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

FIGURE 7.11 Coordination detail for the complex between human CA II and azide (Wat318 also shown) (From Joensson, B.M. et al. FEBS Letters 322, 186–190.); the structure was generated from the pdb file (1RAY) by using the program RasWin for Windows, version 2.7.1.1.

mechanism with respect to CO2 hydration; the very slow rate of the cyanamide reaction is easily explained by considering that the attacking water molecule (Wat-51) is only partially activated by the interaction with the metal (Guerri et al. 2000).

As predicted by spectroscopic techniques (Bertini and Luchinat 1983; Bertini et al. 1982) another species to generate fourto five-coordinated species would be N3–, the halogen and bicarbonate ions. To confirm these facts, Joensson et al. (1993) determined the structure of CA II in complex with bromide and azide. They found that both azide and bromide ions bind into the CA II active site and replace the zincbound water and the deep water (Figure 7.11 and Figure 7.12), generating a distorted tetrahedral coordination for the metal ion. The inhibitors cannot bind close to Thr 199 Oγ1 because of their lack of protons. In the case of azide ion, the N3 atom is

2.0 Å away from the zinc ion and 3.2 Å away from Oγ1 of Thr-199. The N1 atom is positioned 3.4 Å from the Thr-199 backbone N and 3.7 Å from the Thr-199 Oγ1.

The ligand forms a hydrogen bond with Wat-318 (positioned 2.6 Å away from N3 and 3.4 Å away from N2; Figure 7.11).

A similar pattern is observed in the structure of the CA II–bromide adduct; Br– replaces the zinc-bound water and the deep water and coordinates 2.5 Å away from the zinc ion, 3.6 Å away from the Thr-199 Oγ1 and 3.8 Å from the Thr-199 backbone N (Figure 7.12). The difference between the position of the bromide ion and the azide N2 is only 0.2 Å. Also, there are no significant differences between the structure of the bromide adduct at pH 6.0 and at pH 7.8; however, at higher pH, the hydroxide ions compete for coordination to the metal center (Joensson et al. 1993). Iodide is reported to act in a similar way (Brown et al. 1977; Joensson et al. 1993). This behavior can be explained by their softness (greater polarizability) as compared with oxygen-complexing ligands, which might alleviate the repulsion between its electron

Copyright © 2004 CRC Press, LLC

FIGURE 7.12 Coordination detail for the complex between human CA II and bromide (Wat318 also shown) (From Joensson, B.M. et al. FEBS Letters 322, 186–190.); the structure was generated from the pdb file (1RAZ) by using the program RasWin for Windows, version 2.7.1.1.

orbitals and the lone pair of Thr-199 Oγ1, as confirmed by more recent experiments (Lim et al. 1998; Merz and Banci 1996).

The structure of the native CA II–bicarbonate complex is not available to date, despite the efforts of several crystallographic groups. The insights of bicarbonate coordination were first revealed by using Co-CA II (Haakansson and Wehnert 1992). The ligand replaces the deep water molecule, binding at the cobalt ion in a pentacoordinate fashion, with the O3 atom 2.39 Å away from the metal. The O1 atom accepts a hydrogen bond from the Thr-199 backbone NH, whereas the O2 atom is 2.4 Å from the cobalt ion (Figure 7.13). The positions of O1 and O2 are very similar to the corresponding oxygen atoms of the formate–CA II complex (Haakansson et al. 1992). The metal-bound water (Wat-263) is moved 1.7 Å away from its native position, being located at 2.33 Å from the cobalt ion (Figure 7.13; Haakansson and Wehnert 1992).

In the adduct hCA I–HCO3– (Kumar and Kannan 1994), the bicarbonate ion occupies approximately the same position in the active site of the enzyme but coordinates as an essentially monodentate ligand, as proposed previously (Williams and Henkens 1985). An intermediate position between these two extremes is represented by HCO3– coordination in its complex with the Thr-200 His hCA II mutant (Xue et al. 1993), in which a pseudobidentate coordination is observed. Here, the coordination geometry of the zinc ion is somewhere between tetrahedral and trigonal bipyramidal. The O3 atom is 2.2 Å away from the zinc ion and 2.6 Å from the Oγ1 of Thr-199; the O2 atom is 2.5 Å away from the metal, whereas the O1 atom is

3.0Å from the Thr-199 backbone NH (Figure 7.14).

Thus, the crystallographic data confirm that bicarbonate interaction with CA is

strongly influenced by the characteristics of the active site of each isozyme, as predicted by theoretical calculations (Krauss and Garmer 1991; Liang and Lipscomb

Copyright © 2004 CRC Press, LLC

FIGURE 7.13 Coordination detail of the complex between Co-CA II and bicarbonate ion (From Haakansson, K. and Wehnert, A. 1992. Journal of Molecular Biology 228, 1212–1218.); the structure was generated from the pdb file (1CAH) by using the program RasWin for Windows, version 2.7.1.1.

FIGURE 7.14 Coordination detail of the complex between T200H CA II mutant and bicarbonate ion (From Xue, Y. et al. 1993. Proteins: Structure, Function, and Genetics 15, 80–87.); the structure was generated from the pdb file (1BIC) by using the program RasWin for Windows, version 2.7.1.1.

1987; Pullman 1981; Vedani et al. 1989; Zheng and Merz 1992) and by kinetic determinations (Pocker and Sarkanen 1978).

In addition to the x-ray crystallographic studies of different anions binding into the CA active site (all placed in the zinc coordination sphere), the crystal structure of the complex between CA II and phenol was reported (Nair et al. 1994) — its

Copyright © 2004 CRC Press, LLC

FIGURE 7.15 Coordination detail of the complex between CA II and phenol (From Nair, S.K. et al. 1994. Journal of the American Chemical Society 116, 3659–3660.); the structure was generated from the pdb file using the program RasWin for Windows, version 2.7.1.1.

only known competitive inhibitor [KI = 10 mM at pH 8.7 (Simonsson et al. 1982)]. Unexpectedly, two molecules of phenol were observed in the electron density maps of the complex. The first molecule binds to a hydrophobic patch, ca. 15 Å away from zinc, making van der Waals contacts with residues Leu-57, Ile-91and Pro-237 and hydrogen bonds with Asp-72 and with backbone carbonyl of Gly-235. Although this location does not correspond to the molecular trajectory or secondary CO2 binding sites predicted in molecular dynamics calculations (Liang and Lipscomb 1990; Merz 1990, 1991), the second phenol molecule binds in the hydrophobic pocket of the enzyme, the known CO2 precatalytic association site. It displaces the deep water molecule, making van der Waals contacts with hydrophobic pocket residues Val-121, Val-143, Leu-198, Trp-209. Its hydroxyl group does not coordinate to zinc but is hydrogen bonded to the zinc-bound water/hydroxyl ion (2.6 Å away) and also with the backbone NH of Thr-199 (3.2 Å away; Figure 7.15; Nair et al. 1994.

Considering that the CA II–phenol adduct crystals were prepared at pH 10 and also the differences between the pKa values of phenol and zinc-bound water (10 and 7, respectively), it was assumed that the phenol molecule is ionized (Nair et al. 1994), as suggested also by 13C-NMR studies carried out with this inhibitor (Khalifah et al. 1991).

7.3 OTHER TYPES OF NONSULFONAMIDE INHIBITORS

7.3.1 INHIBITORS OF THE PROTON SHUTTLE

A different class of CAIs is represented by the metal ions Cu2+ and Hg2+. The copper ion was found to be a weak inhibitor of CA II (IC50 = 0.5 μM at pH 7.3; Eriksson et al. 1988b; Magid 1967). Subsequent 18O exchange experiments done by Tu et al.

Copyright © 2004 CRC Press, LLC

(1981) have shown that Cu2+ does not affect the rate of either substrate binding or product release. Moreover, lowering the pH to 6 completely abolishes the inhibitory effect. The same effects were observed for Hg2+ ions. The authors suggested that the metals bind to the proton shuttle of the enzyme — His-64 — and inhibit intramolecular proton transfer (Tu et al. 1981). X-ray crystallographic investigations by Ericksson et al. (1988b) confirmed this hypothesis. In the electron density chart of the Hg2+–CA II complex, both nitrogens of His-64 were found to bind mercury at about half occupancy, suggesting that the two imidazolic nitrogen atoms bind the metal with about equal affinity, thereby eliminating the possibility for His-64 to participate in proton transfer (Eriksson et al. 1988b; Eriksson and Liljas 1991).

Linked with these facts is the common finding of different crystallographers that the use of mercuric ions or mercury-containing compounds, such as HgCl2, 4-chloro- benzenesulfonic acid or 4-hydroxymercuribenzoate, to avoid dimerization of cysteine residues of CA II also enhances the hCA II crystal quality (Briganti et al. 1998, 1999; Eriksson et al. 1988b; Mangani and Liljas 1993; Tilander et al. 1965; Xue et al. 1993), probably by blocking the very mobile His-64 in a fixed conformation.

7.3.2 ORGANIC SULFAMATES AND HYDROXAMATES AS CAIS

Besides the shuttle inhibitors, other nonsulfonamide CAIs were discovered or invented in the quest for topical antiglaucoma drugs (Maren 2000; Supuran and Scozzafava 2000, 2002b) or by analogy with other zinc enzymes (Scolnick et al. 1997).

Thus, Lo et al. (1992), following a previous report that topiramate (7.1) and other related alkyl sulfamates can act as weak CAIs (Maryanoff et al. 1987), synthesized a series of imidazolylphenyland imidazolylphenoxyalkyl sulfamates, which were evaluated as topical antiglaucoma CAIs. The best results were obtained with the sulfamic acid ester 7.2 (AHR-16329, KI = 7 nM against CA II), which displayed a peak 2.5 mmHg decrease in intraocular pressure (IOP) 1 h after being applied as a 2 to 5% solution to the eye (Brechue and Maren 1993; Lo et al. 1992).

Continuing their program on antiepileptic drugs, Maryanoff et al. (1998) conducted an extensive structure–activity study on topiramate-related compounds. Besides investigating their anticonvulsant properties, the authors also tested the drugs as CAIs. The cyclic sulfamate 7.3 (RWJ-37947) exhibited eight times greater anticonvulsant activity than did topiramate and also proved to be a potent inhibitor of CA (IC50 = 36 nM). Because of its rather unusual structure for a very efficient CAI, RWJ-37947 was further investigated by x-ray crystallography in complex with CA II (Recacha et al. 2002). The results showed that the sulfamate group of 7.3, which has a pKa value of 8.51 (Dodgson et al. 2000), coordinates to zinc, displacing the water/hydroxide ion and making a hydrogen bond with the Oγ1 of Thr-199, similar to sulfonamides. One sulfamate oxygen accepts a hydrogen bond from the Thr-199 backbone NH, the other 3.1 Å away from the zinc ion. Taking into account the distances and angles of the latter oxygen, it was considered weakly coordinated to the metal. The six-member tetrahydropyran ring of the inhibitor retains the skew conformation (3S0) found in solution (Dodgson et al. 2000; Figure 7.16). The cyclic sulfate group forms a weak hydrogen bond with Pro-202 and Leu-198. No evidence

Copyright © 2004 CRC Press, LLC

FIGURE 7.16 Coordination detail of the complex between CA II and RWJ-37947 (From 7.3; Recacha, R. et al. 2002. Biochemical Journal 361, 437–441.); the structure was generated from the pdb file (1EOU) by using the program RasWin for Windows, version 2.7.1.1.

of irreversible binding was found and the structure of CA II exhibited only minor differences in conformation when compared to the native protein. The coordination pattern is similar, in general terms, to those found for sulfamide [7.4, KI = 1.13 mM (Briganti et al. 1996)] and sulfamic acid [7.5, KI = 390 μM (Briganti et al. 1996)] in their adducts with CA (Abbate et al. 2002).

However, a very recent crystallographic study of the complex CA II–topiramate (Casini et al. 2003) revealed that this drug has a totally different conformation when bound to the active site of the enzyme, as compared with that of RWJ-37947. Although the sulfamate group is anchored in essentially the same way (Figure 7.17), the conformation of the backbone is entirely changed and strongly stabilized by an

extended network of hydrogen bonds between the inhibitor and some amino acid residues inside the cavity: Asn-62–Nε1 to O4 of topiramate, Gln-92–Nε2 to O6 of

topiramate, Wat-1134 to O3 of topiramate and to Oγ1 of Thr-200. Moreover, a full SAR study of the interaction of CA isozymes I, II and IV with related sugar sulfamates/sulfamides was reported and the main finding was that topiramate 1 is actually a very potent CAI, three orders of magnitude more efficient than previously

reported (KI hCA I = 250 nM, KI hCA II = 5 nM, KI hCA IV = 54 nM). It is comparable in effect with acetazolamide, a fact sustained not only by the tightly bound crystal

adduct with the CA II, but also by the side effects observed (Sabers and Gram 2000) in many patients treated with this antiepileptic drug, which are typical for strong systemic CAIs (Supuran and Scozzafava 2000, 2002b).

Hence, the use of sulfamide and sulfamic acid moieties as new anchoring groups for designing CAIs has a solid theoretical base. Other recent investigations (Scozzafava

Copyright © 2004 CRC Press, LLC

FIGURE 7.17 The complex between CA II and topiramate (From 7.1; Casini, A. et al. 2003.

Bioorganic and Medicinal Chemistry Letters 13, 837–840.); the structure was generated from the pdb file by using the program RasWin for Windows, version 2.7.1.1.

et al. 2000, 2001) on these types of compounds confirmed these findings. Thus, when aromatic/heterocyclic scaffolds were used in conjunction with sulfamide and sulfamate anchoring groups, nanomolar levels of CA inhibition were obtained, e.g.,

7.6 [KI hCA II = 16 nM, KI hCA IV = 50 nM (Scozzafava et al. 2001)], 7.7 [KI hCA II = 21 nM, KI hCA IV = 54 nM (Scozzafava et al. 2001)], 7.8 [KI hCA I = 4.6 nM, KI hCA II =

1.1 nM, KI hCA IX = 18 nM (Winum et al. 2003)], associated also by a good selectivity against different CA isozymes, such as in the case of compounds 7.9 [KI hCA II =

10 nM, KI hCA IV = 0.8 μM (Scozzafava et al. 2000)] and 7.10 [KI hCA II = 20 nM, KI hCA IV = 0.4 μM (Scozzafava et al. 2000)].

Christianson’s group (Scolnick et al. 1997) showed that hydroxamic acids, known to be efficient inhibitors of zinc enzymes such as thermolysin (Jin and Kim 1998; Powers and Harper 1986) and matrix metalloproteinases (Supuran and Scozzafava 2002a), can act as CAIs. The three-dimensional structures of CA II complexes with acetohydroxamic acid (7.11, IC50 = 47 μM) and its fluorinated congener (7.12, IC50 = 3.8 μM) revealed an interesting binding mode: instead of forming a chelate with the CO and OH groups of the metal, they bind through the ionized nitrogen atom directly to the zinc ion, displacing the coordinated water/hydroxyl ion. The inhibitors also displace the deep water molecule and form a donor hydrogen bond with the Oγ1 of Thr-199. The complexes are further stabilized by a hydrogen bond between the Thr-199 backbone NH and the C=O group, and by van der Waals contacts with the hydrophobic pocket (Val-121, Val-143, Leu-198, Trp-209; Figure 7.18). Moreover, in the case of trifluoroacetohydroxamic acid 7.12, a weakly polar C-F Zn2+ interaction can be observed; the fluorine and ionized nitrogen atom of the ligand form a five-membered chelate with zinc (Scolnick et al. 1997).

Copyright © 2004 CRC Press, LLC

This finding prompted Scozzafava and Supuran (2000a) to investigate whether the sulfonylated amino acid hydroxamates synthesized within their MMP inhibitors program (Scozzafava and Supuran 2000b, 2000c; Supuran et al. 2000; Supuran and Scozzafava 2001) could act as efficient CAIs. The study confirmed this possibility and revealed some very potent inhibitors bearing this anchoring groups, such as 7.13

(KI hCA I = 7 nM, KI hCA II = 8 nM, KI hCA IV = 10 nM), 7.14 (KI hCA I = 7 nM, KI hCA II =

8 nM, KI hCA IV = 10 nM) and 7.15 [KI hCA I = 50 nM, KI hCA II = 5 nM, KI hCA IV = 39 nM; Scozzafava and Supuran 2000a]. This new anchoring group was considered

recently in a successful virtual screening experiment for identifying novel inhibitors of CA (Gruneberg et al. 2002).

All these new insights on the coordination patterns of different inhibitors within the active site of CAs contribute to a better understanding of the role of CAs in different organisms and provide a solid ground for the design of drugs with improved selectivity and reduced side effects.

Copyright © 2004 CRC Press, LLC

FIGURE 7.18 Coordination detail of the complex between CA II and acetohydroxamic acid (From 7.10; Scolnick, L.R. et al. 1997. Journal of the American Chemical Society 119,

850–851.); the structure was generated from the pdb file (1AM6) by using the program RasWin for Windows, version 2.7.1.1.

REFERENCES

Abbate, F., Supuran, C.T., Scozzafava, A., Orioli, P., Stubbs, M.T., and Klebe, G. (2002) Nonaromatic sulfonamide group as an ideal anchor for potent human carbonic anhydrase inhibitors: Role of hydrogen-bonding networks in ligand binding and drug design. Journal of Medicinal Chemistry 45, 3583–3587.

Alberti, G., Bertini, I., Luchinat, C., and Scozzafava, A. (1981) A new class of inhibitors capable of binding both the acidic and alkaline forms of carbonic anhydrase. Biochimica et Biophysica Acta 668, 16–26.

Armitage, I.M., Schoot Uiterkamp, A.J.M., Chlebowski, J.F., and Coleman, J.E. (1978) Cad- mium-113 NMR as a probe of the active sites of metalloenzymes. Journal of Magnetic Resonance 29, 375–392.

Baird, T.T., Jr., Waheed, A., Okuyama, T., Sly, W.S., and Fierke, C.A. (1997) Catalysis and inhibition of human carbonic anhydrase IV. Biochemistry 36, 2669–2678.

Banci, L., Bertini, I., Luchinat, C., Donaire, A., Martinez, M.J., and Moratal Mascarell, J.M. (1990) The factors governing the coordination number in the anion derivatives of carbonic anhydrase. Comments on Inorganic Chemistry 9, 245–261.

Banci, L., Bertini, I., Luchinat, C., Monnanni, R., and Moratal Mascarell, J. (1989) Proton NMR spectra of cobalt(II)-substituted carbonic anhydrase isoenzymes. Gazzetta Chimica Italiana 119, 23–29.

Bencini, A., Bertini, I., Canti, G., Gatteschi, D., and Luchinat, C. (1981) The EPR spectra of the inhibitor derivatives of cobalt carbonic anhydrase. Journal of Inorganic Biochemistry 14, 81–93.

Copyright © 2004 CRC Press, LLC

Bertini, I., Borghi, E., Canti, G., and Luchinat, C. (1979a) Investigation of the system cobalt(II) bovine carbonic anhydrase B-trichloroacetaldehyde. Journal of Inorganic Biochemistry 11, 49–56.

Bertini, I., Canti, G., Luchinat, C., and Mani, F. (1981) Hydrogen-1 NMR spectra of the coordination sphere of cobalt-substituted carbonic anhydrase. Journal of the American Chemical Society 103, 7784–7788.

Bertini, I., Canti, G., Luchinat, C., and Romanelli, P. (1980a) Cyanometallates and cobalt(II) bovine carbonic anhydrase B: Five coordination with dicyanoaurate(I). Inorganica Chimica Acta 46, 211–214.

Bertini, I., Canti, G., Luchinat, C., and Scozzafava, A. (1978a) Characterization of cobalt(II) bovine carbonic anhydrase and of its derivatives. Journal of the American Chemical Society 100, 4873–4877.

Bertini, I., Canti, G., Luchinat, C., and Scozzafava, A. (1979b) Characterization of oxovanadium(IV) substituted bovine carbonic anhydrase B. Inorganica Chimica Acta 36, 9–12.

Bertini, I., and Luchinat, C. (1983) Cobalt(II) as a probe of the structure and function of carbonic-anhydrase. Accounts of Chemical Research 16, 272–279.

Bertini, I., and Luchinat, C. (1984) The structure of cobalt(II)-substituted carbonic anhydrase and its implications for the catalytic mechanism of the enzyme. Annals of the New York Academy of Sciences 429, 89–98.

Bertini, I., Luchinat, C., Pierattelli, R., and Vila, A.J. (1992) A multinuclear ligand NMR investigation of cyanide, cyanate, and thiocyanate binding to zinc and cobalt carbonic anhydrase. Inorganic Chemistry 31, 3975–3979.

Bertini, I., Luchinat, C., and Scozzafava, A. (1977a) Interaction of cobalt(II) bovine carbonicanhydrase with aniline, benzoate, and anthranilate. Journal of the American Chemical Society 99, 581–583.

Bertini, I., Luchinat, C., and Scozzafava, A. (1977b) Interactions between alpha-amino acids and cobalt(II) bovine-carbonic anhydrase. Bioinorganic Chemistry 7, 225–231.

Bertini, I., Luchinat, C., and Scozzafava, A. (1978b) Evidence of exchangeable protons in the acidic form of manganese(II) bovine carbonic anhydrase B. FEBS Letters 87, 92–94.

Bertini, I., Luchinat, C., and Scozzafava, A. (1980b) The acid-base equilibriums of carbonic anhydrase. Inorganica Chimica Acta 46, 85–89.

Bertini, I., Luchinat, C., and Scozzafava, A. (1982) Carbonic anhydrase: An insight into the zinc binding site and into the active cavity through metal substitution. Structure and Bonding 48, 45–92.

Brechue, W.F., and Maren, T.H. (1993) Carbonic anhydrase inhibitory activity and ocular pharmacology of organic sulfamates. Journal of Pharmacology and Experimental Therapeutics 264, 670–675.

Briganti, F., Iaconi, V., Mangani, S., Orioli, P., Scozzafava, A., Vernaglione, G., and Supuran, C. T. (1998) A ternary complex of carbonic anhydrase: x-ray crystallographic structure of the adduct of human carbonic anhydrase II with the activator phenylalanine and the inhibitor azide. Inorganica Chimica Acta 275/276, 295–300.

Briganti, F., Mangani, S., Orioli, P., Scozzafava, A., Vernaglione, G., and Supuran, C.T. (1997) Carbonic anhydrase activators: x-ray crystallographic and spectroscopic investigations for the interaction of isozymes I and II with histamine. Biochemistry 36, 10384–10392.

Briganti, F., Mangani, S., Scozzafava, A., Vernaglione, G., and Supuran, C.T. (1999) Carbonic anhydrase catalyzes cyanamide hydration to urea: is it mimicking the physiological reaction? Journal of Biological Inorganic Chemistry 4, 528–536.

Copyright © 2004 CRC Press, LLC