Human Carbonic Anhydrase Inhibitors Research Articles

Catalysis and inhibition of human carbonic anhydrase IV.

Carbonic anhydrase IV (CA IV) is a membrane-bound form of carbonic anhydrase. We have characterized the catalytic activity and inhibition of recombinant human CA IV. CA IV is a high-activity isozyme in CO2 hydration with a pH-independent kcat value (1.1 x 10(6) s(-1)) comparable to that of CA II (8 x 10(5) s(-1)). Furthermore, CA IV is more active in HCO3- dehydration than is CA II as illustrated by the nearly 3-fold increase in kcat/K(M) to 3 x 10(7) M(-1) s(-1). However, the esterase activity of CA IV is decreased 150-fold compared to CA II. The catalytic mechanisms of CA II and CA IV are nearly identical. Both isozymes show similar dependence on buffer concentration with the rate-limiting step at high buffer concentration being intramolecular proton transfer, although the intramolecular proton transfer for CA IV is 3 times faster than that observed with CA II. Additional positive charges in the active site of CA IV stabilize anions as indicated by a decreased pKa for the Zn-bound water compared to CA II (6.2 vs 6.9), as well as lower inhibition constants for a variety of anions, including halides, sulfate, formate, acetate, and bicarbonate. CA IV is also activated by low concentrations (<20 mM) of chloride, bromide, and phosphate. Activation by phosphate suggests that the phospholipid anchor may be acting both as an extracellular tether and as a protein activator. Finally, the affinity of CA IV for sulfonamide inhibitors is decreased up to 65-fold compared to CA II as demonstrated by fluorescence titration. The increased bicarbonate activity and altered pH profile are consistent with the proposed physiological role of CA IV in renal bicarbonate reabsorption.

Benzenesulfonamide-peptide conjugates as probes for secondary binding sites near the active site of carbonic anhydrase

Libraries of N-(4-sulfamoylbenzoyl)oligoglycines terminated with different L-amino acids were screened to identify tight binding inhibitors of human carbonic anhydrase II. Inhibitors terminated with hydrophobic amino acids showed significant enhancements in binding compared to the corresponding glycine derivatives. No enhancements were observed due to polar interactions.

Application of the free energy perturbation method to human carbonic anhydrase II inhibitors.

An analysis of the free energy perturbation (FEP) method is presented that attempts to evaluate the efficacy of the FEP method in the drug discovery process. To accomplish this we have evaluated whether the FEP technique can accurately predict energetic and structural quantities relating to the inhibition of human carbonic anhydrase II (HCAII) by sulfonamides. Three well-characterized (both structurally and energetically) sulfonamide inhibitors of HCAII were examined in this study, 1a, 1b, and 1c. Results from FEP simulations on these compounds indicate that the FEP method can predict energetic trends reasonably well; however, the FEP method was less successful in reproducing detailed structural data. In particular, an expected movement of His-64 when inhibitor 1c was bound did not occur. We conclude that the FEP method can be used to determine relative free energies of binding but cannot be relied upon to reproduce subtle geometric changes.

Differences in anionic inhibition of human carbonic anhydrase I revealed from the structures of iodide and gold cyanide inhibitor complexes.

The crystal structures of two anionic inhibitor complexes of human carbonic anhydrase I (HCAI), namely, HCAI-iodide and HCAI-Au(CN)(2)(-), have been refined by the restrained least-squares method at 2.2 and 2 A nominal resolution, respectively, with good stereochemistry for the final models. The R values have improved from 30.3 to 16.6% for HCAI-iodide and from 28.8 to 17.1% for HCAI-Au(CN)(2)(-). The sites of inhibitor binding as elucidated are totally different in the two structures. The iodide anion replaces the zinc-bound H(2)O/OH(-) ligand and renders the enzyme inactive. This result confirms that the zinc-bound H(2)O/OH(-) is the activity-linked group in carbonic anhydrase enzymes. Au(CN)(2)(-) binds at a different and new site near the zinc ion, without liganding to the metal. The N atom of Au(CN)(2)(-) is within hydrogen-bonding distance of the zinc-bound H(2)O/OH(-) group which shifts by about 0.4 A away from the zinc ion in relation to its position in the native HCAI. It is proposed that the presence of the inhibitor Au(CN)(2)(-) results in a conformational reorientation of the activity-linked group, due to hydrogen-bond formation with the inhibitor, which in turn sterically hinders the binding of the substrate CO(2) molecule in the active site, leading to the inhibition of HCAI enzyme.

4-Substituted thiophene- and furan-2-sulfonamides as topical carbonic anhydrase inhibitors

A series of 4-substituted thiophene- and furan-2-sulfonamides was prepared and was found to possess nanomolar-level potency for inhibition of human carbonic anhydrase II in vitro. Selected examples from this group were further evaluated for their potential to act as topically effective ocular hypotensive agents in the ocular normotensive albino rabbit and the ocular alpha-chymotrypsinized rabbit. Solubility studies in water and pH 7.4 buffer were carried out to estimate the ability of compounds to be formulated in solution. The sensitization potential of key representative structures was determined by in vitro glutathione reactivity studies and guinea pig maximization testing.

Thieno[2,3-b]furan-2-sulfonamides as topical carbonic anhydrase inhibitors.

Novel 5-[(alkylamino)methyl]thieno[2,3-b]furan-2-sulfonamides were prepared and evaluated in vitro for inhibition of human carbonic anhydrase II (CA II) and ex vivo for their ability to inhibit Ca II in the albino rabbit eye after topical administration. Compound 11a was found to lower intraocular pressure (IOP) in both the alpha-CT ocular hypertensive albino rabbit and the normal albino rabbit, but was ineffective at lowering IOP in a hypertensive, pigmented monkey model. Since 11a was highly bound to ocular pigment, a series of less basic analogs was prepared. Examples in this series were both less extensively bound to ocular pigment and more active at reducing IOP in pigmented rabbits after topical dosing. Key examples displayed moderate reactivity toward glutathione.

The effect of bile salts on carbonic anhydrase

Bile salts are potent inhibitors of bovine carbonic anhydrase and human carbonic anhydrase I and human carbonic anhydrase II. To further characterize the binding of bile salts to carbonic anhydrase, rate constants for the CO2 hydration reaction in the presence of deoxycholate, cholate, glycocholate and taurocholate were determined using stop-flow experiments. Values for the Michaelis-Menton dissociation constant for bovine carbonic anhydrase, human carbonic anhydrase I and human carbonic anhydrase II were found to be 5.2, 9.2 and 13.2 mmol/L, respectively. The inhibition constant values for the various bile salts tested ranged from 0.1 to 1 mmol/L for bovine carbonic anhydrase, 1.6 to 2.4 mmol/L for human carbonic anhydrase I and 0.09 to 0.7 mmol/L for human carbonic anhydrase II. Our results suggest a mechanism of noncompetitive carbonic anhydrase inhibition for bile salts. Bile-salt binding to carbonic anhydrases as measured by scanning molecular sieve chromatography resulted in an increase in partition radius, molecular volume and surface area. The partition radius increased from 24 A to 28 A in the presence of 2.5 mmol/L sodium deoxycholate at critical micelle concentration. As determined by sedimentation equilibrium measurements, approximately 1 gm of carbonic anhydrase will bind 0.03 gm of deoxycholate, suggesting three to six binding sites for bile salt on the carbonic anhydrase molecule. The conformational changes and inhibition of carbonic anhydrases resulting from bile-salt binding may be important to the regulation of enzymatic activity in tissues along the enterohepatic circulation; by limiting bicarbonate availability this interaction may also contribute to the metabolic derangements seen in patients with cholestatic liver disease.

MK-507 (L-671,152), a Topically Active Carbonic Anhydrase Inhibitor, Reduces Aqueous Humor Production in Monkeys

An investigation was carried out to determine the mechanism by which MK-507 (L-671,152), a water-soluble inhibitor of human carbonic anhydrase II in vitro, reduces intraocular pressure when applied topically to monkey eyes. Intraocular pressure, tonographically measured outflow facility, and fluorophotometrically determined aqueous humor flow were measured before and after therapy in eight normal cynomolgus monkeys. Fifty microliters of 2% MK-507 was instilled in one eye and diluent in the contralateral eye. Baseline values for intraocular pressure, outflow facility, and aqueous humor flow were similar in the drug-treated and diluent-treated control eyes. After therapy, intraocular pressure was significantly (P less than .05) reduced from 1 to 7 hours (eg, 14.0 +/- 1.0 and 15.9 +/- 0.9 mm Hg [mean +/- SEM], treated and control eyes, respectively, at 3 hours). Outflow facility was not significantly (P greater than .40) changed at 3 hours, and aqueous humor flow measured over 5 hours was significantly (P less than .05) reduced (38%) in treated (0.9 +/- 0.1 microL/min) as compared with control eyes (1.5 +/- 0.1 microL/min). The results suggest that MK-507 reduces intraocular pressure by decreasing aqueous humor production.

The Ocular Hypotensive Effect of the Topical Carbonic Anhydrase Inhibitor L-671,152 in Glaucomatous Monkeys

L-671,152, a new potent water-soluble inhibitor of human carbonic anhydrase II in vitro, was applied topically to cynomolgus monkey eyes in which glaucoma had been produced by argon laser photocoagulation of the trabecular mesh-work. Intraocular pressure was measured at 0 hours, 0.5 hours, and hourly for 8 hours in eight eyes for 2 baseline days, 1 day receiving the vehicle and 5 days receiving therapy with 2% L-671,152 twice a day, after initial single-dose trials of various concentrations. Intraocular pressure was not significantly different comparing baseline and vehicle-treated days. Significant intraocular pressure reductions occurred from 1 to 8 hours after the first dose, and lasted for at least 16 hours after the second dose. The reduction in intraocular pressure became more pronounced from day 1 to day 5 at each time interval. The mean (+/- SEM) maximum reduction in intraocular pressure was 7.8 +/- 2.1 mm Hg on day 1 and 10.1 +/- 2.4 mm Hg on day 5 at 3 hours after administration, comparing the intraocular pressure in drug-treated and vehicle-treated eyes. L-671,152 has a longer duration of action than does previously studied MK-927 in glaucomatous monkeys. It appears to have great clinical potential.

The effect of MK-927, a topical carbonic anhydrase inhibitor, on IOP in glaucomatous monkeys

MK-927 is a water soluble, potent inhibitor of human carbonic anhydrase (CA) II in vitro. Topical administration of MK-927 reduces intraocular pressure (IOP) in rabbits. Elevated IOP was produced in cynomolgus monkey eyes by argon laser photocoagulation of the trabecular meshwork. IOP was measured at 0 hr, 0.5 hr and hourly for 8 hrs in 8 eyes for two baseline days, one day on vehicle and five days of therapy with 2% MK-927 b.i.d., after initial single-dose trials of various concentrations. IOP was not significantly different comparing baseline and vehicle treated days. Significant (p less than 0.05) reductions of IOP occurred for five days lasting at least 8 hrs after each dosing. At 3 hrs after treatment with vehicle the IOP was 31.6 +/- 3.4 (SE) mm Hg. Maximum reduction of IOP occurred at 3 hrs after application of MK-927, the IOP decreasing from day 1 (19.9 +/- 1.0 mm Hg) to day 5 (16.5 +/- 1.6 mm Hg). MK-927 appears to have great clinical potential.

ChemInform Abstract: Lone‐Pair Directionality in Hydrogen Bond Potential Functions for Molecular Mechanics Calculations: The Inhibition of Human Carbonic Anhydrase II by Sulfonamides

ChemInform Abstract: Lone‐Pair Directionality in Hydrogen Bond Potential Functions for Molecular Mechanics Calculations: The Inhibition of Human Carbonic Anhydrase II by Sulfonamides

Lone-pair directionality in hydrogen-bond potential functions for molecular mechanics calculations: the inhibition of human carbonic anhydrase II by sulfonamides

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTLone-pair directionality in hydrogen-bond potential functions for molecular mechanics calculations: the inhibition of human carbonic anhydrase II by sulfonamidesAngelo Vedani and Jack D. DunitzCite this: J. Am. Chem. Soc. 1985, 107, 25, 7653–7658Publication Date (Print):December 1, 1985Publication History Published online1 May 2002Published inissue 1 December 1985https://doi.org/10.1021/ja00311a071RIGHTS & PERMISSIONSArticle Views650Altmetric-Citations147LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (762 KB) Get e-Alerts Get e-Alerts

The inhibition of human carbonic anhydrase II by some organic compounds

The inhibition of human carbonic anhydrase II (carbonate hydro-lyase, EC 4.2.1.1) by tetrazole, 1,2,4-triazole, 2-nitrophenol, and chloral hydrate has been investigated. These inhibitors, together with phenol which has been studied previously (Simonsson, I., Jonsson, B.-H. and Lindskog, S. (1982) Biochem. Biophys. Res. Commun. 108, 1406–1412), can be classified in three groups depending upon the kinetic patterns of inhibition of CO 2 hydration at pH near 9. The first group, represented by tetrazole and 2-nitrophenol, yields predominantly uncompetitive inhibition under these conditions in analogy with simple, inorganic anions. The second group, represented by 1,2,4-triazole and chloral hydrate gives rise to essentially noncompetitive inhibition patterns, whereas phenol, representing the third group, is a competitive inhibitor of CO 2 hydration. These diverse inhibition patterns are discussed in terms of the kinetic mechanism scheme originally proposed by Steiner et al. (Steiner, H., Jonsson, B.-H. and Lindskog, S. (1975) Eur. J. Biochem. 59, 253–259).

Kinetics and inhibition of carbonic anhydrase-catalyzed hydrolysis of 2-hydroxy-5-nitro-α-toluenesulfonic acid sultone: Comparison with reactions of other substrates

The catalytic activities of human red cell carbonic anhydrase (EC 4.2.1.1) isozymes B and C for the hydrolysis of 2-hydroxy-5-nitro-α-toluenesulfonic acid sultone have been compared with their activities towards three other substrates. The substrate specificity (measured as K cat/ K m) for either isozyme decreases in this order: CO 2 > 2-hydroxy-5-nitro-α-toluenesulfonic acid sultone > acetaldehyde > p-nitrophenyl acetate. Unlike CO 2 hydration, enzyme B is slightly more active towards sultone hydrolysis than C. Despite these widely differing activities of both isozymes with regard to different substrates, the inhibition constants for anion and sulfonamide inhibitors are nearly independent of the substrate used. This suggests that the binding sites of these substrates in the enzyme are the same or nearly the same. Earlier studies on 2-hydroxy-5-nitro-α-toluenesulfonic acid sultone from this and other laboratories had underestimated both the intrinsic activity and the susceptibility to anion inhibition of human carbonic anhydrase B. We now find that this was due to the use of acetonistrile as the substrate solvent, which is often contaminated with cyanide, a powerful inhibitor of carbonic anhydrase. The inhibition of human carbonic anhydrase B by several industrial batches of acetonitrile agrees completely with the spectrophotometrically determined cyanide content of these batches.

Inhibition of human carbonic anhydrase II by anions and some ‘neutral’ compounds

Anions inhibit carbonic anhydrase-catalyzed reactions by binding to the zinc ion in the active center. The pH dependence of the inhibition of the esterase activity shows that anions predominantly bind to enzymes having a protonated catalytic group. Formally, anion binding can be described as a competition with OH − for a coordination site on the metal ion [1]. Pocker and Deits [2] recently showed that anions inhibit the CO 2 hydration catalyzed by bovine carbonic anhydrase at high pH values in an uncompetitive fashion, and they presented a kinetic scheme to explain this phenomenon. We have studied the anion inhibition of human carbonic anhydrase II (or C). We have confirmed the ▪ observations of Pocker and Deits that at least certain anions, such as SCN − [3], OCN −, and N − 3 , give rise to uncompetitive inhibition patterns of CO 2 hydration at high pH. This is illustrated for N − 3 in Fig. 1. However, we offer an alternative interpretation to that of Pocker and Deits. We will show that the uncompetitive pattern is a direct consequence of the kinetic mechanism which we have proposed previously [3, 4]. The crucial point is that a strongly anion binding enzyme species, HisEZn 2+OH 2 (see ref. [5]), precedes the rate-limiting step in catalysis. Recently, some organic compounds that bind at or near the metal ion have received attention. One of these is phenol which has been shown to act as a competitive inhibitor of CO 2 hydration and as a mixed noncompetitive inhibitor of HCO − 3 dehydration [6]. We have also studied o-nitrophenol, 1,2,4-triazole and tetrazole. However, these inhibitors behave kinetically like anions and give rise to mixed uncompetitive-noncompetitive patterns in the CO 2 hydration reaction at high pH.

Interaction of the unique competitive inhibitor imidazole with human carbonic anhydrase B

Imidazole was previously found to be unique among the inhibitors of human carbonic anhydrase B (HCAB) in that it binds competitively with the CO2 substrate (Khalifah, R. G. (1971), J. Biol. Chem. 246, 2561). We report here an aromatic ultraviolet difference spectral study of its interaction with HCAB and compare it with a variety of other inhibitors. Imidazole is found to be unique in that: (1) it generates a different spectrum upon binding that is also much supressed in intensity; (2) its affinity for HCAB is maximal at high pH, being abolished upon its protonation and being independent of active-site ionizations. Imidazole differs from CO2 in that it binds competitively with the anionic inhibitor iodide. The unique properties of imidazole binding are consistent with the recently determined crystal structure of its complex with HCAB showing it to bind as a weak and distant fifth ligand of the essential zinc atom, rather than displacing the solvent molecule in the fourth ligand position (Kannan, K.K., Petef, M., Fridborg, K., Cid-Dresdner, H., and Lövgren, S. (1977), FEBS Lett 73, 115).

Carbonic anhydrase: chemistry, physiology, and inhibition.

Carbonic anhydrase: chemistry, physiology, and inhibition.