 880 794 93 94 2,170 16 18 98 689

15 54,000 43 7.8 × 104 1,340 912 88 572 3,900 27 13 425 107

16 18,540 5,950 1.0 × 106 7,920 10,060 7,210 935 10 103 633 12,100 773

17 1,300 45 1.3 × 106 650 134 76 145 18 24 5 76 33

18 4,000 21 3.1 × 105 60 88 70 65 15 14 7 21 13

19a 328 290 7.9 × 105 427 4,225 603 3,655 5,010 367 355 3,885 4,105

20 35,000 1,260 NT NT NT NT NT NT NT NT NT NT

21 54,000 2,000 6.1 × 105 216 750 312 1,714 2.1 320 5.4 15 5,432

22 348 138 1.1 × 104 196 917 9 1,347 2.8 23 4.5 15 4,130

23 51,900 2,520 2.3 × 105 213 890 274 1,606 0.23 36 10 13 4,950

24 62 65 3.2 × 106 564 499 322 245 513 420 261 550 52

25 4,930 6,980 3.4 × 106 303 700 NT NT NT 25.8 21.2 2,570 250

*The isoforms CA VIII, X and XI are devoid of catalytic activity and probably do not bind sulphonamides as they do not contain Zn2+ ions. ‡Full-length enzyme. §Catalytic domain. ||The data against the full-length enzyme is of 1,590 nM. NT, not tested, data not available.

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E–Zn2+–OH– E–Zn2+–OH2 + HCO3–

H2OE–Zn2+–HCO3

– (2)

Nature Reviews | Drug DiscoveryE–Zn2+–OH2 E–Zn2+–HO– + H+ (3)

Nature Reviews | Drug Discovery


HCO3-–

CO2+

B

+ H2O

a b

cd

BH– +

Zn2+

OH

His119His96

His94

-

Zn2+

His119His96

His94

OH-O

O

Zn2+

OO

H

His119His96

His94

O

-Zn2+

OH2

His119His96

His94

Gln92Phe131

His119His94

His96His64

His4

His3

His10

His15

His17

CO2 + H2O (1)HCO3– + H+


there are sulphonamide‑ and sulphamate‑avid isoforms, such as CA II, VI, VII, IX, XII and XIII, which generally show low nanomolar affinity for most of these inhibitors. Other isozymes, however, such as CA I, IV, VA, VB and XIV show less propensity to be inhibited by these com‑pounds, with inhibition constants in the nanomolar to micromolar range. This leaves CA III as the only isoform that is not susceptible to inhibition by sulphonamides or sulphamates.

Few of the derivatives 1–16 (FIG. 1; TABLE 1) show selectivity for a specific isoform: the classical inhibi‑tors, such as compounds 1–5, and the topically acting antiglaucoma sulphonamides 6 and 7 together with indisulam 8, are promiscuous CAIs, with strong affini‑ties for isoforms II, VA, VB, VI, VII, IX, XII, XIII and XIV7–18. Topiramate (compound 9) is a subnanomolar CA VII inhibitor but it also effectively inhibits isoforms II, VB and XII7–18. Zonisamide (compound 10) shows a good affinity for CA IX but appreciably inhibits also CA II and VA, while having lower affinities for the other isoforms. Sulpiride (compound 11) is a potent CA VI and CA XII inhibitor and shows lower affinity for other isoforms. Valdecoxib (compound 15) is a strong CA XII inhibitor7–18, whereas saccharin (compound 16) is a CA VII‑specific inhibitor (KI of 10 nM against this isoform and much higher for the other CAs)19.

Progress in the design of CA‑selective and isozyme‑specific CAIs has recently been made. Owing to the extracellular location of some CA isozymes, such as CA IV, IX, XII and XIV (TABLE 2), it is possible to design membrane‑impermeant CAIs, which would therefore specifically inhibit membrane‑associated CAs without interacting with the cytosolic or mitochondrial isoforms. This possibility has been explored through the design of positively charged sulphonamides that generally incorporate pyridinium moieties, of which compound 18 is a representative20–22. The inhibitors obtained in this way showed nanomolar affinities for CA II as well as CA IV and CA IX, and, more importantly, they were unable to cross the plasma membranes in vivo20–22. This new class of potent, positively charged CAIs, was able to discriminate between the membrane‑bound and the cytosolic isozymes, selectively inhibiting only CA IV, in two model systems20–22. Another approach for the design of isoform‑selective CAIs exploited the presence of an Ala65 amino‑acid residue, which is present only in the ubiquitous CA II mammalian isoform23. Compared with topiramate, its sulphamide analogue is a 210‑times less potent inhibitor of isozyme CA II, but effectively inhibits isozymes CA VA, VB, VII, XIII and XIV (KIs in the range of 21–35 nM). The weak binding of the sulphamide analogue of topiramate to CA II was shown to be due to a clash between one methyl group of the inhibitor with the Ala65 amino‑acid residue, which might therefore be exploited for the design of compounds with lower affinity for this isoform23.

A further approach for selectively inhibiting the tumour‑associated isoforms CA IX (and XII) present in hypoxic tumour tissues envisaged bioreductive prodrugs that are activated by hypoxia24,25. The chosen strategy was to use the disulphide bond as a bioreducible

Box 1 | Mechanism of action of carbonic anhydrases

Carbonic anhydrases (CAs) catalyse the following reaction:

The metal ion (which is a Zn2+ ion in all α‑CAs investigated up to now) is essential for catalysis1–8. X‑ray crystallographic data show that the ion is situated at the bottom of a 15 Å deep active‑site cleft (shown as a pink sphere in the figure below; left panel), and is coordinated by three histidine residues (His94, His96 and His119; shown in green in the figure) and a water molecule/hydroxide ion1–8. The histidine cluster involved in the proton‑shuttling processes between the active site and the environment, comprising residues His64, His4, His3, His17, His15 and His10, is also evidenced. Amino‑acid residues 92 and 131 involved in the binding of many sulphonamide/sulphamate inhibitors are shown in yellow2,3.

The zinc‑bound water is also engaged in hydrogen‑bond interactions with the hydroxyl moiety of Thr199, which in turn is bridged to the carboxylate moiety of Glu106; these interactions enhance the nucleophilicity of the zinc‑bound water molecule, and orient the substrate (CO2) in a favourable location for nucleophilic attack1–8 (figure, right panel). The active form of the enzyme is the basic one, with hydroxide bound to Zn2+ (a). This strong nucleophile attacks the CO2 molecule that is bound in a hydrophobic pocket in its neighbourhood (the elusive substrate‑binding site comprises residues Val121, Val143 and Leu198 in the case of the human isozyme CA II) (b), leading to the formation of bicarbonate coordinated to Zn2+ (c). The bicarbonate ion is then displaced by a water molecule and liberated into solution, leading to the acid form of the enzyme, with water coordinated to Zn2+ (d), which is catalytically inactive1–8.

To regenerate the basic form (a), a proton transfer reaction from the active site to the environment takes place, which may be assisted either by active‑site residues (such as His64, the proton shuttle in isozymes I, II, IV, VI, VII, IX and XII–XIV among others) or by buffers present in the medium. The process may be schematically represented by reactions 2 and 3:

The rate‑limiting step in catalysis is reaction 3, that is, the proton transfer that regenerates the zinc‑hydroxide species of the enzyme1–8. In the catalytically very active isozymes, such as CA II, IV, VI, VII, IX, XII, XIII and XIV, the process is assisted by a histidine residue placed at the entrance of the active site (His64), or by a cluster of histidines (figure, left panel), which protrudes from the rim of the active site to the surface of the enzyme, thus assuring efficient proton‑transfer pathways1–8. This may explain why CA II is one of the most active enzymes known (with a kcat/Km of 1.5 x 108 M–1 s–1), approaching the limit of diffusion‑controlled processes1–8.

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2 31 4

8 11

5 6 7

10

12 13 1514

16 17 18

S

N

CH3CON SO2NH2

H C3

N

S

N

SO2NH2EtOS

NN

CH3CONH SO2NH2

SO2NH2

S

OO

HN

NH

Cl

9

O

O

OO

O

O

SNH2

O

O

N

SO2NH2

OMe

NH

O

SO2NH2

Cl

SO2NH2Cl S S

NHEt

SO2NH2

O OMe

NS S

NHEt

SO2NH2

O OMeO(CH2)3

SO2NH2

ON

OO

SO2NH2

O O

SO2NH2

O

NN

S

O

NH2

CH3

O

F

FF

SNH

O

O O

N SO2NH2S

O O

19

abcde

R2 = R3 = H, R6 = Cl, HydrochlorothiazideR2 = R3 = H, R6=CF3 , HydroflumethiazideR2=H, R3 = PhCH2, R

6 = CF3, BendroflumethiazideR2 = H, R3 = CHCl2, R

6 = Cl, TrichloromethiazideR2 = Me, R3 = CH2SCH2CF3, R

6 = Cl, Polythiazide

20 21

22 23 24 25

HN

NS SO2NH2

R6

R2

R3

O O

HN

HNSO2NH2

ClEt

O

HN

NSO2NH2

ClMe

O

Me

SO2NH2

Cl

HN

OHO SO2NH2

Cl

HN

O

N

Me

SO2NH2

ClHN

O

HOOC SO2NH2

O

HOOC

NH

OHO O

HN

COOH

NH

S SO2NH2

N+

SO2NH2

ClO4-

NO

S

O

NH2O

H3C

Figure 1 | structures of carbonic anhydrase inhibitors 1–25.

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Zn 2 +

HN S

R

O

O

His119His96His94

-

Zn 2 +

OH N

His119His96His94

-

CS

Tetrahedral adduct (sulphonamide)

Trigonal-bipyramidal adduct (thiocyanate)

a

b

E–Zn2+–OH2 + I (substitution reaction)

E–Zn2+–I + H2O

E–Zn2+–OH2 + I (addition reaction)

E–Zn2+–OH2(I)

High-ceiling (loop) diureticDiuretics are drugs that increase the rate of urine excretion. A high-ceiling (loop) diuretic inhibits the kidney’s ability to reabsorb sodium in the ascending loop. This leads to an increased loss of sodium and water in the urine, as water normally follows sodium back into the extracellular fluid.

function. The reducing conditions present in hypoxic tumours, in combination with the presence of the redox protein thioredoxin 1, mediates the reduction of the disulphide bond with the formation of thiols24,25. The reduced compounds (thiols) are less bulky and show excellent CA inhibitory activity (in the low nanomolar range) compared with the corresponding sterically hin‑dered disulphides, which have difficulty entering the limited space of the enzyme active site24,25.

CAIs as diureticsCAs are highly abundant in the kidney, and the isoforms present in this organ play a crucial function in at least three physiological processes: the acid–base homeostasis balance (by secreting and excreting protons, due to the CO2 hydration reaction catalysed by these enzymes); the bicarbonate reabsorption process; and the nH4

+ out‑put42,43. Acetazolamide (compound 1) was the first non‑mercurial diuretic to be used clinically in 1956 (REF. 2). It represents the prototype of a class of pharmacological agents with relatively limited therapeutic use, but which played a major role in the development of fundamental renal physiology and pharmacology, and in the design of many of the current widely used diuretic agents, such as the thiazide and the high-ceiling (loop) diuretic. Following the administration of a CAI, such as acetazolamide, the urine volume increases and becomes alkaline2,42. Increased bicarbonate is eliminated into the urine, together with na+ and K+ as accompanying cations, whereas the amount of chloride excreted is diminished. This sequence of events is due to the inhibition of CA in the proximal tubule, which leads to the inhibition of H+ secretion by this segment of the nephron. Inhibition of cytosolic (CA II) and membrane‑bound (CA IV, XII and CA XIV) enzymes seems to be involved in the diuretic effects of the sulphonamides2,43. The net effect of these processes is the transport of sodium bicarbonate from the tubular lumen to the interstitial space, followed by movement of the isotonically obligated water, and augmented diuresis. Acetazolamide, methazolamide (compound 2), ethox‑zolamide (compound 3) and dichlorophenamide (com‑pound 5) are used to treat oedema due to congestive heart failure and for drug‑induced oedema42,43. Many of the other diuretics, such as the benzothiadiazines (compounds 19a–e; for example, chlorothiazide and hydrochlorothiazide); quinethazone (compound 20); metolazone (compound 21); chlorthalidone (compound 22); indapamide (compound 23); furosemide (compound 24); and bumetanide (compound 25) act as CA inhibi‑tors with varying efficiencies2,3,42 (FIG. 1). This is to be expected, as all of them have unsubstituted primary sulphonamide moieties acting as effective zinc-binding moieties19.

CAIs as drugs for eye disordersGlaucoma is a chronic, degenerative eye disease, char‑acterised by high intraocular pressure (IOP) that causes irreversible damage to the optic nerve head, resulting in the progressive loss of visual function and eventually blindness44–46. Studies on the chemistry and dynamics of aqueous humour have identified the main constituent

of this secretion to be sodium bicarbonate1,2,44–46. CAs were identified in the anterior uvea of the eye and were shown to be responsible for the bicarbonate secretion. CAIs represent the most physiological treatment of glaucoma, as by inhibiting the ciliary‑process enzyme — the sulphonamide susceptible isozyme CA II (TABLE 1) — the rate of bicarbonate and aqueous humour secretion is reduced, resulting in a 25–30% decrease in IOP44–51.

Indeed, systemic acetazolamide (compound 1), meth‑azolamide (compound 2), ethoxzolamide (compound 3) or dichlorophenamide (compound 4) are extensively used to treat this disease2,45. The best‑studied drug is acetazolamide, which is frequently administered long‑term owing to its efficient reduction of IOP, minimal toxicity and ideal pharmacokinetic properties. However, as CAs are ubiquitously expressed in vertebrates, the systemic administration of sulphonamides leads to nonspecific CA inhibition and is associated with unde‑sired side effects, including numbness and tingling of extremities; metallic taste; depression; fatigue; malaise; weight loss; decreased libido; gastrointestinal irritation; metabolic acidosis; renal calculi; and transient myo‑pia. The development of water‑soluble sulphonamide CAIs to be used as eye drops began in the 1990s, and by 1995 the first such pharmacological agent, dorzola‑mide (compound 6), was launched45 by Merck under the tradename Trusopt. A second structurally similar compound, brinzolamide (compound 7), has also been approved (by Alcon under the tradename Azopt) for the topical treatment of glaucoma45.

Dorzolamide and brinzolamide are potent water‑soluble CAIs that are sufficiently liposoluble to penetrate the cornea, and may be administered topically as the hydro‑chloride salt (at a pH of 5.5) or as the free base, respec‑tively45. The two drugs are effective in reducing IOP and show fewer side effects as compared with systemically

Figure 2 | Mechanisms of inhibition of carbonic anhydrase. a | Unsubstituted sulphonamides and their bioisosteres bind to the Zn2+ ion of the enzyme by substituting the non-protein zinc ligand to generate a tetrahedral adduct. b | Anionic inhibitors add to the metal coordination sphere, generating trigonal-bipyramidal species.

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Macular oedemaSwelling of the retina due to the leakage of fluid from blood vessels within the central macula region of the retina, causing blurred vision and loss of visual function.

applied drugs. The observed side effects include sting‑ing, burning or reddening of the eye, blurred vision and pruritus, which are probably due to the acidic pH of the dorzolamide eyedrops solution. Also, a bitter taste is experienced with both systemic as well as topical CAIs, which is probably due to drug‑laden lachrymal fluid draining into the oropharynx and inhibiting CAs present in the saliva (CA VI) and taste buds (CA II and CA VI) with the consequent accumulation of bicarbo‑nate. Therefore, novel, topically effective CAIs as possible antiglaucoma agents are being investigated46. One recent approach involved attaching water‑solubilizing moieties and ring‑system derivatives to aromatic and heterocyclic sulphonamides, which produced compounds that were two to three times more effective than dorzolamide in lowering IOP in rabbits46–51. These compounds were water‑soluble (as hydrochlorides, triflates or trifluoro‑acetates), potent human CA II inhibitors, penetrated the cornea and significantly lowered IOP in both normo‑tensive and glaucomatous rabbits46–51.

The use of CAIs in the treatment of macular oedema is based on the observation that systemically administered acetazolamide (sodium salt) is effective in the treatment of this condition52. Similar efficiency has also recently been reported for topical administration of dorzolamide and brinzolamide45. It is generally assumed that the dis‑appearance of oedema and improvement of visual func‑tion are independent of the hypotensive activity of the sulphonamide, due to direct effects on the circulation in the retina. Acetazolamide, dorzolamide or brinzolamide probably act as local vasodilators, improving blood flow in this organ and consequently clearing metabolic waste products and drusen. Vision following such treatment (in early phases of the disease) is markedly improved45,52.

Gao et al.53 recently reported that the slow cytosolic isozyme CA I mediates haemorrhagic retinal and cerebral vascular permeability through activation of prekallikrein and generation of the highly active serine protease factor XIIa. These phenomena contribute to the pathogenesis of proliferative diabetic retinopathy

Table 2 | Kinetic parameters for CO2 hydration reaction catalysed by the 16 vertebrate α-CA isozymes*

Isozyme Kcat (s–1) Km (mM) Kcat/Km (M–1 s–1) KI (nM) subcellular

localizationTissue/organ localization

refs

hCA I 2.0 × 105 4.0 5.0 × 107 250 Cytosol Erythrocytes, GI tract 1–3

hCA II 1.4 × 106 9.3 1.5 × 108 12 Cytosol Erythrocytes, eye, GI tract, bone osteoclasts, kidney, lung, testis, brain

1–3

hCA III 1.0 × 104 33.3 3.0 × 105 2.1 × 105 Cytosol Skeletal muscle, adipocytes

7,8

hCA IV 1.1 × 106 21.5 5.1 × 107 74 Membrane-bound

Kidney, lung, pancreas, brain capillaries, colon, heart muscle

6

hCA VA 2.9 × 105 10.0 2.9 × 107 63 Mitochondria Liver 9

hCA VB 9.5 × 105 9.7 9.8 × 107 54 Mitochondria Heart and skeletal muscle, pancreas, kidney, spinal cord, GI tract

10

hCA VI 3.4 × 105 6.9 4.9 × 107 11 Secreted (saliva, milk)

Salivary and mammary glands

11

hCA VII 9.5 × 105 11.4 8.3 × 107 2.5 Cytosol CNS 12

hCA VIII ND ND ND ND Cytosol CNS 13

hCA IX 3.8 × 105 6.9 5.5 × 107 25 Transmembrane Tumours, GI mucosa 14,18

hCA X ND ND ND ND Cytosol CNS 13

hCA XI ND ND ND ND Cytosol CNS 13

hCA XII 4.2 × 105 12.0 3.5 × 107 5.7 Transmembrane Renal, intestinal, reproductive epi- thelia, eye, tumours

15

hCA XIII 1.5 × 105 13.8 1.1 × 107 16 Cytosol Kidney, brain, lung, gut, reproductive tract

16

hCA XIV 3.1 × 105 7.9 3.9 × 107 41 Transmembrane Kidney, brain, liver 17

mCA XV 4.7 × 105 14.2 3.3 × 107 72 Membrane-bound

Kidney Unpublished observations

*At 20°C and pH 7.5, their inhibition data with acetazolamide 1 (5-acetamido-1,3,4-thiadiazole-2-sulphonamide), and their subcellular localization. α-CA, α-carbonic anhydrase; h, human; GI, gastrointestinal; m, mouse; ND, not determined.

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[Pyruvate]m

[Pyruvate]c

Acetyl-CoA

CoA

[Citrate]m [Citrate]c

Krebs cycle

– CO2

Acetyl-CoA

Malonyl-CoA

HCO3– ACC

Fatty acids

– CO2

– CO2

Oxaloacetate Oxaloacetate

HCO3–

PC

Mitochondrion Cytosol

Pyruvate transporter

Tricarboxylate transporter

and diabetic macular oedema, which represent leading causes of vision loss, and for which there are no phar‑macological treatments currently available. Therefore, as suggested by the authors, CA I inhibition might be a therapeutic target for the treatment of these conditions53. In fact, potent CA I inhibitors are currently available54.

Some of the membrane‑associated isoforms, such as CA IV, IX and XII, have also been considered as possible targets of the antiglaucoma sulphonamides55–57. The bovine CA IV isozyme was shown to be susceptible to inhibition by most sulphonamides and sulphamates, with KIs in the low nanomolar range55. However, the corresponding human isoform, CA IV, shows different behaviour, with some drugs, such as acetazolamide, ethoxzolamide and sulthiame, acting as effective inhibitors, whereas other antiglaucoma agents, such as methazolamide, dichlorophenamide, dorzolamide and brinzolamide, act as weak inhibitors56 (TABLE 1), which suggests that it is improbable that CA IV plays a role in aqueous humor secretion, as it is so weakly inhibited by most antiglaucoma sulphonamides. However, CA XII (but not CA IX) was recently shown to be highly

overexpressed in the eyes of patients with glaucoma, and probably plays an important role in the elevated IOP characteristic of the disease57. we have subsequently shown that CA XII is highly inhibited by all the antiglau‑coma sulphonamides used clinically15 (TABLE 1), and this is probably the membrane‑bound isoform involved in glaucoma that is targeted by these agents. Further work is warranted to better understand the involvement of various CA isoforms in eye pathologies such as glaucoma, retinopathy and macular degeneration.

CAIs as potential anti-obesity drugsAmong the α‑CA isoforms found in animals, two CA isozymes, VA and VB, are present in mitochondria (TABLE 2). These isozymes are involved in several biosyn‑thetic processes, such as ureagenesis, gluconeogenesis and lipogenesis, in vertebrates (for example, rodents) and in invertebrates (for example, the locust)58. The pro‑vision of enough of the substrate, bicarbonate, in several biosynthetic processes involving pyruvate carboxylase (PC), acetyl‑CoA carboxylase (ACC) and carbamoyl phosphate synthetases I and II, is assured mainly by the catalytic reaction involving the mitochondrial isozymes CA VA and VB — probably assisted by the high activity cytosolic isozyme CA II58 (FIG. 3).

Several studies have provided evidence that CAIs have potential as anti‑obesity drugs, which might be due to their effects on CA isozymes. Topiramate (compound 9) is an anti‑epileptic drug possessing potent anticonvulsant effects due to a multifactorial mechanism of action: block‑ade of sodium channels and kainate/AMPA (α‑amino‑3‑hydroxy‑5‑methyl‑4‑isoxazole propionic acid) receptors, CO2 retention secondary to inhibition of the red blood cell and brain CA isozymes, as well as enhancement of GABA (γ‑aminobutyric acid)‑ergic transmission59. A side effect of this drug observed in obese patients was the loss of body weight, although no pharmacological explanation of this phenomenon has been provided60. Furthermore, topiramate was shown to reduce energy and fat gain in lean (Fa/?) and obese (fa/fa) Zucker rats60. It was recently demonstrated that topiramate is also a potent inhibitor of several CA isozymes, such as II, VA, VB, VI, VII, XII and XIII (TABLE 1), and the X‑ray crystal structure of its complex with human CA II has been determined, revealing the molecular interactions that explain the high affinity of this compound for the CA active site59. As topiramate also acts as an efficient inhib‑itor of the human mitochondrial isozymes CA VA and VB, this inhibition of both mitochondrial and cytosolic CA isozymes involved in lipogenesis may provide a new approach to control weight loss58–60.

Zonisamide (compound 10) is another anti‑epileptic drug used as adjunctive therapy for refractory partial seizures1,61,62. It has multiple mechanisms of action, and exhibits a broad spectrum of anticonvulsant activity. Similar to topiramate, recent clinical studies have dem‑onstrated additional potential for therapeutic use for neuropathic pain, bipolar disorder, migraine, obesity, eating disorders and Parkinson’s disease1. Zonisamide is an aliphatic sulphonamide, which also potently inhibits cytosolic and mitochondrial CAs involved in lipogenesis61

Figure 3 | Fatty-acid biosynthesis and the role of carbonic anhydrase isozymes. Mitochondrial pyruvate carboxylase (PC) is needed for the efflux of acetyl groups from the mitochondria to the cytosol where fatty-acid biosynthesis takes place1,58. Pyruvate is carboxylated to oxaloacetate in the presence of bicarbonate under the catalytic influence of the mitochondrial isozymes (carbonic anhydrase (CA) VA and/or CA VB). The mitochondrial membrane is impermeable to acetyl-CoA, which reacts with oxaloacetate, leading to the formation of citrate, which is then translocated to the cytoplasm by means of the tricarboxylic acid transporter. As oxaloacetate is unable to cross the mitochondrial membrane, its decarboxylation regenerates pyruvate, which can then be transported into the mitochondria by means of the pyruvate transporter. The acetyl-CoA thus generated in the cytosol is in fact used for de novo lipogenesis, by carboxylation in the presence of acetyl-CoA carboxylase (ACC) and bicarbonate, with formation of malonyl-CoA, the conversion between CO2 and bicarbonate being assisted by CA II. Subsequent steps involving the sequential transfer of acetyl groups lead to longer-chain fatty acids. Therefore, CA isozymes are critical to the entire process of fatty-acid biosynthesis: VA and/or VB within the mitochondria (to provide enough substrate to PC), and CA II within the cytosol (for providing sufficient substrate to ACC).

R E V I E W S



OH VHL

VHL

HIFα HIFβ

HIFα

HIFα

HIFα

HIFα

HIFα

HIFβ HIFβ

HRE GLUT1/3VEGFEPO1CA IX

(anaerobic glycolysis)(angiogenesis)(erythropoesis)(pH regulation)

Active transcription factor

Constitutive subunit

Stabilization


HIFα

HIFα

O2 PHD Hydroxylation

Interaction with VHL

OH HIFα Ubiquitylation

Degradation

Ubiquitin

Hypoxia Normoxia

Cytoplasm

Nucleus

(TABLE 1). Furthermore, zonisamide in conjunction with a reduced‑calorie diet (deficit of 500 kcal per day), resulted in an additional mean 5 kg (11 pound) weight loss compared with diet alone in obese female patients62. Thus, inhibition of mitochondrial isoforms CA VA and VB, probably in conjunction with that of the ubiquitous cytosolic isoform CA II, may represent targets for novel anti‑obesity drugs that reduce lipogenesis by inhibiting CA58.

Anticancer CAIsA key feature of many tumours is hypoxia63–68. The inadequate supply of oxygen is primarily a pathophysio‑logical consequence of structurally and functionally disturbed microcirculation and deteriorated oxygen diffusion processes5,63–68. Tumour hypoxia appears to

be strongly associated with tumour propagation, malig‑nant progression, and resistance to chemotherapy and radiotherapy5,63–68. Hypoxia regulates the expression of several genes, including a CA isozyme, CA IX, through the hypoxia inducible factor 1 (HIF1) cascade5,63–71. The expression of CA IX is strongly upregulated by hypoxia and is downregulated by the wild‑type von Hippel‑lindau tumour suppressor protein (pVHl) (FIG. 4).

CA IX expression is strongly increased in many types of tumours, such as gliomas/ependymomas64, mesothelio‑mas64, papillary/follicular carcinomas64, carcinomas of the bladder72, uterine cervix73,74, nasopharyngeal carcinoma75, head and neck76, breast70,77,78, oesophagus64, lungs79, brain64, vulva64, squamous/basal cell carcinomas64, and kidney80 tumours, among others. In some cancer cells, the VHL gene is mutated leading to the strong upregulation of CA IX (up to 150‑fold) as a consequence of constitutive HIF activation5,64,77.

CA IX belongs to the highly active human α‑CAs, its catalytic properties for the CO2 hydration reaction being comparable with those of the highly evolved catalyst CA II5,64. As for all α‑CAs, CA IX is suscep‑tible to inhibition by anions and sulphonamides and sulphamates1,2,5,14,81–89, with the inhibitors coordinating directly to the zinc ion within the active‑site cavity and participating in various other favourable interactions with amino‑acid residues that are situated in the hydro‑phobic and hydrophilic halves of the active site. Many low nanomolar CA IX inhibitors have been identified in the past several years1,2,5,14 (TABLE 1). Among them, some sulphamates81,88,89 and sulphonamides82–86 were characterized by X‑ray crystallography and homology modelling. Such studies also evidenced compounds that are membrane impermeable (and thus specifically inhibit CA IX in vivo)90–93 or act as dual CA IX–COX2 (cyclooxygenase 2) inhibitors82,93. Both heterocyclic94,95, aromatic sulphonamides96,97 as well as aliphatic98 sulphon‑amides/sulphamates/sulphamides99–101 possessing low nanomolar inhibitory activity against CA IX have been detected so far. Some sulphonamides incorporating various sugar moieties were also reported102,103, but up until now the most useful CAIs for understanding the function of this protein in vivo were the fluorescent com‑pounds of type 17 (see later in the text)84,94,104.

As described previously, hypoxia, through the HIF cascade, leads to a strong overexpression of CA IX in many tumours. The overall consequence of this is a pH imbalance, with most hypoxic tumours having acidic pH values around 6, in contrast to normal tissue, which has characteristic pH values around 7.4 (REFS 84,85,104). Constitutive expression of human CA IX was recently shown to decrease extracellular pH (pHe) in Madin‑Darby canine kidney (MDCK) epithelial cells84. CA IX‑selective sulphonamide inhibitors (of type 17 and 18) reduced the medium acidity by inhibiting the catalytic activity of the enzyme, and thus the generation of H+ ions, bind‑ing specifically only to hypoxic cells expressing CA IX84. Deletion of the CA active site was also shown to reduce the acidity of the medium, but a sulphonamide inhibitor did not bind to the active site of such mutant proteins. Therefore, tumour cells decrease their pHe both by

Figure 4 | Mechanism of hypoxia-induced gene expression mediated by the HIF transcription factor. At normal oxygen levels (normoxia), prolyl-4-hydroxylase (PHD) hydroxylates the P564 on hypoxia inducible factor-α (HIFα). The von Hippel-Lindau protein (VHL) binds hydroxylated HIFα and targets it for degradation by the ubiquitin–proteasome system. Under hypoxia, HIFα is not hydroxylated, because PHD is inactive in the absence of dioxygen. Non-hydroxylated HIFα is not recognized by the VHL protein, it is stabilized and accumulates. After translocation to the nucleus, HIFα dimerizes with the HIFβ constitutive subunit to form an active transcription factor. The HIF transcription factor then binds the hypoxia response element (HRE) in target genes and activates their transcription. Target genes include glucose transporters (GLUT1 and GLUT3) that participate in glucose metabolism, vascular endothelial growth factor (VEGF) that triggers neoangiogenesis, erythropoietin (EPO1) involved in erythropoiesis, carbonic anhydrase (CA) IX involved in pH regulation and tumorigenesis, and additional genes with functions in cell survival, proliferation, metabolism and other processes64–69.

R E V I E W S




Glucose

Lactate

Glycolysis

H+

H+

Cl–

H+

Na+

Na+

K+

H+

a

d

c

b

e f g

pH ~7.2

pH ~6.8

pH ~7.4Endothelial cells

CO2 + H2O ↔ H+ + HCO3–

CA IX CA XII

CO2 + H2O ↔ H+ + HCO3–

CA II

Cancer cell

production of lactic acid (due to the high glycolysis rates), and by CO2 hydration catalysed by the tumour‑associated CA IX, possessing an extracellular catalytic domain5,84,104

(FIG. 5). low pHe has been associated with tumorigenic transformation, chromosomal rearrangements, extracell‑ular matrix breakdown, migration and invasion, induc‑tion of the expression of cell growth factors and protease activation5,64,104. CA IX probably also plays a role in providing bicarbonate to be used as a substrate for cell growth, as it is established that bicarbonate is required in the synthesis of pyrimidine nucleotides5,64.

Indisulam (compound 10), a sulphonamide deriva‑tive (originally called E7070) with powerful anticancer activity, was recently shown to act as a nanomolar inhibi‑tor of CA IX105–108 (TABLE 1). Its detailed mechanism of action is not clear, but it is known to be involved in the perturbation of the cell cycle in the G1 and/or G2 phases, the downregulation of cyclins, the reduction of cyclin‑dependent kinase 2 (CDK2) activity, the inhibition of retinoblastoma protein (prb) phosphorylation and differential expression of molecules known to participate in cell adhesion, signalling and immune response, in addition to its CA IX inhibitory properties. Indisulam showed in vivo efficacy against human tumour xenografts in nude mice, exhibiting a significant antitumour effect and progressing to Phase I and II clinical trials for the treatment of solid tumours105–108.

Among the many derivatives reported so far, some of the most interesting potent CA IX inhibitors are the compounds investigated by Svastova et al.84 (structures 17 and 18). Derivative 17 is a fluorescent sulphonamide that binds only to CA IX under hypoxic conditions in vivo84,85,104. This compound may therefore be used as a fluorescent probe in hypoxic tumour imaging. Compound 18 belongs to a class of positively charged, membrane‑impermeable compounds. Therefore, as such compounds do not inhibit intracellular CAs, they may exhibit fewer side effects as compared with the presently available compounds (such as acetazolamide), which indiscriminately inhibit all CAs48–50. The X‑ray crystal structure of compound 18 in adduct with CA II (the active site of which is similar to that of CA IX)6 has been reported recently. The positively charged pyridinium derivative 18 favourably binds within the enzyme active site, coordinating with the deprotonated sulphonamide moiety to the catalytically critical Zn2+ ion. It also par‑ticipates in many other favourable interactions with amino‑acid residues present in the active‑site cavity, including stacking between the trimethylpyridinium ring of the inhibitor 18 and the phenyl ring of Phe131, an amino acid important for the binding of inhibitors to CAs86. A similar binding mechanism was subsequently reported for the fluorescein derivative 17 (REF. 18). Thus, such structures can be used for the rational drug design of more selective and potent isozyme IX inhibitors. As the X‑ray structure of CA IX is not yet available, most studies have used the CA II structure for modelling and designing CA IX inhibitors.

In summary, biochemical, physiological and pharmacological data indicate that inhibition of the tumour‑associated CA isozyme IX may be useful in the

management of hypoxic tumours that do not respond to classical chemotherapy or radiotherapy5,64,84,104. Therefore, the use of the CAIs described above provide possibilities of developing both diagnostic tools for the non‑invasive imaging of these tumours, as well as thera‑peutic agents that probably perturb the extratumoral acidification in which CA IX is involved5,64,84,104. Many types of highly effective in vitro CA IX inhibitors have been developed and evaluated in vitro80–104. However, although a large number of derivatives with enhanced affinity for the tumour‑associated isozyme IX over the ubiquitously expressed CA I and II isozymes have been discovered94, further studies are warranted in order to understand the behaviour of such compounds in vivo, in cell cultures or animal models of the disease.

CAIs as potential drugs for osteoporosisThe highly active CA II is abundant in the bone, and is present only in osteoclasts at concentrations of the same order of magnitude as those present in the kidneys109. Its role there is to provide hydrogen ions, formed from the hydration of CO2, to an ATP‑dependent proton pump, which uses them in the mobilization of calcium from the bone. These activities are required for inorganic matrix dissolution that precedes the enzymatic removal of organic bone matrix.

Figure 5 | Proteins and processes involved in pH regulation within the tumour cell. CO2 hydration to bicarbonate and protons is catalysed in these cells by the transmembrane isozymes possessing an extracellular active site, carbonic anhydrase (CA) IX and/or CA XII. Other involved proteins include monocarboxylate carrier (a); Na+– H+ antiporter (b); ATP-dependent Na+–K+ antiporter (c); H+ channels (d); plasma-membrane proton pump H+–ATPase (e); acquaporins (f); and anion exchangers (AE1-AE3 isoforms) (g)5,64–69.

R E V I E W S



Transport metabolonsCarbonic anhydrase isozymes that interact with anion exchanger proteins to form transport metabolons that regulate intracellular and/or extracellular pH.

To evaluate the physiological role of membrane‑bound CAs in osteoclasts, a novel membrane‑impermeable CA inhibitor structurally related to compound 18 was used. Increased osteoclast number and bone resorption activity was observed in rat osteoclast cultures exposed to a low concentration of inhibitor, whereas higher con‑centrations affected cell survival109. Inhibitor treatment also disturbed intracellular acidification in osteoclasts. Membrane‑bound isoenzymes CA IV and CA XIV are expressed in osteoclasts in vivo and in vitro. In addition, the inhibitor experiments provide novel evidence to sup‑port the hypothesis that intracellular pH regulation in osteoclasts may involve transport metabolons and that a possible use of such inhibitors would be in the design of novel anti‑osteoporosis therapies109.

Non-vertebrate CAs and their inhibitionSequencing of eukaryotic/prokaryotic genomes, in par‑ticular of pathogens causing widespread diseases (for example, malaria, tuberculosis, as well as other bacterial and fungal infections), has revealed that CAs are also present in these organisms1–4. However, although verte‑brates possess only CAs belonging to the α‑class9–16, such ‘simpler’ organisms have enzymes belonging to several CA families, for example the α‑ and β‑CAs, β‑ and γ‑CAs, γ‑ and δ‑CAs, or even representatives from three such gene families (α–γ‑CAs)27–34,110–112.

recently, representatives of the α‑ or β‑CA class have been cloned and characterized in several pathogens, such as the protozoan P. falciparum26, the bacteria H. pylori27,28 and M. tuberculosis29, and the fungi C. albicans30 and C. neoformans31,32. As it has been proved that these CAs are critical for the growth or virulence of these patho‑gens, their capacity to be inhibited has also been investi‑gated (TABLE 3). As many such organisms are highly pathogenic, and present different degrees of resistance to the currently available drugs targeting them, inhibition of their CAs may constitute novel approaches to fighting these diseases26–34,110–112.

P. falciparum is responsible for the majority of life‑threatening cases of human malaria. The global emer‑gence of drug‑resistant malarial parasites necessitates the identification and characterization of novel drug targets. The CAs present in P. falciparum belongs to the α‑CA class (pfCA) and possess catalytic properties that

are distinct to that of the human host enzymes CA I and II. 4‑(3,4‑Dichlorophenylureido‑ethyl)‑benzene‑sulphonamide was the most effective antimalarial sulphonamide CAI against growth of P. falciparum in vitro, with a KI in the nanomolar range (80 nM) and EC50 (ex vivo) in the low micromolar range (20 µM). Thus, sulphonamide CAIs targeting the protozoan enzyme may have the potential for the development of novel antimalarial drugs26.

H. pylori α‑class CA (hpαCA) has been cloned and sequenced from patients with different gastric mucosal lesions, including gastritis, ulcer and cancer27,28. Some potent hpαCA inhibitors were detected (KIs of 12–84 nM), among which were acetazolamide, 4‑amino‑6‑chloro‑ 1,3‑benzenedisulphonamide and some newly designed compounds incorporating lipophilic tails. As hpαCA is essential for the survival of the pathogen in acid, its inhibition might be used as a new pharmaco‑logical tool in the management of drug‑resistant H. pylori infection27. DnA clones for the β‑class CA of H. pylori (hpβCA) were isolated from independent strains obtained from patients with various gastric mucosal lesions, including patients with gastritis, gas‑tric ulcer and gastric cancer28. hpβCA was also highly inhibited (KIs in the range of 24–45 nM) by many sul‑phonamides/sulphamates, including the clinically used drugs acetazolamide, ethoxzolamide, topiramate and sulpiride. The dual inhibition of α‑ and/or β‑class CAs of H. pylori could therefore be a useful alternative for the management of gastritis/gastric ulcers, as well as gastric cancer. This was also the first study showing that a bacterial β‑CA can be a druggable target28. The crucial role played by these two CAs present in H. pylori in acid acclimatization of the pathogen within the stomach is shown schematically in FIG. 6, and this also helps to understand why inhibition of the two enzymes leads to the death of the bacteria and a possible eradication of H. pylori from the stomach27,28.

The M. tuberculosis rv3588c gene, which has been shown to be required for in vivo growth of the pathogen, was discovered to encode a β‑CA with activity that is highly dependent on pH, being active at pH 8.4 but not at pH 7.5 (REF. 29). This β‑CA was observed to be a dimeric protein with a blocked active site, being able to switch between two states with an opened or closed active site.

Table 3 | Catalytic activity of non-vertebrate CAs belonging to the α-, β- and γ-classes*

enzyme Class Activity level Kcat (s–1) Km (mM) Kcat/Km (M–1 s–1) KI (nM) subcellular

localizationrefs

pfCA‡ α Very low 0.173 3.7 46.75 99 Cytoplasm 26

hpαCA§ α Low 2.5 × 105 16.6 1.5 × 107 21 Periplasm 27

hpβCA|| β Medium 7.1 × 105 14.7 4.8 × 107 40 Cytoplasm 28

cab¶ β Low 3.1 × 104 1.7 1.8 × 106 12,100 Unknown 110

cam# γ Low 7.1 × 104 1.8 3.9 × 106 63 Unknown 111

*Their inhibition by acetazolamide; most carbonic anhydrases (CAs) show low activity as esterases1,2. ‡Plasmodium falciparum CA enzyme; esterase activity with 4-nitrophenyl acetate as substrate at 25°C. §Helicobacter pylori α-class CA enzyme; at pH 8.9 and 25°C (CO2 hydration reaction). ||Helicobacter pylori β-class CA enzyme; at pH 8.3 and 20°C (CO2 hydration reaction). ¶β-CA from the archaeon Methanobacterium thermoautotrophicum; at pH 7.5 and 20°C (CO2 hydration reaction). #γ-CA from the archaeon Methanosarcina thermophila; at pH 7.1 and 25°C (CO2 hydration reaction).

R E V I E W S




NH4+ ← NH3 + H+ ← H+ + HCO3

–

NH4+ ← NH3 + H+ ← H+ + HCO3

–

NH3

2NH3 + CO2 Urea

Urea

H+ Urea

CO2

H2O

H2O

α-CA

β-CA

Urease

Porin

Urea channel

Gastric lumen

Periplasm

Cytoplasm

Although no inhibition studies were performed with this β‑CA, it is probable that potent inhibitors may be detected and designed. This could lead to a completely new class of antituberculosis drugs, a disease for which resistance to the presently available drugs is a worldwide problem29.

The nCE103 gene of the yeast Saccharomyces cerevisiae also encodes a CA34. The main physiological role of CA during growth of S. cerevisiae in glucose‑ammonium salts media is the provision of inorganic carbon for the bicarbonate‑dependent carboxylation reactions catalysed by PC, ACC and CPSase (carbamoyl‑phosphate syn‑thetase)30–32. However, the most interesting findings in this field regard the signalling role of CAs that is important for the virulence of fungal pathogens such as C. albicans and C. neoformans, as identified by Muhlschlegel’s and Heitman’s groups30–32. It has been demonstrated that physiological concentrations of CO2/HCO3

– induce fila‑mentation in C. albicans by a direct stimulation of the adenylyl cyclase activity, as shown schematically in FIG. 7. Furthermore, it has been shown that CO2/HCO3

– equili‑bration by the β‑CAs present in the organism is essential for the pathogenesis of C. albicans in niches where the available CO2 is limited. Muhlschlegel’s group also dem‑onstrated that adenylyl cyclase from C. neoformans is sensitive to physiological concentrations of CO2/HCO3

–. Thus, the link between cyclic AMP signalling and CO2/HCO3

– sensing is conserved in fungi, with CO2 sensing being an important mediator of fungal pathogenesis. Although no inhibitors of these new β‑CAs have yet been tested, novel therapeutic agents targeting this pathway at several levels could act as new antifungals30–32.

CA activators in drug designAlthough CAIs have been extensively studied, and exploited clinically for the prevention and treatment of several diseases, the field of CA activators (CAAs) is largely unexplored. However, in the past decade, by means of electronic spectroscopy, X‑ray crystallography and kinetic measurements, activators have been shown to bind within the CA active cavity, at a site distinct to that of the inhibitor or substrate‑binding sites, to facilitate the proton‑transfer step of the catalytic cycle. recently, activation of some members of the α‑CA family (human CA I and CA II) was shown to constitute a possible therapeutic approach for the enhancement of synaptic efficacy, which may represent a conceptually new treatment for Alzheimer’s disease, ageing and other conditions in which the achievement of spatial learn‑ing and memory therapy is necessary40,113. Sun and Alkon reported that phenylalanine, an activator first investigated by our group to target isozymes I and II114, when administered to experimental animals produces a relevant pharmacological enhancement of synaptic efficacy, spatial learning and memory, proving that this class of unexplored enzyme modulators may be used for the management of conditions in which learning and memory are impaired40. A multitude of physiologically relevant compounds such as biogenic amines (histamine, serotonin, catecholamines), amino acids, oligopeptides or small proteins, among others, act as efficient CAAs for many of the human CA isozymes35–41,115–121.

Several X‑ray crystallographic structures of adducts of the main human isoforms, CA I and II, with activators were reported recently, in addition to that of histamine, reported in 1997 (REFS 35–41). The X‑ray crystal struc‑tures of the human CA II–l‑His/d‑His adducts and the CA II–l‑Phe/d‑Phe adducts showed the activators to be anchored at the entrance of the active site, participating in extended networks of hydrogen bonds and hydro‑phobic interactions with specific amino‑acid residues or water molecules present in the cavity, thus explaining their different potency and interaction patterns with vari‑ous isozymes35–39,41. Many drug design studies of CAAs have also been reported, and histamine or carnosine as lead molecules has been considered114–121.

CAAs may lead to the design of pharmacologically useful derivatives for the enhancement of synaptic effi‑cacy, which may represent a conceptually new approach for the treatment of Alzheimer’s disease, ageing and other conditions in which spatial learning and memory therapy need to be enhanced.

ConclusionsIn the field of CA research, the past few years have been highly dynamic and productive. The presumed last mam‑malian α‑CA isoform (CA XV) has now been isolated, characterized and purified; along with representatives of the α‑, β‑, γ‑ and δ‑classes from various pathogenic organisms from across the phylogenetic tree. Many of these new CAs are druggable targets, and therefore pro‑vide potential for the development of pharmacological

Figure 6 | A model for the role of urease and α- and β-CA in the maintenance of periplasmic pH in Helicobacter pylori. Under acidic conditions, urea moves into the cytoplasm through the urea channel. In the cytoplasm, 2NH3 and CO2 are produced from urea owing to the activity of urease. CO2 in the periplasm and cytoplasm is hydrated by H. pylori α- and β-carbonic anhydrases (CAs), respectively, resulting in production of H+ and bicarbonate. The proton is then consumed by NH3 to form NH4

+ in the periplasm and cytoplasm, respectively27,28.

R E V I E W S




Cytoplasm

CO2

CO2

ATP cAMP

Regulationof capsulebiosynthesis

HCO3– + H+

H2O

Can2

Cac1

Capsule

1. Scozzafava, A., Mastrolorenzo, A. & Supuran, C. T. Carbonic anhydrase inhibitors and activators and their use in therapy. Expert Opin. Ther. Pat. 16, 1627–1664 (2006).

2. Supuran, C. T., Scozzafava, A. & Conway, j. Carbonic Anhydrase — Its inhibitors and Activators 1–363 (CRC, Boca Raton, 2004).

3. Supuran, C. T., Scozzafava, A. & Casini, A. Carbonic anhydrase inhibitors. Med. Res. Rev. 23, 146–189 (2003).A comprehensive review on the development of CAIs.

4. Smith, K. S. & Ferry, j. G. Prokaryotic carbonic anhydrases. FEMS Microbiol. Rev. 24, 335–366 (2000).

5. Thiry, A. et al. Targeting tumor-associated carbonic anhydrase IX in cancer therapy. Trends Pharmacol. Sci. 27, 566–573 (2006).An up-to-date review regarding the targeting of the tumour-associated CA IX by small-molecule inhibitors.

6. Stams, T. & Christianson, D. W. in The Carbonic Anhydrases — New Horizons (eds Chegwidden, W. R., Carter, N. D. & Edwards, Y. H.) 159–174 (Birkhauser, Basel, 2000).

7. Pastorekova, S. et al. Carbonic anhydrases: current state of the art, therapeutic applications and future prospects. J. Enzyme Inhib. Med. Chem. 19, 199–229 (2004).

8. Nishimori, I. et al. Carbonic anhydrase inhibitors. Cloning, characterization and inhibition studies of the cytosolic isozyme III with sulfonamides. Bioorg. Med. Chem. 15, 7229–7236 (2007).

9. Vullo, D. et al. Carbonic anhydrase inhibitors. Inhibition of mitochondrial isozyme V with aromatic and heterocyclic sulfonamides. J. Med. Chem. 47, 1272–1279 (2004).

10. Nishimori, I. et al. Carbonic anhydrase inhibitors. The mitochondrial isozyme VB as a new target for sulfonamide and sulfamate inhibitors. J. Med. Chem. 48, 7860–7866 (2005).

11. Nishimori, I. et al. Carbonic anhydrase inhibitors. DNA cloning, characterization and inhibition studies of the human secretory isoform VI, a new target for sulfonamide and sulfamate inhibitors. J. Med. Chem. 50, 381–388 (2007).

12. Vullo, D. et al. Carbonic anhydrase inhibitors. Inhibition of the human cytosolic isozyme VII with aromatic and heterocyclic sulfonamides. Bioorg. Med. Chem. Lett. 15, 971–976 (2005).

13. Nishimori, I. in Carbonic Anhydrase — Its Inhibitors and Activators (eds Supuran, C. T., Scozzafava, A. & Conway j.) 25–43 (CRC, Boca Raton, 2004).

14. Vullo, D., et al. Carbonic anhydrase inhibitors. Inhibition of the tumor-associated isozyme IX with aromatic and heterocyclic sulfonamides. Bioorg. Med. Chem. Lett. 13, 1005–1009 (2003).First CA IX inhibition study reported showing the enzyme to be a druggable target.

15. Vullo, D. et al. Carbonic anhydrase inhibitors. Inhibition of the transmembrane isozyme XII with sulfonamides — a new target for the design of antitumor and antiglaucoma drugs? Bioorg. Med. Chem. Lett. 15, 963–969 (2005).First CA XII inhibition study reported. This isozyme is involved in glaucoma and cancer.

16. Lehtonen, j. et al. Characterization of CA XIII, a novel member of the carbonic anhydrase isozyme family. J. Biol. Chem. 279, 2719–2727 (2004).

17. Nishimori, I. et al. Carbonic anhydrase inhibitors. Inhibition of the transmembrane isozyme XIV with sulfonamides. Bioorg. Med. Chem. Lett. 15, 3828–3833 (2005).

agents with a new mechanism of action. Indeed, resist‑ance to currently available drugs constitutes a serious medical problem for infections caused by P. falciparum, H. pylori, M. tuberculosis and C. albicans, and CAIs tar‑geting enzymes from these pathogens may overcome this owing to their novel mechanism of action. Over the past few years, the inhibitory profile of most of the important classes of CAIs — the sulphonamides, sulphamates and sulphamides — against the catalytically active isozymes have been investigated in detail. This is valuable in the search for isozyme‑selective compounds and for reducing side effects of the presently available drugs. However, few compounds have been identified that exhibit selectivity

for any CA isoform with clinical relevance, although important advances have been reported in the design of compounds with high selectivity for CA VA, IX and XIII over CA II (CA II is the ubiquitous, catalytically highly effective isoform, and so its inhibition is often regarded as detrimental).

Significant advances have been reported in the design of fluorescently labelled and membrane‑impermeable compounds inhibiting only the membrane‑associated CA isoforms (such as CA IX) and not those present in the cytosol (such as CA I and II). The use of fluores‑cent CAIs has indicated the important role of CA IX in tumour acidification processes, and the possibility of reversing this phenomenon by inhibiting the catalytic activity of the enzyme with a CA IX‑selective and potent inhibitor.

Various ophthalmologic and anti‑obesity applications of the CAIs have also been reported, together with novel derivatives that act as anticonvulsants. The reported X‑ray crystal structures of many human CA II and I adducts with sulphonamides and sulphamates has pro‑vided a better understanding of the molecular interac‑tions between the inhibitor and the enzyme, which may lead to a rational drug design of inhibitors with reduced side effects and selectivity for the target isoform.

Finally, important progress has been achieved in the field of the CAAs, with a large number of X‑ray crystallo‑graphic structures of adducts of isozymes I and II with amino‑acid activators reported. Interesting differences in the binding of enantiomeric activators such as l‑/ d‑Phe and l‑/d‑His to human CA II have been observed; and the first X‑ray crystal structure of an adduct of human CA I with an activator (l‑His) has recently been reported, showing great differences between the bind‑ing of activators within the active site of the two main isoforms, CA I and II. For the first time, isoforms other than CA I and II, such as CA IV, VA, VII, XIII and XIV have been investigated for their interaction with a large number of amine and amino‑acid activators. Owing to positive effects on spatial learning and memory, CAAs may provide a novel mechanism for treating disorders such as Alzheimer’s disease.

Figure 7 | A model of the regulation of filamentation and fungal capsule biosynthesis. CO2 is catalytically converted to bicarbonate in the presence of the β-carbonic anhydrase isozyme Can2 from Candida albicans. This bicarbonate directly stimulates Candida adenylate cyclase (Cac1) activity, which converts ATP to cyclic AMP, which regulates filamentation and fungal capsule biosynthesis30,31.

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AcknowledgementsResearch from the author’s laboratory was financed in part by two EU grants of the 6th framework programme (EUROXY and DeZnIt projects).

Competing interests statementThe authors declare competing financial interests: see web version for details.

DATABASESUniProtKB: http://ca.expasy.org/sprotCA I | CA II | CA III | CA IV | CA VA | CA VB | CA VI | CA VII | CA IX | CA XII | CA XIII | CA XIV | CAXV

All lInKs Are ACTIVe In THe onlIne PdF

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http://www.nature.com/nrd/journal/v7/n2/box/nrd2467_audecl.html

http://ca.expasy.org/sprot






http://www.expasy.org/uniprot/Q9Y2D0



http://www.expasy.org/uniprot/Q16790

http://www.expasy.org/uniprot/O43570

http://www.expasy.org/uniprot/Q8N1Q1

http://www.expasy.org/uniprot/Q9ULX7

http://www.expasy.org/uniprot/Q99N23

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