Carbonic anhydrase inhibitors: X-ray crystal structure of a benzenesulfonamide strong CA II and CA IX inhibitor bearing a pentafluorophenylaminothioureido tail in complex with isozyme II

N-1-(4-Sulfamoylphenyl)-N-4-pentafluorophenyl-thiosemicarbazide was prepared by the reaction of 4-isothiocyanato-benzenesulfonamide with pentafluorophenyl hydrazine, and proved to be an effective inhibitor of several isozymes of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), such as CA I, II, and IX. Against the physiologically relevant isozymes hCA II and hCA IX, the compound showed inhibition constants in the range of 15-19 nM, whereas it was less effective as a hCA I inhibitor (K(I) of 78 nM). The high-resolution X-ray crystal structure of its adduct with hCA II showed the inhibitor to bind within the hydrophobic half of the enzyme active site, making extensive and strong van der Waals contacts with amino acid residues Gln92, Val121, Phe131, Leu198, Thr200, Pro202, in addition to the coordination of the sulfonamide nitrogen to the Zn(II) ion of the active site, and participation of the SO(2)NH(2) group to a network of hydrogen bonds involving residues Thr199 and Glu106. These results are helpful for the design of better CA II or CA IX inhibitors based on the thioureido-benzenesulfonamide motif, with potential applications as anti-glaucoma or anti-cancer drugs.     

Carbonic anhydrase inhibitors: binding of an antiglaucoma glycosyl-sulfanilamide derivative to human isoform II and its consequences for the drug design of enzyme inhibitors incorporating sugar moieties

N-(4-Sulfamoylphenyl)-alpha-d-glucopyranosylamine, a promising topical antiglaucoma agent, is a potent inhibitor of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). The high resolution X-ray crystal structure of its adduct with the target isoform involved in glaucoma, CA II, is reported here. The sugar sulfanilamide derivative binds to the enzyme in a totally new manner as compared to other CA-inhibitor adducts investigated earlier. The sulfonamide anchor was coordinated to the active site metal ion, and the phenylene ring of the inhibitor filled the channel leading to the active site cavity. The glycosyl moiety responsible for the high water solubility of the compound was oriented towards a hydrophilic region of the active site, where no other inhibitors were observed to be bound up to now. A network of seven hydrogen bonds with four water molecules and the amino acid residues Pro201, Pro202 and Gln92 further stabilize the enzyme-inhibitor adduct. Topiramate, another sugar-based CA inhibitor, binds in a completely different manner to CA II as compared to the sulfonamide investigated here. These findings are useful for the design of potent, sugar-derived enzyme inhibitors.     

Carbonic anhydrase inhibitors: X-ray crystallographic structure of the adduct of human isozyme II with the perfluorobenzoyl analogue of methazolamide. Implications for the drug design of fluorinated inhibitors

The X-ray crystal structure for the adduct of human carbonic anhydrase (hCA) II with 4-methyl-5-perfluorophenylcarboximido-delta2-1,3,4-thiadiazoline-2-sulfonamide (PFMZ), a topically acting antiglaucoma sulfonamide, has been resolved at a resolution of 1.8 A. This compound is almost 10 times more effective as a hCA II inhibitor (KI of 1.5 nM) compared to the lead molecule, methazolamide, a clinically used drug (KI of 14 nM). Its binding to the enzyme active site is similar to that of other sulfonamide inhibitors, considering the interactions of the sulfonamide zinc anchoring group and thiadiazoline ring contacts, but differs considerably when the perfluorobenzoylimino fragment of the molecule is analyzed. Indeed, several unprecedented strong hydrogen bonds involving the imino nitrogen, carbonyl oxygen, a fluorine atom in the ortho position of the inhibitor, and two water molecules, as well as Gln 92 of the enzyme active site were seen. A stacking interaction of the perfluorophenyl ring of the inhibitor and the aromatic ring of Phe 131 was also observed for the first time in a CA-sulfonamide adduct. All these findings prove that more potent CA inhibitors incorporating perfluoroaryl/alkyl tails may be designed, with potentially improved antiglaucoma properties, in view of the new types of interactions seen here between the enzyme and the perfluorobenzoylated analogue of methazolamide.     

Carbonic anhydrase inhibitors: X-ray crystallographic structure of the adduct of human isozyme II with a bis-sulfonamide-two heads are better than one?

The X-ray crystal structure for the adduct of human carbonic anhydrase II (hCA II) with 4-(4-sulfamoylphenylcarboxamidoethyl)benzenesulfonamide, a topically acting antiglaucoma sulfonamide has been resolved at a resolution of 1.8 A. Its binding to the enzyme is similar with that of other sulfonamides, considering the interactions of the sulfonamide zinc anchoring group, but differs considerably when the organic part of the inhibitor is analyzed. This part of the inhibitor interacts only within the hydrophobic half of the CA active site, leaving the hydrophilic half able to accomodate several water molecules not present in the uncomplexed enzyme. Furthermore, the second head (sulfonamide moiety) participates in two strong hydrogen bonds with amino acid residues (Gly 132 and Gln 136) situated on the rim of the entrance to the active site cleft. Thus, the answer to the question in the title of this paper is that two heads are better than one, since the two sulfamoyl moieties of the inhibitor allow its proper orientation within the active site, with only one head binding in ionized form to the zinc ion, the organic part lying within the hydrophobic half of the active site, and the terminal, carboxamido containing phenylsulfamoyl head participating in strong hydrogen bonds with amino acid residues located at the entrance of it. All these findings are important for the design of better carboxamido CA inhibitors with applications in clinical medicine.     

Carbonic anhydrase inhibitors: X-ray crystallographic structure of the adduct of human isozyme II with a topically acting antiglaucoma sulfonamide

The X-ray crystal structure for the adduct of human carbonic anhydrase (hCA) II with a topically acting antiglaucoma sulfonamide (the 2-N,N-diethylaminoethylamide of 5-(4-carboxybenzenesulfonamido-1,3,4-thiadiazole-2-sulfonamide), has been resolved at a resolution of 1.6A. This compound is a very potent inhibitor of the physiologically most relevant isozyme hCA II for the secretion of aqueous humor within the eye K(I) of 1.4 nM), and in animal models of glaucoma showed very effective intraocular pressure (IOP) lowering after topical administration. Surprisingly, the inhibitor bound within the enzyme active site is in the sulfonylimido-4H- delta(2)-1,3,4-thiadiazoline tautomeric form. The inhibitor is directly bound to the Zn(II) ion of the enzyme through the deprotonated primary sulfonamide moiety, participating to the classical hydrogen bond network involving residues of the zinc-binding function and Thr 199 and Glu 106. The 1,3,4-thiadiazoline fragment of the inhibitor makes two hydrogen bonds with the active site residue Thr 200, the secondary sulfonamide moiety makes two hydrogen bonds involving a water molecule and the residue Gln 92, whereas the phenyl ring of the inhibitor participates to an edge-to-face interaction with the phenyl ring of Phe 131, the two cycles being almost perfectly perpendicular to each other. The tertiary amine fragment of the carboxamido tail and the carboxamido moiety itself make hydrogen bonds with water molecules present at the rim of the active site entrance and van der Waals contacts with His 4, Trp 5, and Phe 20. All these multiple interactions never evidenced previously in CA-sulfonamide complexes, explain the very high affinity of this inhibitor for the hCA II active site and may allow further optimization of this class of inhibitors.     

Carbonic anhydrase inhibitors: SAR and X-ray crystallographic study for the interaction of sugar sulfamates/sulfamides with isozymes I,II and IV

A series of sugar sulfamate/sulfamide derivatives were prepared and assayed as inhibitors of three carbonic anhydrase (CA) isozymes, hCA I, hCA II and bCA IV. Best inhibitory properties were observed for the clinically used antiepileptic drug topiramate, which is a low nanomolar CA II inhibitor, and possesses good inhibitory properties against the other two isozymes investigated here, similarly with acetazolamide, methazolamide or dichlorophenamide. The X-ray structure of the complex of topiramate with hCA II has been solved and it revealed a very tight association of the inhibitor, with a network of seven strong hydrogen bonds fixing topiramate within the active site, in addition to the Zn(II) coordination through the ionized sulfamate moiety. Structural changes in this series of sugar derivatives led to compounds with diminished CA inhibitory properties as compared to topiramate.     

Carbonic anhydrase inhibitors: X-ray crystallographic structure of the adduct of human isozyme II with the antipsychotic drug sulpiride

The X-ray crystal structure for the adduct of human carbonic anhydrase (hCA) II with sulpiride, a sulfonamide derivative clinically used as antipsychotic drug, has been resolved at a resolution of 1.6 A. This compound is an effective inhibitor of the physiologically most relevant isozyme hCA II (K(i) of 40 nM), being only a moderate or moderate-weak inhibitor of the cytosolic isozyme hCA I (K(i) of 1200 nM) and the membrane-bound isozyme hCA IV (K(i) of 620 nM). Sulpiride shows CA inhibitory properties of the same magnitude as dichlorophenamide, a clinically used antiglaucoma sulfonamide, or valdecoxib, a COX-2 selective inhibitor recently shown to inhibit CA. The binding of sulpiride to the hCA II active site is similar to that of other sulfonamide inhibitors, considering the interactions of the sulfonamide zinc anchoring group, but differs considerably when the organic scaffold of the molecule is analyzed. Indeed, one unprecedented hydrogen bond involving the imino moiety of the carboxamido group of sulpiride and a water molecule was observed, together with a unique stacking interaction of the N-methyl-pyrrolidine ring of the inhibitor and the aromatic ring of Phe 131 of the enzyme active site, which has been observed only recently in another CA-sulfonamide complex.     

Carbonic anhydrase inhibitors: synthesis and inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, IX, and XII with N-hydroxysulfamides--a new zinc-binding function in the design of inhibitors

A small library of N-hydroxysulfamides was synthesized by an original approach in order to investigate whether this zinc-binding function is efficient for the design of inhibitors targeting the cytosolic (hCA I and II) and transmembrane, tumor-associated (hCA IX and XII) isozymes of carbonic anhydrase (CA, EC 4.2.1.1). The parent derivative, N-hydroxysulfamide was a more potent inhibitor as compared to sulfamide or sulfamic acid against all isozymes, with inhibition constants in the range of 473 nM-4.05 microM. Its substituted n-decyl-, n-dodecyl-, benzyl-, and biphenylmethyl-derivatives were less inhibitory against hCA I (K(I)s in the range of 5.8-8.2 microM) but more inhibitory against hCA II (K(I)s in the range of 50.5-473 nM). The same situation was true for the tumor-associated isozymes, with K(I)s in the range of 353-790 nM against hCA IX and 372-874 nM against hCA XII. Some sulfamides/sulfamates possessing similar substitution patterns have also been investigated for the inhibition of these isozymes, being shown that in some particular cases sulfamides are more efficient inhibitors as compared to the corresponding sulfamates. Potent CA inhibitors targeting the cytosolic or tumor-associated CA isozymes can thus be designed from various classes of sulfonamides, sulfamides, or sulfamates and their derivatives, considering the extensive interactions in which the inhibitor and the enzyme active site are engaged, based on recent X-ray crystallographic data.     

Carbonic Anhydrase Inhibitors: X-ray Crystallographic Structure of the Adduct of Human Isozyme II with the Perfluorobenzoyl Analogue of Methazolamide. Implications for the Drug Design of Fluorinated Inhibitors

The X-ray crystal structure for the adduct of human carbonic anhydrase (hCA) II with 4-methyl-5-perfluorophenylcarboximido-δ2-1,3,4-thiadiazoline-2-sulfonamide (PFMZ), a topically acting antiglaucoma sulfonamide, has been resolved at a resolution of 1.8?Å. This compound is almost 10 times more effective as a hCA II inhibitor (KI of 1.5?nM) compared to the lead molecule, methazolamide, a clinically used drug (KI of 14?nM). Its binding to the enzyme active site is similar to that of other sulfonamide inhibitors, considering the interactions of the sulfonamide zinc anchoring group and thiadiazoline ring contacts, but differs considerably when the perfluorobenzoylimino fragment of the molecule is analyzed. Indeed, several unprecedented strong hydrogen bonds involving the imino nitrogen, carbonyl oxygen, a fluorine atom in the ortho position of the inhibitor, and two water molecules, as well as Gln 92 of the enzyme active site were seen. A stacking interaction of the perfluorophenyl ring of the inhibitor and the aromatic ring of Phe 131 was also observed for the first time in a CA–sulfonamide adduct. All these findings prove that more potent CA inhibitors incorporating perfluoroaryl/alkyl tails may be designed, with potentially improved antiglaucoma properties, in view of the new types of interactions seen here between the enzyme and the perfluorobenzoylated analogue of methazolamide.     

Carbonic anhydrase inhibitors: X-ray crystallographic structure of the adduct of human isozyme II with EMATE, a dual inhibitor of carbonic anhydrases and steroid sulfatase

The X-ray crystal structure for the adduct of human carbonic anhydrase II (hCA II) with estrone-3-O-sulfamate (EMATE), an antiendocrine agent showing both CA and estrone sulfatase inhibitory properties, has been resolved at a resolution of 1.5A. Its binding to the enzyme is similar to that of other sulfamates/sulfonamides, considering the interactions of the zinc anchoring group, but differs considerably when the steroidal scaffold of the inhibitor is analyzed. This part of the inhibitor interacts only within the hydrophobic half of the CA active site, interacting with residues Val 121, Phe 131, Val 135 and Pro 202, and leaving the hydrophilic half able to accommodate several water molecules not present in the uncomplexed enzyme. In addition, a very short bond of 1.78A between the zinc ion and the coordinated nitrogen atom of the sulfamate moiety is observed, which may explain the high affinity of this inhibitor for hCA II (K(i) of 10nM).     

Hydroxyurea is a carbonic anhydrase inhibitor

The interaction of hydroxyurea with the cytosolic isozymes of carbonic anhydrase (CA), hCA I and hCA II has been investigated by means of kinetic and spectroscopic techniques. Hydroxyurea acts as a weak, non-competitive inhibitor of both isozymes, for the 4-nitrophenyl acetate esterase activity, with inhibition constants around 0.1 mM for both isozymes. The spectrum of the adduct of hydroxyurea with Co(II)-hCA II is similar to the spectra of tetrahedral adducts (such as those with sulfamide, acetazolamide or cyanamide), proving a direct interaction of the inhibitor molecule with the metal center of the enzyme, whose geometry remains tetrahedral. Based on the X-ray crystal structure of the adducts of hCA II with ureate and hydroxamate inhibitors, the hypothetical binding of hydroxyurea is proposed to be achieved in deprotonated state, with the nitrogen atom coordinated to Zn(II), and the OH group of the inhibitor making a hydrogen bond with Thr 199. This binding may be exploited for the design of both CA as well as matrix metalloproteinase (MMP) inhibitors, since hydroxyurea is the simplest compound incorporating a hydroxamate functionality in its molecule. Indeed, such inhibitors of the sulfonylated amino acid hydroxamate type have been generated, with potencies in the low nanomolar range for both type of enzymes, CAs and MMPs.     

Carbonic anhydrase activators: the first X-ray crystallographic study of an adduct of isoform I

The X-ray crystallographic structure for the adduct of an activator with human carbonic anhydrase isozyme I (hCA I) is reported. L-Histidine binds deep within the enzyme active site, participating in a network of hydrogen bonds involving its carboxylate moiety and the zinc-bound water molecule, as well as the imidazole of His200, being in van der Waals contacts with Thr199, His200, His64, and His67. This binding is very different from that to the other major cytosolic isozyme hCA II.     

Carbonic anhydrase inhibitors. Zonisamide is an effective inhibitor of the cytosolic isozyme II and mitochondrial isozyme V: solution and X-ray crystallographic studies

The antiepileptic drug zonisamide was considered to act as a weak inhibitor of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) (with a K(I) of 4.3 microM against the cytosolic isozyme II). Here we prove that this is not true. Indeed, testing zonisamide in the classical assay conditions of the CO2 hydrase activity of hCA II, with incubation times of enzyme and inhibitor solution of 15 min, a K(I) of 10.3 microM has been obtained. However, when the incubation between enzyme and inhibitor was prolonged to 1 h, the obtained K(I) was of 35.2 nM, of the same order of magnitude as that of the clinically used sulfonamides/sulfamates acetazolamide, methazolamide, ethoxzolamide and topiramate (K(I)s in the range of 5.4-15.4 nM). The inhibition of the human mitochondrial isozyme hCA V with these compounds has been also tested by means of a dansylamide competition binding assay, which showed zonisamide and topiramate to be effective inhibitors, with K(I)s in the range of 20.6-25.4 nM. The X-ray crystal structure of the adduct of hCA II with zonisamide has also been solved at a resolution of 1.70 A, showing that the sulfonamide moiety participates in the classical interactions with the Zn(II) ion and the residues Thr199 and Glu106, whereas the benzisoxazole ring is oriented toward the hydrophobic half of the active site, establishing a large number of strong van der Waals interactions (<4.5 A) with residues Gln92, Val121, Phe131, Leu198, Thr200, Pro202.     

Carbonic anhydrase inhibitors: Valdecoxib binds to a different active site region of the human isoform II as compared to the structurally related cyclooxygenase II "selective" inhibitor celecoxib

The high resolution X-ray crystal structure of the adduct of human carbonic anhydrase (CA, EC 4.2.1.1) isoform II (hCA II) with the clinically used painkiller valdecoxib, acting as a potent CA II and cyclooxygenase-2 (COX-2) inhibitor, is reported. The ionized sulfonamide moiety of valdecoxib is coordinated to the catalytic Zn(II) ion with a tetrahedral geometry. The phenyl-isoxazole moiety of the inhibitor fills the active site channel and interacts with the side chains of Gln92, Val121, Leu198, Thr200, and Pro202. Its 3-phenyl group is located into a hydrophobic pocket, simultaneously establishing van der Waals interactions with the aliphatic side chain of various hydrophobic residues (Val135, Ile91, Val121, Leu198, and Leu141) and a strong offset face-to-face stacking interaction with the aromatic ring of Phe131 (the chi1 angle of which is rotated about 90 degrees with respect to what was observed in the structure of the native enzyme and those of other sulfonamide complexes). Celecoxib, a structurally related COX-2 inhibitor for which the X-ray crystal structure was reported earlier, binds in a completely different manner to hCA II as compared to valdecoxib. Celecoxib completely fills the entire CA II active site, with its trifluoromethyl group in the hydrophobic part of the active site and the p-tolyl moiety in the hydrophilic one, not establishing any interaction with Phe131. In contrast to celecoxib, valdecoxib was rotated about 90 degrees around the chemical bond connecting the benzensulfonamide and the substituted isoxazole ring allowing for these multiple favorable interactions. These different binding modes allow for the further drug design of various CA inhibitors belonging to the benzenesulfonamide class.     

Carbonic anhydrase inhibitors: Crystallographic and solution binding studies for the interaction of a boron-containing aromatic sulfamide with mammalian isoforms I–XV

We investigated the inhibition of carbonic anhydrase (CA, EC 4.2.1.1) isoforms I–XV with 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylsulfamide and other simple or sugar sulfamides, a class of less investigated CA inhibitors (CAIs). The crystal structure of the adduct of hCA II with the boron-substituted sulfamide shows the organic scaffold of this compound bound in the hydrophilic half of the active site where it makes a large number of van der Waals contacts with Ile91, Gln92, Val121, Phe131, Leu198, and Thr200. The data here reported provide further insights into sulfamide binding mechanism confirming that this zinc-binding group could be usefully exploited for obtaining new potent and selective CAIs.     

New thiopyrimidine-benzenesulfonamide conjugates as selective carbonic anhydrase II inhibitors: synthesis,in vitro biological evaluation,and molecular docking studies

In the present work, a new series of thiopyrimidine-benzenesulfonamide conjugates was designed, synthesized and tested as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. Our design strategy was based on the molecular hybridization of the benzenesulfonamide moiety as a zinc binding group (ZBG), an alkylated thiopyrimidine moiety as a spacer and (un)substituted phenyl moieties with various electronic and hydrophobic environments as a tail. The designed and synthesized compounds were evaluated against four human (h) CA isoforms hCA I, hCA II, hCA IX and hCA XII. Series 6 showed promising activity and selectivity toward the cytosolic isoforms hCA I and hCA II versus the membrane bound isoforms hCA IX and hCA XII. Compounds 6e and 6f showed K_i of 0.04 µM against hCA II with a selectivity of 15.8- to 980-fold towards hCA II over hCA I, hCA IX, hCA XII isoforms. Molecular docking in the hCA II active site attributed the promising inhibitory activity of series 6 to the interaction of their sulfonamide moiety with the active site Zn²⁺ ion as well as its hydrogen bonding with the key amino acids Thr199 and Thr200. Through hydrophobic interaction, the benzenesulfonamide and the thiopyrimidine moieties interact with the hydrophobic side chains of the amino acids Val121/Leu198 and Ile91/Phe131, respectively. These results indicated that the designed and synthesized series is an interesting scaffold that can be further optimized for the development of selective antiglaucoma drugs.     

Acipimox inhibits human carbonic anhydrases

Acipimox, a nicotinic acid derivative in clinical use for the treatment of hyperlipidaemia, incorporates a free carboxylic acid and an N-oxide moiety, functionalities known to interact with the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) and inhibit its activity. Herein we report that acipimox acts as a low micromolar CA inhibitor (CAI) against most human (h) isoforms possessing catalytic activity, hCA I – XIV. By using computational techniques (docking and molecular dynamics simulations), we propose that acipimox coordinates through its carboxylate group to the zinc ion from the enzyme active site cavity, whereas the N-oxide group is hydrogen-bonded to the proton shuttle His residue in some isoforms (hCA I) or to active site Thr or Gln residues in other isoforms (hCA II, III, IV, VII, etc). As some CA isoforms are involved in lipogenesis, these data may be useful for the design of more effective CAIs with antiobesity activity.     

Carbonic anhydrase catalyzes cyanamide hydration to urea: is it mimicking the physiological reaction?

 The interaction of human carbonic anhydrase (hCA) isozymes I and II with cyanamide, a linear molecule isoelectronic with the main physiological substrate of the enzyme, CO₂, was investigated through spectroscopic, kinetic, and X-ray crystallographic studies. We show here that cyanamide is hydrated to urea in the presence of CAs, and that it also acts as a weak non-competitive inhibitor (K _I=61±3 mM and 238±9 mM for hCA II and hCA I, respectively) towards the esterasic activity of these enzymes, as tested with 4-nitrophenyl acetate. Changes in the spectrum of the Co(II)-hCA II derivative observed in the presence of cyanamide suggest that it likely binds the metal ion within the CA active site, adding to the coordination sphere, not substituting the metal-bound solvent molecule. It thereafter undergoes a nucleophilic attack from the metal-bound hydroxide ion, forming urea which remains bound to the metal, as observed in the X-ray crystal structure of hCA II soaked in cyanamide solutions for several hours. The urea molecule is directly coordinated to the active site Zn(II) ion through a protonated nitrogen atom. Several hydrogen bonds involving active site residues Thr199 and Thr200 as well as three water molecules (Wat99, Wat122, and Wat123) further stabilize the urea-hCA II adduct. Kinetic studies in solution further proved that urea acts as a tight binding inhibitor of the two isozymes hCA I and hCA II, with very slow binding kinetics (k _on=2.5×10^–5s^–1M^–1). A mechanism to explain the hydration process of cyanamide by CAs, as well as the tight binding of urea in the active site, is also proposed based on the hypothesis that urea is deprotonated when bound to the enzyme. Cyanamide is thus the first true suicide substrate of this enzyme for which binding has been documented by means of X-ray crystallographic and spectroscopic studies. Received: 26 February 1999 / Accepted: 25 May 1999     

Phosph(on)ate as a zinc-binding group in metalloenzyme inhibitors: X-ray crystal structure of the antiviral drug foscarnet complexed to human carbonic anhydrase I

Foscarnet (phosphonoformate trisodium salt), an antiviral used for the treatment of HIV and herpes virus infections, also acts as an activator or inhibitor of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). Interaction of the drug with 11 CA isozymes has been investigated kinetically, and the X-ray structure of its adduct with isoform I (hCA I-foscarnet complex) has been resolved. The first CA inhibitor possessing a phosphonate zinc-binding group is thus evidenced, together with the factors governing recognition of such small molecules by a metalloenzyme active site. Foscarnet is also a clear-cut example of modulator of an enzyme activity which can act either as an activator or inhibitor of a CA isozyme.     

Carbonic anhydrase inhibitors: E7070, a sulfonamide anticancer agent, potently inhibits cytosolic isozymes I and II, and transmembrane, tumor-associated isozyme IX

E7070 [N-(3-chloro-7-indolyl)-1,4-benzenedisulfonamide] is an anticancer drug candidate under clinical development for the treatment of several types of cancers. We prove here that this compound also acts as a potent carbonic anhydrase (CA) inhibitor. Similarly to the clinically used drugs acetazolamide, methazolamide and topiramate, E7070 showed inhibition constants in the range of 15-31nM against isozymes I, II and IX, being slightly less effective as a CA IV inhibitor (K(i) of 65nM). The X-ray crystal structure of the adduct of hCA II with E7070 revealed unprecedented interactions between the inhibitor and the active site, with three different conformations of the chloroindole fragment of the inhibitor interacting with different amino acid residues/water molecules of the enzyme. A superimposition of these conformations with those of other sulfonamide/sulfamate CA inhibitors indicated that similar regions of the hCA II active site could be involved in the interaction with inhibitors.