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 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: 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.     

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. 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: 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 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: 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: 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. Interaction of 2-(hydrazinocarbonyl)-3-phenyl-1H-indole-5-sulfonamide with 12 mammalian isoforms: kinetic and X-ray crystallographic studies

2-(Hydrazinocarbonyl)-3-phenyl-1H-indole-5-sulfonamide was tested for its interaction with 12 carbonic anhydrase (CA, EC 4.2.1.1) isoforms in the search of compounds with good inhibitory activity against isozymes with medicinal chemistry applications, such as CA I, II, VA, VB, VII, IX, and XII among others. This sulfonamide is a potent inhibitor of CA I and II (K(I)s of 7.2-7.5 nM), a medium potency inhibitor of CA VII, IX, XII, and XIV, and a weak inhibitor against the other ubiquitous isoforms, making it thus a very interesting clinical candidate for situations in which a strong inhibition of CA I and II is needed. The crystal structure of the hCA II adduct of this sulfonamide revealed many favorable interactions between the inhibitor and the enzyme which explain its strong low nanomolar affinity for this isoform but may also be exploited for the design of effective inhibitors incorporating bicyclic moieties.     

Kinetic and X-ray crystallographic investigations on carbonic anhydrase isoforms I,II, IX and XII of a thioureido analog of SLC-0111

SLC-0111 (4-(4-fluorophenylureido)-benzenesulfonamide) is the first carbonic anhydrase (CA, EC 4.2.1.1) IX inhibitor to reach phase I clinical trials as an antitumor/antimetastatic agent. Here we report a kinetic and X-ray crystallographic study of a congener of SLC-0111 which incorporates a thioureido instead of ureido linker between the two aromatic rings as inhibitor of four physiologically relevant CA isoforms. Similar to SLC-0111, the thioureido derivative was a weak hCA I and II inhibitor and a potent one against hCA IX and XII. X-ray crystallography of its adduct with hCA II and comparison of the structure with that of other five hCA II—sulfonamide adducts belonging to the SLC-0111 series, afforded us to understand the particular inhibition profile of the new sulfonamide. Similar to SLC-0111, the thioureido sulfonamide primarily interacted with the hydrophobic side of the hCA II active site, with the tail participating in van der Waals interactions with Phe131 and Pro202, in addition to the coordination of the deprotonated sulfonamide to the active site metal ion. On the contrary, the tail of other sulfonamides belonging to the SLC-0111 series (2-isopropyl-phenyl; 3-nitrophenyl) were orientated towards the hydrophilic half of the active site, which was correlated with orders of magnitude better inhibitory activity against hCA II, and a loss of selectivity for the inhibition of the tumor-associated CAs.     

Carbonic anhydrase inhibitors. Inhibition of red blood cell ostrich (Struthio camelus) carbonic anhydrase with a series of aromatic and heterocyclic sulfonamides

The purification of red blood cell carbonic anhydrase (CA, EC 4.2.1.1) from ostrich (scCA) blood is reported, as well as an inhibition study of this enzyme with a series of aromatic and heterocylic sulfonamides. The ostrich enzyme showed a high activity, comparable to that of the human isozyme II, with kcat, of 1.2 x 10(6) s(-1) and kcat/KM of 1.8 x 10(7) M(-1)s(-1), and an inhibition profile quite different from that of the human red blood cell cytosolic isozymes hCA I and II. scCA has generally a lower affinity for sulfonamide inhibitors as compared to hCA I and II. The only sulfonamide which behaved as a very potent inhibitor of this enzyme was ethoxzolamide (KI = 3.9 nM) whereas acetazolamide and sulfanilamide behaved as weaker inhibitors (inhibition constants in the range 303-570 nM). Several other aromatic and heterocyclic sulfonamides, mostly derivatives of sulfanilamide, homosulfanilamide, 4-aminoethylbenzenesulfonamide or 5-amino-1,3,4-thiadiazole-2-sulfonamide, showed good affinities for the ostrich enzyme, with KI values in the range 25-72 nM.     

Crystal structure of the human carbonic anhydrase II adduct with 1-(4-sulfamoylphenyl-ethyl)-2,4,6-triphenylpyridinium perchlorate,a membrane-impermeant,isoform selective inhibitor

Pyridinium containing sulfonamides have been largely investigated as carbonic anhydrase inhibitors (CAIs), showing interesting selectivity features. Nevertheless, only few structural studies are so far available on adducts that these compounds form with diverse CA isoforms. In this paper, we report the structural characterization of the adduct that a triphenylpyridinium derivative forms with hCA II, showing that the substitution of the pyridinium ring plays a key role in determining the conformation of the inhibitor in the active site and consequently the binding affinity to the enzyme. These findings open new perspectives on the basic structural requirements for designing sulfonamide CAIs with a selective inhibition profile.     

5-Substituted-(1,2,3-triazol-4-yl)thiophene-2-sulfonamides strongly inhibit human carbonic anhydrases I,II, IX and XII: Solution and X-ray crystallographic studies

We report here a series of 2-thiophene-sulfonamides incorporating 1-substituted aryl-1,2,3-triazolyl moieties, prepared by click chemistry from 5-ethynylthiophene-2-sulfonamide and substituted aryl azides. The new sulfonamides were investigated as inhibitors of the zinc metalloenzyme CA (EC 4.2.1.1), and more specifically against the human (h) cytosolic isoforms hCA I and II and the transmembrane, tumor-associated ones hCA IX and XII: The new compounds were medium–weak hCA I inhibitors (K_Is in the range of 224–7544 nM), but were compactly, highly effective, low nanomolar hCA II inhibitors (K_Is of 2.2–7.7 nM). The tumor-associated hCA IX was inhibited with K_Is ranging between 5.4 and 811 nM, whereas hCA XII with inhibition constants in the range of 3.4–239 nM. The X-ray crystal structure of the adducts of two such compounds bound to hCA II (one incorporating 1-naphthyl, the other one 3-cyanophenyl moieties) evidenced the reasons of the high affinity for hCA II. Highly favorable, predominantly hydrophobic interactions between the sulfonamide scaffold and the hCA II active site were responsible for the binding, in addition to the coordination of the sulfamoyl moiety to the zinc ion. The tails of the two inhibitors adopted very diverse orientations when bound to the active site, with the naphthyltriazolyl moiety orientated towards the hydrophobic half of the active site, and the 3-cyanophenyl one pointing towards the hydrophilic half. These data may be used for the structure-based drug design of even more effective hCA II inhibitors, with potential use as antiglaucoma agents or as diuretics.     

Carbonic anhydrase inhibitors. Inhibition of the transmembrane isozyme XII with sulfonamides—a new target for the design of antitumor and antiglaucoma drugs?

The inhibition of a newly cloned human carbonic anhydrase (CA, EC 4.2.1.1), isozyme XII (hCA XII), has been investigated with a series of sulfonamides, including some clinically used derivatives (acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, brinzolamide, benzolamide, and sulpiride, or indisulam, a compound in clinical development as antitumor drug), as well as the sulfamate antiepileptic drug topiramate. Some simple amino-/hydrazine-/hydroxy-substituted aromatic/heterocyclic sulfonamides have also been included in the study. All types of activity have been detected, with several medium potency inhibitors (K(I)s in the range of 34-220 nM), whereas ethoxzolamide and several halogenated sulfanilamides showed stronger potency, with K(I)s in the range of 11-22 nM. The antiglaucoma sulfonamides used clinically, except dichlorophenamide, which is a moderate inhibitor (K(I) of 50 nM), as well as topiramate, indisulam, and sulpiride behave as very potent hCA XII inhibitors, with K(I)s in the range of 3.0-5.7 nM. Several subnanomolar inhibitors (K(I)s in the range of 0.30-0.85 nM) have also been detected. Compounds with excellent selectivity against hCA XII over hCA II have been found, showing selectivity ratios in the range of 177.7-566.7. Apparently, hCA XII is a target of the antiglaucoma sulfonamides, and potent hCA XII inhibitors may be developed/used for the management of hypoxic tumors, together with inhibitors of the other tumor-associated isozyme, CA IX.     

The human carbonic anhydrase isoenzymes I and II (hCA I and II) inhibition effects of trimethoxyindane derivatives

Carbonic anhydrases (CAs, EC 4.2.1.1) had six genetically distinct families described to date in various organisms. There are 16 known CA isoforms in humans. Human CA isoenzymes I and II (hCA I and hCA II) are ubiquitous cytosolic isoforms. Acetylcholine esterase (AChE. EC 3.1.1.7) is a hydrolase that hydrolyzes the neurotransmitter acetylcholine relaying the signal from the nerve. In this study, some trimethoxyindane derivatives were investigated as inhibitors against the cytosolic hCA I and II isoenzymes, and AChE enzyme. Both hCA isozymes were inhibited by trimethoxyindane derivatives in the low nanomolar range. These compounds were good hCA I inhibitors (Kis in the range of 1.66–4.14?nM) and hCA II inhibitors (Kis of 1.37–3.12?nM) and perfect AChE inhibitors (Kis in the range of 1.87–7.53?nM) compared to acetazolamide as CA inhibitor (Ki: 6.76?nM for hCA I and Ki: 5.85?nM for hCA II) and Tacrine as AChE inhibitor (Ki: 7.64?nM).     

Carbonic anhydrase inhibitors. Interaction of 2-N,N-dimethylamino-1,3,4-thiadiazole-5-methanesulfonamide with 12 mammalian isoforms: kinetic and X-ray crystallographic studies

2-N,N-Dimethylamino-1,3,4-thiadiazole-5-methanesulfonamide was tested for its interaction with the 12 catalytically active mammalian carbonic anhydrase (CA, EC 4.2.1.1) isozymes, CA I-XIV. The compound is a potent inhibitor of CA IV, VII, IX, XII, and XIII (K(I)s of 0.61-39 nM), a medium potency inhibitor of CA II and VA (K(I)s of 121-438 nM), and a weak inhibitor against the other isoforms (CA III, VB, VI, and XIV), making it a very interesting candidate for situations in which a strong/selective inhibition of certain isozymes is needed. The crystal structure of the hCA II adduct of this sulfonamide revealed interesting interactions between the inhibitor and the enzyme which are quite different from those observed in the adducts of CA II with the structurally related aliphatic derivatives zonisamide, 2-amino-1,3,4-thiadiazolyl-5-difluoromethanesulfonamide, and 2-dimethylamino-5-[sulfonamido-(aminomethyl)]-1,3,4-thiadiazole reported earlier.     

Carbonic anhydrase inhibitors. Inhibition of the membrane-bound human and bovine isozymes IV with sulfonamides

An inhibition study of the human and bovine membrane-associated isozymes of carbonic anhydrase (CA, EC 4.2.1.1), hCA IV and bCA IV, with a series of sulfonamides and sulfamates, some of which are widely clinically used, such as acetazolamide, methazolamide, ethoxzolamide, topiramate, dorzolamide, dichlorophenamide, celecoxib, and valdecoxib among others, is reported. In contrast to bCA IV, which is generally strongly inhibited by most of these derivatives, hCA IV has a rather different inhibition profile. Several of these compounds such as acetazolamide, ethoxzolamide, and bromosulfanilamide are potent hCA IV inhibitors (K(i)'s of 74-93 nM), others, such as celecoxib and some halogenated sulfanilamides are medium potency inhibitors (K(i)'s of 450-880 nM) whereas most of them are weak hCA IV inhibitors (methazolamide: 6.2 microM; dorzolamide 8.5 microM; topiramate 4.9 microM; dichlorophenamide: 15.3 microM). The hCA IV/bCA IV inhibition ratios for all the investigated compounds ranged between 1.05 (for acetazolamide) and 198.37 (for dorzolamide). Based on these results, we doubt that hCA IV is indeed one of the main contributors to the intraocular pressure (IOP) lowering effects of sulfonamide CA inhibitors, in addition to hCA II, as hypothesized earlier by Maren et al. (Mol. Pharmacol.1993, 44, 901-906). Indeed, both the very good hCA IV inhibitors (acetazolamide and ethoxzolamide) as well as the quite weak hCA IV inhibitors (methazolamide, dorzolamide, or dichlorophanamide) are effective in lowering IOP when administered either systemically or topically. The membrane-associated isozyme which probably is critical for aqueous humor secretion is hCA XII and not hCA IV.     

An inhibitor-like binding mode of a carbonic anhydrase activator within the active site of isoform II

The 2,4,6-trimethylpyridinium derivative of histamine is an effective activator of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). However, unlike other CA activators, which bind at the entrance of the active site cavity, an X-ray crystal structure of hCA II in complex with the 1-[2-(1H-imidazol-4-yl)-ethyl]-2,4,6-trimethylpyridinium salt evidenced a binding mode never observed before either for activators or inhibitors of this enzyme, with the 2,4,6-trimethylpyridinium ring pointing towards the metal ion deep within the enzyme cavity, and several strong hydrophobic interactions stabilizing the adduct. Indeed, incubation of the activator with the enzyme for several days leads to potent inhibitory effects. This is the first example of a CA activator which after a longer contact with the enzyme behaves as an inhibitor.     

Carbonic anhydrase inhibitors. Interaction of the antiepileptic drug sulthiame with twelve mammalian isoforms: kinetic and X-ray crystallographic studies

Sulthiame, a clinically used antiepileptic, was investigated for its interaction with 12 catalytically active mammalian carbonic anhydrase (CA, EC 4.2.1.1) isoforms. The drug is a potent inhibitor of CA II, VII, IX, and XII (K(I)s of 6-56 nM), and a medium potency inhibitor against CA IV, VA, VB, and VI (K(I)s of 81-134 nM). The high resolution crystal structure of the hCA II-sulthiame adduct revealed a large number of favorable interactions between the drug and the enzyme which explain its strong low nanomolar affinity for this isoform and may also be exploited for the design of effective inhibitors incorporating sultam moieties.