Carbonic Anhydrase

Its Inhibitors and Activators

Claudiu T. Supuran, Andrea Scozzafava, Janet Conway, Claudiu T. Supuran, Andrea Scozzafava, Janet Conway

• Publisher: CRC Press
• Year: 2004
• ISBN:
  9781134400157
• Edition: 1
• Topic: Medicina
• Subtopic: Biochimica in medicina

Carbonic Anhydrase: Its Inhibitors and Activators provides a state-of-the-art overview of the latest developments and challenges in carbonic anhydrase research. Authors describe the mechanisms of action of specific inhibitors in relation to physiological function, and present previously unpublished research on CA activators. Written by a team of in

1 Carbonic Anhydrases: Catalytic and Inhibition Mechanisms, Distribution and Physiological Roles

Claudiu T. Supuran

CONTENTS

1.1 Introduction

1.2 Catalytic and Inhibition Mechanisms of CAs

1.2.1 α-CAs

1.2.2 β-CAs

1.2.3 γ-CAs

γ-1.2.4 Cadmium CA

1.3 Distribution of CAs

1.4 Physiological Functions of CAs

References

At least 14 different α-carbonic anhydrase (CA, EC 4.2.1.1) isoforms have been isolated in higher vertebrates, wherein these zinc enzymes play crucial physiological roles. Some of these isozymes are cytosolic (CA I, CA II, CA III and CA VII), others are membrane bound (CA IV, CA IX, CA XII and CA XIV), one is mito-chondrial (CA V) and one is secreted in the saliva (CA VI). Three acatalytic forms are also known, designated CA-related proteins (CARPs): CARP VIII, CARP X and CARP XI. Representatives of the β-and γ-CA family are highly abundant in plants, bacteria and archaea. These enzymes are very efficient catalysts for the reversible hydration of carbon dioxide to bicarbonate, and at least α-CAs possess a high versatility, being able to catalyze other different hydrolytic processes, such as the hydration of cyanate to carbamic acid or of cyanamide to urea; aldehyde hydration to gem-diols; hydrolysis of carboxylic or sulfonic acids esters; as well as other less investigated hydrolytic processes, such as hydrolysis of halogeno derivatives and arylsulfonyl halides. It is not known whether the reactions catalyzed by CAs other than the hydration of CO/dehydration of HCO^– have physiological relevance in ₂3 systems in which these enzymes are present. The catalytic mechanism of α-CAs is understood in great detail. The active site consists of a Zn(II) ion coordinated by three histidine residues and a water molecule/hydroxide ion. The latter is the active species, acting as a potent nucleophile. For β-and γ-CAs, the zinc hydroxide mechanism is valid too, although at least some β-class enzymes do not have water directly coordinated to the metal ion. CAs are inhibited primarily by two main classes of inhibitors: the metal-complexing inorganic anions (such as cyanide, cyanate, thiocyanate, azide and hydrogensulfide) and the unsubstituted sulfonamides possessing the general formula RSO₂NH₂ (R = aryl, hetaryl, perhaloalkyl). Several important physiological and physiopathological functions are played by the CA isozymes, which are present in organisms at all levels of the phylogenetic tree. Among these functions are respiration and transport of CO₂/bicarbonate between metabolizing tissues and lungs, pH and CO₂ homeostasis, electrolyte secretion in a variety of tissues and organs, biosynthetic reactions, such as the gluconeogenesis and urea synthesis (in animals) and CO₂ fixation (in plants and algae). The presence of these ubiquitous enzymes in so many tissues and in so many different isoforms makes them useful to design inhibitors or activators that have biomedical applications.

1.1 INTRODUCTION

The carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metalloenzymes present in prokaryotes and eukaryotes and encoded by three distinct evolutionarily unrelated gene families: (1) α-CAs (in vertebrates, bacteria, algae and cytoplasm of green plants), (2) β-CAs (predominantly in bacteria, algae and chloroplasts of both mono-and dicotyledons) and (3) γ-CAs (mainly in archaea and some bacteria) (Hewett-Emmett 2000; Krungkrai et al. 2000; Chirica et al. 1997; Smith and Ferry 2000; Supuran and Scozzafava 2000, 2002; Supuran et al. 2003). In higher vertebrates, including humans, 14 α-CA isozymes or CA-related proteins (CARPs) have been described (Table 1.1), with very different subcellular localizations and tissue distributions (Hewett-Emmett 2000; Supuran and Scozzafava 2000, 2002; Supuran et al. 2003). There are several cytosolic forms (CAs I–III, CA VII), four membrane-bound iso-zymes (CA IV, CA IX, CA XII and CA XIV), one mitochondrial form (CA V) and a secreted CA isozyme (CA VI) (Supuran and Scozzafava 2000, 2002; Supuran et al. 2003). These enzymes catalyze a very simple physiological reaction, the intercon-version of the carbon dioxide and the bicarbonate ion, and are thus involved in crucial 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 and organs, biosynthetic reactions (such as gluconeogenesis and lipoid and urea synthesis), bone resorption, calcification, tumor-igenicity and many other physiological or pathological processes (Hewett-Emmett 2000; Supuran and Scozzafava 2000, 2002; Supuran et al. 2003). Many of these isozymes are important targets for the design of inhibitors with clinical applications.

In addition to the physiological reaction — the reversible hydration of CO₂ to bicarbonate (Equation 1.1, Figure 1.1) — α-CAs catalyze a variety of other reactions, such as hydration of cyanate to carbamic acid or of cyanamide to urea (Equation 1.2 and Equation 1.3, Figure 1.1); aldehyde hydration to gem-diols (Equation 1.4, Figure 1.1); hydrolysis of carboxylic or sulfonic acid esters (Equation 1.5 and Equation 1.6, Figure 1.1); as well as other less-investigated hydrolytic processes, such as those described by Equation 1.7 to Equation 1.9 in Figure 1.1 (Briganti et al. 1999; Guerri et al. 2000; Supuran et al. 1997, 2003; Supuran and Scozzafava 2000, 2002). The previously reported phosphatase activity of CA III was recently proved to be an artefact (Kim et al. 2000). It is unclear whether α-CA catalyzed reactions other than CO2 hydration have physiological significance. To date, x-ray crystal structures have been determined for six α-CAs (isozymes CA I to CA V and CA XII; Stams and Christianson 2000) as well as for representatives of the β-(Mitsuhashi et al. 2000) and γ-CA families (Kisker et al. 1996; Smith and Ferry 2000; Iverson et al. 2000).

TABLE 1.1

1.2CATALYTIC AND INHIBITION MECHANISMS OF CAs

1.2.1 α-CAS

The Zn(II) ion of CAs is essential for catalysis (Lindskog and Silverman 2000; Christianson and Fierke 1996; Bertini et al. 1982; Supuran et al. 2003). X-ray crystallographic data show that the metal ion is situated at the bottom of a 15-Ådeep active-site cleft (Figure 1.2), coordinated by three histidine residues (His 94, His 96 and His 119) and a water molecule/hydroxide ion (Christianson and Fierke 1996; Stams and Christianson 2000). The zinc-bound water is also engaged in hydrogen bond interactions with the hydroxyl moiety of Thr 199, which in turn is bridged to the carboxylate moiety of Glu 106. These interactions enhance the nucleophilicity of the zinc-bound water molecule and orient the substrate (CO2) in a location favorable for nucleophilic attack (Figure 1.3; Lindskog and Silverman 2000; Stams and Christianson 2000; Supuran et al. 2003). The active form of the enzyme is the basic one, with hydroxide bound to Zn(II) (Figure 1.3A; Lindskog and Silverman 2000). This strong nucleophile attacks the CO2 molecule bound in a hydrophobic pocket in its neighborhood (the substrate-binding site comprises residues Val 121, Val 143 and Leu 198 in human isozyme CA II — Christianson and Fierke 1996; Figure 1.3B), leading to the formation of bicarbonate coordinated to Zn(II) (Figure 1.3C). The bicarbonate ion is then displaced by a water molecule and liberated into solution, forming the acid form of the enzyme, with water coordinated to Zn(II) (Figure 1.3D), which is catalytically inactive (Lindskog and Silverman 2000; Christianson and Fierke 1996; Bertini et al. 1982; Supuran et al. 2003). To regenerate the basic form A, a proton transfer reaction from the active site to the environment occurs, which might be assisted either by active-site residues (such as His 64 — the proton shuttle in isozymes I, II, IV, VII and IX, among others; see Figure 1.2 for isozyme II) or by buffers present in the medium. The process is schematically represented by Equation 1.10 and Equation 1.11:

FIGURE 1.1 Reactions catalyzed by α-CAs. (Reproduced from Supuran, C.T. et al. (2003) Medicinal Research Reviews 23, 146–189, John Wiley & Sons. With permission.)

FIGURE 1.2(See color insert following page 148.) hCA II active site. The Zn(II) ion (central pink sphere) and its three histidine ligands (in green, His 94, His 96, His 119) are shown. The histidine cluster, comprising residues His 64, His 4, His 3, His 17, His 15 and His 10, is also shown, as this is considered to play a critical role in binding activators of the types 6 to 14 reported in the chapter as well as the carboxyterminal part of the anion exchanger AE1. The figure was generated from the x-ray coordinates reported by Briganti et al. (1997) (PDB entry 4TST). (Reproduced from Scozzafava, A. and Supuran, C.T. (2002) Biorganic Medicinal Chemistry Letters 12, 1177–1180. With permission from Elsevier.)

(1.10)

(1.11)

The rate-limiting step in catalysis is the second reaction, i.e., the proton transfer that regenerates the zinc hydroxide species of the enzyme (Lindskog and Silverman 2000; Christianson and Fierke 1996; Bertini et al. 1982; Supuran et al. 2003). In the catalytically very active isozymes, such as CA II, CA IV, CA V, CA VII and CA IX, the process is assisted by a histidine residue (His 64) at the entrance of the active site, as well as by a cluster of histidines (Figure 1.2) that protrudes from the rim of the active site to the surface of the enzyme, assuring a very efficient proton transfer process for CA II, the most efficient CA isozyme (Briganti et al. 1997). This also explains why CA II is one of the most active enzymes known (with a kcat/Km = 1.5 × 108 M–1s–1), approaching the limit of diffusion control (Lindskog and Silverman 2000; Christianson and Fierke 1996; Supuran et al. 2003), and also has important consequences for designing inhibitors that have clinical applications.

FIGURE 1.3 Schematic representation of the catalytic mechanism for the CA-catalyzed CO2 hydration. The hypothesized hydrophobic pocket for the binding of substrates is shown schematically at Step B.

Two main classes of carbonic anhydrase inhibitors (CAIs) are known: the metal-complexing anions and the unsubstituted sulfonamides, which bind to the Zn(II) ion of the enzyme either by substituting the nonprotein zinc ligand (Equation 1.12, Figure 1.4) or add to the metal coordination sphere (Equation 1.13, Figure 1.4), generating trigonal-bipyramidal species (Bertini et al. 1982; Lindskog and Silverman 2000; Supuran et al. 2003; Supuran and Scozzafava 2000, 2002). Sulfonamides, the most important CAIs, bind in a tetrahedral geometry of the Zn(II) ion (Figure 1.4), in a deprotonated state, with the nitrogen atom of the sulfonamide moiety coordinated to Zn(II) and an extended network of hydrogen bonds involving the residues Thr 199 and Glu 106, also participating in anchoring the inhibitor molecule to the metal ion. The aromatic/heterocyclic part of the inhibitor (R) interacts with hydrophilic and hydrophobic residues of the cavity. Anions might bind either in tetrahedral geometry of the metal ion or as trigonal-bipyramidal adducts, such as the thiocyanate adduct shown in Figure 1.4B (see also Chapter 7 for a detailed description of CA inhibition by anions; Lindahl et al. 1991; Stams and Christianson 2000; Abbate et al. 2002; Supuran et al. 2003).

X-ray crystallographic structures are available for many adducts of sulfonamide inhibitors with isozymes CA I, CA II and CA IV (see also Chapter 3; Lindahl et al. 1991; Stams et al. 1996; Stams and Christianson 2000; Abbate et al. 2002, 2003;

(1.12)

Tetrahedral adduct

(1.13)

FIGURE 1.4 CA inhibition mechanism by sulfonamide (A) and anionic (B) inhibitors. In the case of sulfonamides, in addition to the Zn(II) coordination, an ext...

Table of contents

• COVER PAGE
• TITLE PAGE
• COPYRIGHT PAGE
• PREFACE
• CONTRIBUTORS
• 1: CARBONIC ANHYDRASES: CATALYTIC AND INHIBITION MECHANISMS, DISTRIBUTION AND PHYSIOLOGICAL ROLES
• 2: ACATALYTIC CAs: CARBONIC ANHYDRASE-RELATED PROTEINS
• 3: MULTIPLE BINDING MODES OBSERVED IN X-RAY STRUCTURES OF CARBONIC ANHYDRASE INHIBITOR COMPLEXES AND OTHER SYSTEMS: CONSEQUENCES FOR STRUCTURE-BASED DRUG DESIGN JOCHEN ANTEL, ALEXANDER WEBER, CHRISTOPH A.
• 4: DEVELOPMENT OF SULFONAMIDE CARBONIC ANHYDRASE INHIBITORS
• 5: QSAR STUDIES OF SULFONAMIDE CARBONIC ANHYDRASE INHIBITORS
• 6: METAL COMPLEXES OF HETEROCYCLIC SULFONAMIDES AS CARBONIC ANHYDRASE INHIBITOR
• 7: NONSULFONAMIDE CARBONIC ANHYDRASE INHIBITORS
• 8: CLINICAL APPLICATIONS OF CARBONIC ANHYDRASE INHIBITORS IN OPHTHALMOLOGY
• 9: CANCER-RELATED CARBONIC ANHYDRASE ISOZYMES AND THEIR INHIBITION
• 10: ROLE OF CARBONIC ANHYDRASE AND ITS INHIBITORS IN GASTROENTEROLOGY, NEUROLOGY AND NEPHROLOGY
• 11: CARBONIC ANHYDRASE INHIBITORS IN DERMATOLOGY
• 12: CARBONIC ANHYDRASE ACTIVATORS