Ординатура / Офтальмология / Английские материалы / Carbonic Anhydrase Its Inhibitors and Activators_Supuran, Scozzafava, Conway_2004

development of CAAs as target-synthons in the projecting of drug diagnostic tools to manage some specific disorders.

12.2 MECHANISM OF ACTION OF CAAs

The model proposed for the mechanism of CA activation (Rowlett et al. 1991; Supuran 1992; Briganti et al. 1997a, 1997b, 1998) is closely related with that for the catalytic cycle. As discussed in Chapter 1, catalysis of α-CAs occurs in two well-defined stages that follow a ping-pong type of kinetics. The rate-limiting second step (Equation 12.1) is the intramolecular proton transfer between the zinc-bound water molecule and the reaction medium through the so-called proton shuttle residue, widely assumed to be the imidazole ring of His 64 in isozyme II (and several other efficient isozymes), as shown in Figure 12.1.

Crystallographic data (Nair and Christianson 1991) show that the His 64 imidazole side chain exhibits a pH-dependent conformational mobility, changing gradually its orientation related to the metal site through a 64° ring-flipping. Thus, at pH 8.5, conformer “in” predominates, the imidazole moiety being directed toward the zinc ion and linked to it through hydrogen bond bridges, whereas after proton catching the “out” conformation, in which the heterocyclic ring points toward the enzyme surface and engages no hydrogen bond contacts, becomes increasingly predominant as the pH decreases, reaching the maximum conformational percentage at a pH of 5.7. The high flexibility between the two conformations is crucial for the catalytic proton shuttling (Christianson and Fierke 1996; Duda et al. 2001; An et al. 2002).

It has been proposed that a CAA interferes directly in the proton transfer step of the catalytic cycle, facilitating such an intramolecular transfer by a transient enzyme–activator complex (Equation 12.1). Because intramolecular reactions are much faster than the intermolecular ones (Page and Williams 1989), the result is a significantly enhanced catalytic rate:

EZn2+-OH2 + A [EZn2+-OH2 ----- A EZn2+-OH– ----- AH+] EZn2+-OH– + AH+ (12.1) enzyme–activator complexes

The first x-ray structure obtained for a CAA complex was that of the adduct between human isozyme II (hCA II) and histamine (Briganti et al. 1997b). This structure has confirmed the previous hypothesis on the activation mechanism (Supuran 1991, 1992), revealing that histamine binds in the hydrophilic region located at the entrance of the active site, establishing through the nitrogen atoms of the imidazole ring new hydrogen bonds with water molecules and polar amino acids residues, whereas the aliphatic amino group remains free in the solvent (Figure 12.2 and Figure 12.3).

The supplementary hydrogen bond pathways generated by the activator binding have two key consequences for the rate-determining step of catalysis: (1) stabilize the His 64 “in” conformation, which is a steric requirement for the proton shuttling, and (2) offer adjacent routes for the proton transport from the zinc-bound water molecule to the external medium. Furthermore, histamine shows few contacts with

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FIGURE 12.2 hCA II–histamine adduct. Zn2+ (central sphere) and its three His ligands are placed in the center of the active site. Histamine (numbered as His 264) is anchored at the active site entrance, between residues His 64 and Gln 92. The figure was generated with the program RasMol for Windows 2.6, by using the x-ray crystallographic coordinates available in the Brookhaven Protein Database (PDB entry 4TST). (Reproduced from Briganti, F. et al. (1997b) Bioorganic and Medicinal Chemistry Letters 9, 2043–2048. With permission from Elsevier.)

the enzyme, limited to the imidazole ring that interacts with three protein amino acids residues (Asn 62, Asn 67, Gln 92). This interaction is favorable for the CA–histamine complex dissociation in the last step of the activation mechanism, in the same way by which the conformational flexibility of His 64 confers to this residue the ability to easily pendulate between the cavity and the opening of the active site during the catalytic mechanism. It can thus be concluded that the activator actually acts as an efficient second proton shuttle, besides the native one, His 64.

hCA II binds histamine with the displacement of at least three water molecules from the active-site cavity, followed by a substantial rearrangement of the hydrogen bond network within the cavity. The entropic contribution to the histamine free energy of binding provided by the release of water molecules favors energetically CAA complex formation, in addition to its kinetic and steric stabilization through the new hydrogen bond bridges.

Structural details of active sites of different CA isozymes have provided a further interesting explanation for the activation effect (Briganti et al. 1997b). The CA II active site possesses a well-defined histidine cluster, which starts from His 64 and

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FIGURE 12.3 Scheme of hydrogen bond bridges between histamine, histidine 64 and zincbound water molecule (Wat 150); the hydrogen bonds lengths are in Å. (Reproduced from Briganti, F. et al. (1997a) Biochemistry 36, 10384–10392. With permission.)

extends to the vicinity of the active-site entrance through the His residues in positions 4, 3, 10, 15 and 17, such that it surrounds the entrance and the edge of the active site (Figure 12.4). This cluster acts as a kind of rapid proton release channel, and might explain the high catalytic efficiency of the isozyme. Histamine binds within the hCA II active site at the beginning of this channel, adding a particular extension to it and thereby accentuating the tunnelling effect.

The CA I active site lacks the histidine cluster. Moreover, the histidine residues able to participate in the proton transfer are placed at bifurcating positions, creating divergent pathways for the proton release and thereby significantly slowing down the overall catalytic process (Briganti et al. 1997a). It is supposed that these totally different structural features explain the experimental results according to which, at low concentrations, histamine modulates more strongly the hCA I activity than the hCA II one; isozyme II seems to be already activated by its unique histidine cluster.

The ternary adduct of hCA II with the activator phenylalanine and the inhibitor azide is the only other structure of the CA activation complex reported up to the present (Briganti et al. 1998). From the analysis of the refined atomic model of this adduct by electronic spectroscopy and x-ray crystallography, it is clear that azide replaces the hydroxide anion from the Zn(II) native enzyme coordination sphere, binding directly to the metal site (with the maintenance of the overall tetrahedral geometry) and extending into the hydrophobic half of the active-site cavity. Unlike the inhibitor molecule and similarly to histamine, phenylalanine experiences no contacts with the zinc ion, being anchored through hydrogen bond bridges in the

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FIGURE 12.4 (See color insert following page 148.) hCA II active site. Zinc ion (pink); its three histidine ligands — His 94, His 96, His 119 (green); and the histidine cluster — His 64, His 4, His 3, His 17, His 15, His 10 (orange) are shown. The figure was generated from the diffraction coordinates reported by Briganti et al. (1997a) (PDB entry 4TST). (Reproduced from Scozzafava and Supuran (2002a). Bioorganic and Medicinal Chemistry Letters 12, 1177–1180. With permission from Elsevier.)

proximity of the active-site entrance, with the phenyl ring oriented toward this entrance. A water molecule (Wat 73) links simultaneously the inhibitor and activator molecules by two strong hydrogen bonds involving the zinc-bound nitrogen atom and the amino group, respectively (Figure 12.5).

Not surprisingly, as in the hCA II–histamine complex, the His 64 imidazole side chain appears with the “in” conformation. The strong, charge-assisted hydrogen bond (2.4 Å) between the phenylalanine carboxylate group and the Nε1 hydrogen of His 64 is undoubtedly one of the most important factors stabilizing this conformation.

Although belonging to diverse classes of compounds, both histamine and phenylalanine have a similar location when bound to the enzyme, being fixed at the activesite entrance. It is assumed that this particular active-site opening stop experienced by the activator molecules is also a consequence of their weak Lewis base character, which makes them unable to replace the stronger HO– zinc ligand. All these findings led to the conclusion that, in addition to the CO2 substrate binding site (the hydrophobic pocket consisting of the amino acid residues Val 121, Val 143, Leu 198, Thr 199, Val 207 and Trp 209) and the inhibitor binding site (the zinc ion), CAs possess a third site, the activators binding site, located at the entrance of the active-site cavity, between the His 64, Gln 92, Asn 62 and Asn 67 side chains. This site, although an enzymatic one, does not match strictly with the lock and key principle that generally governs the physicochemical interactions of proteins; that is, it is able to accommodate

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FIGURE 12.5 Scheme of hydrogen bond network that links azide, water molecule at position 73, phenylalanine, and the His 64 residue (hydrogen bonds lengths in Å also indicated). (Reproduced from Briganti, F. et al. (1998) Inorganica Chimica Acta 275/276, 295–300. With permission from Elsevier.)

molecules from diverse classes of compounds, with no a priori substrate specificity. The only thing these molecules have in common is the presence in their structure of one or more functional groups by which they can be efficiently involved in hydrogen bonds with well-defined strength and orientation so that they can participate in proton transfer processes between the active site and the environment. As previously shown, the activator binding mode or, in other words, stability of the enzyme–activator complex, is precisely tuned kinetically (through the binding affinity between the protein and its activator), sterically (through an advanced stabilization of His 64 “in” conformation) and energetically (through the rearrangement of the active-site water structure) to achieve the most appropriate alternative hydrogen bond network for proton transfer from the zinc-bound water molecule to the bulk solvent.

Although this activation mechanism was initially proposed and then investigated in great detail for isozyme II only, subsequent activation studies for isozymes III, IV and V (see later) have suggested its general validity for all CA isoforms investigated until the present time.

12.3CAAs TYPES: DESIGN STRATEGIES, STRUCTURE–ACTIVITY RELATIONSHIPS AND ISOZYME SPECIFICITY

The drug design of CAAs has three major aims: (1) clarifying aspects of the catalytic mechanism; (2) elucidating the in vivo role of CAAs both in physiological and pathological conditions; and (3) obtaining biologically active compounds possessing isozyme specificity, or, at least, organ and tissue selectivity, which might be used in the clinical development of pharmacological agents devoid of severe side effects.

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The first goal has been achieved almost completely through detailed studies that have explained the activation mechanism (see Section 12.2), but the other two still represent an open challenge, although much progress has been made in the past few years.

12.3.1 BACKGROUND

The biogenic amine histamine and the corresponding amino acid histidine were the first reported CAAs (Leiner and Leiner 1941), but no progress was made in this area in the following 30 years (Supuran and Scozzafava 2000a). The early 1970s marked the beginning of a revival in the investigation of activating properties of this type of compound. Narumi and Kanno (1973) reported in vivo CA II activation by some gastric acid stimulants (such as histamine, tetraand pentagastrin and carbachol). These results were supported later by the finding that catecholamines enhance the activity of this red cell isozyme (Igbo et al. 1994). It was presumed (Narumi and Miyamoto 1974) that a cAMP-dependent protein kinase induced the phosphorylation of an active-site threonine residue, being hypothesized that the phosphorylated enzyme is more active than the unphosphorylated one. It is now clear that the explanation for the CA activating effect with such compounds is different and that phosporylation of CA active-site residues does not occur (Supuran and Scozzafava 2000a).

The CAA behavior of histamine continued to be further investigated. Puscas (1978) showed that histamine is strongly involved in gastric ulcers and proved its in vitro and in vivo activating properties against CA II.

Silverman’s laboratory reported hCA II activation by histidine (Silverman et al. 1978), but subsequently considered their discovery an artefact owing to the ethylenediaminotetraacetic acid (EDTA) added in the buffer, which might form complexes with the adventitious Cu2+ possibly present in the protein preparation, thus restoring the activity of the enzyme (heavy metal ions act as micromolar inhibitors of most CAs; Tu et al. 1981).

Seeking for structural requirements that could determine CA activation, Supuran and coworkers (Supuran et al. 1991; Puscas et al. 1990; Supuran 1991, 1992) investigated CA activity in the presence of various natural and synthetic compounds of biological relevance, such as biogenic amines, amino acids and their derivatives, oligopeptides and several pharmacological agents (vide infra). It was shown (Supuran 1991, 1992) that all these compounds fit well in a general structure of type 12.1, incorporating as main structural elements a proton-accepting moiety (a primary or secondary amino group) attached to a bulky aromatic/heterocyclic ring through an aliphatic carbon chain linker. Table 12.1 presents the activation data with some of these compounds and their detailed structural identity.

In the light of this study (Supuran et al. 1991), the initial report by Silverman’s group regarding hCA II activation with histidine (Silverman et al. 1978) was proposed to be authentic. It was explained that the controversial results of Tu et al. (1981) were because of the experimental protocols used, EDTA probably possessing a competitive suppressing effect for the histidine activating effect (Supuran and Puscas 1994).

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TABLE 12.1

hCAs I and II Activation by Different Biologically Active Compounds for CO2 Hydration at 10–5 M of Activator

aAmino acids were L-enantiomers.

bCA activity without activator added is taken as 100%.

Source: Adapted from Supuran, C.T., and Puscas, I. (1994) In Carbonic Anhydrase and Modulation of Physiologic and Pathologic Processes in the Organism, Puscas, I. (Ed.), Helicon, Timisoara, Romania, pp. 113–145.

The data given in Table 12.1 afforded the first structure–activity relationships (SARs) for CAAs. Thus, consistent with the activation mechanism previously described, substitution of the parent structure (12.1) with polar moieties able to engage in hydrogen bonds (such as hydroxyl and carboxyl groups or fluorine atoms) increased activator potency. Most of the arylalkylamines and all the amino acids from Table 12.1 were also included in a quantitative structure–activity relationship (QSAR) study (Clare and Supuran 1994). This is the only QSAR investigation on hCA II activators available until the present time. By regression analysis and partial least squares method, equations for quantitative correlation were derived, which

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translated into mathematical terms the interdependence between biological activity and two main physicochemical parameters — the molecular charge distribution and the molecular volume. The activating properties were expressed as log A, where A is the hCA II activation percentage. The electronic density and the size of the activator molecule were expressed as a large number of different descriptors calculated by the complete neglect of differential overlap (CNDO) approximation. Such optimized equations show that the efficiency of CAAs is determined both by steric and electronic factors. Thus, consideration of a molecular model projection on a three-dimensional axis clearly showed that a decrease in the two smaller orthogonal dimensions of the CAA molecule yields an intensification of its biological activity. This fact has forwarded for the first time the concept that CAAs bind to the enzyme in a site of limited size, as has been subsequently confirmed through x-ray crystallographic studies (Briganti et al. 1997b). Alternatively, the higher the charge of the most electronegative atom in the molecule, the more active the compound is as a CAA. The second theoretical result explained experimental data that showed that molecules bearing highly charged oxygen or nitrogen atoms (such as amino acids and amino-azole derivatives) were more powerful CAAs than were analogous compounds in which the amino group is attached to a molecular skeleton containing only carbon atoms (Supuran et al. 1991, 1993a). This first QSAR of CAAs afforded two main conclusions: (1) the activating properties are conditioned by a relatively compact structure of the molecule, strictly depending on the dimensions of the binding site of activators, and (2) the activating properties are significantly increased by the presence in the molecule of some oxygen-/nitrogen-containing moieties that are strongly polarized. These conclusions, together with the chemical structure of compounds given in Table 12.1, constituted the starting point for different directions in the design and synthesis of CAAs.

12.3.2 CA ACTIVATION WITH AMINE DERIVATIVES

Three premises have been considered in the development of CAAs of this type:

(1) aliphatic amines with the general formula 12.2 have no activating effect on CA II (Puscas et al. 1990); (2) 2-amino-5-(2-aminoethyl)-1,3,4-thiadiazole (12.3), reported as a histamine agonist and proved to possess gastric-acid-stimulating effects, was later recognized as a powerful CAA (Supuran and Puscas 1994); (3) the pyridinium ring presents several features (i.e., aromatic character, permanent positive charge, strong chemical stability, especially when it is alkyl/aryl substituted) that recommend it as a candidate moiety for obtaining biologically active compounds with pharmacological applications. Efficient positively charged CAAs of type 12.4 to 12.6 were thus designed and synthesized (Supuran et al. 1993b, 1996a), by condensing different 2,4,6-trisubstituted pyrylium salts with the corresponding amine derivatives under the classical Baeyer–Piccard reaction conditions.

The following SAR conclusions were derived: (1) The polymethylene bridge between the amino group and the heterocyclic moiety might incorporate either two or three carbon atoms. A higher number than this leads to a decrease in activating properties. (2) The derivatives bearing methyl or t-butyl groups in the 2- and 6-positions of the pyridinium ring are much stronger CAAs than the 2,6-diphenylsubstituted

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