The effect of caffeic acid phenethyl ester (CAPE) on metabolic enzymes including acetylcholinesterase,butyrylcholinesterase, glutathione S-transferase,lactoperoxidase, and carbonic anhydrase isoenzymes I,II, IX,and XII

Caffeic acid phenethyl ester (CAPE) is an active component of honeybee propolis extracts. Carbonic anhydrases (CAs, EC 4.2.1.1) are widespread and intensively studied metalloenzymes present in higher vertebrates including humans as many diverse isoforms. Acetylcholinesterase (AChE) is responsible for acetyl choline (ACh) hydrolysis and plays a fundamental role in nerve impulse transmission by terminating the action of the ACh neurotransmitter at cholinergic synapses and neuromuscular junctions. Butyrylcholinesterase (BChE) is another enzyme abundantly present in the liver and released into blood in a soluble form. Lactoperoxidase (LPO) is an enzyme involved in fighting pathogenic microorganisms whereas glutathione S-transferases (GSTs) are dimeric proteins present both in prokaryotic and eukaryotic organisms and involved in cellular detoxification mechanisms. In the present study, the inhibition effect of CAPE on human carbonic anhydrase (hCA) isoforms I, II, IX, and XII, AChE, BChE, LPO, and GST was evaluated. CAPE inhibited these enzymes with K_is in the range between micromolar to picomolar. The best inhibitory effect was observed against AChE and BChE.     

Synthesis and bioactivity studies on new 4-(3-(4-Substitutedphenyl)-3a,4-dihydro-3H-indeno[1,2-c]pyrazol-2-yl) benzenesulfonamides

A series of new 4-(3-(4-substitutedphenyl)-3a,4-dihydro-3H-indeno[1,2-c]pyrazol-2-yl) benzenesulfonamides (7–12) was synthesized starting from 2-(4-substitutedbenzylidene)-2,3-dihydro-1H-inden-1-one (1–6) and 4-hydrazinobenzenesulfonamide. The substituted benzaldehydes from which the key intermediate was prepared by introducing 2- or 4-substituents such as fluorine, hydroxy, methoxy, or the 3,4,5-trimethoxy moieties. The compounds were tested for their cytotoxicity, tumor-specificity and potential as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. The 3,4,5-trimethoxy and the 4-hydroxy derivatives showed interesting cytotoxic activities, which may be crucial for further anti-tumor activity studies, whereas some of these sulfonamides strongly inhibited both human (h) cytosolic isoforms hCA I and II.     

Carbonic Anhydrase Inhibitors. Synthesis of Topically Effective Intraocular Pressure Lowering Agents Derived from 5-(omega-Amino- alkylcarboxamido)-1,3,4-Thia- diazole-2-Sulfonamide

Reaction of the acyl chlorides of phthalimido-glycine or phthalimido-beta-alanine with 5-amino-1,3,4-thiadiazole-2-sulfonamide afforded after hydrazinolysis and deprotection of the phthalimido group the corresponding 5-(omega-aminoalkylcarboxamido)-1,3,4-thiadiazole-2-sulfonamides. Reaction of 5-(beta-aminoethylcarboxamido)-1,3,4-thiadiazole-2-sulfonamide with sulfonyl halides or acyl halides afforded a series of compounds possessing beta-alkyl/arylsulfonyl/carbonylamidoethylcarboxamido moieties in the 5 position of the thiadiazole-2-sulfonamide ring. The new derivatives were efficient inhibitors of three carbonic anhydrase (CA) isozymes, CA I, II (cytosolic forms) and IV (membrane-bound form), but especially against CA II and CA IV (in nanomolar range), the two isozymes known to play an important role in aqueous humor secretion within the ciliary processes of the eye. Some of the synthesized inhibitors possessed good water solubility (as hydrochlorides or sodium salts) and were applied as 2% solutions directly into the eye of normotensive or glaucomatous albino rabbits. Very strong intraocular pressure (IOP) lowering was observed for many of them for prolonged periods of 1-2 h, and the active drug was detected in eye tissues and fluids indicating that the antiglaucoma effect is due to CA inhibition within the eye.     

Carbonic anhydrase inhibitors: synthesis and inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, and IX with sulfonamides derived from 4-isothiocyanato-benzolamide

A series of sulfonamides incorporating 4-thioureido-benzolamide moieties have been prepared from aminobenzolamide and thiophosgene followed by the reaction of the thiocyanato intermediate with aliphatic/aromatic amines or hydrazines. The new derivatives have been investigated as inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), and more precisely of the cytosolic isozymes hCA I and II, as well as the tumor-associated isozyme hCA IX (all of human origin). The new compounds showed excellent inhibitory properties against all three isozymes with inhibition constants in the range of 0.6-62 nM against hCA I, 0.5-1.7 nM against hCA II and 3.2-23 nM against hCA IX, respectively. These derivatives are interesting candidates for the development of novel therapies targeting hypoxic tumors.     

Carbonic anhydrase inhibitors. Interaction of isozymes I, II, IV, V, and IX with phosphates, carbamoyl phosphate, and the phosphonate antiviral drug foscarnet

A detailed inhibition study of five carbonic anhydrase (CA, EC 4.2.1.1) isozymes with inorganic phosphates, carbamoyl phosphate, the antiviral phosphonate foscarnet as well as formate is reported. The cytosolic isozyme hCA I was weakly inhibited by neutral phosphate, strongly inhibited by carbamoyl phosphate (K(I) of 9.4 microM), and activated by hydrogen- and dihydrogenphosphate, foscarnet and formate (best activator foscarnet, K(A)=12 microM). The cytosolic isozyme hCA II was weakly inhibited by all the investigated anions, with carbamoyl phosphate showing a K(I) of 0.31 mM. The membrane-associated isozyme hCA IV was the most sensitive to inhibition by phosphates/phosphonates, showing a K(I) of 84 nM for PO(4)(3-), of 9.8 microM for HPO(4)(2-), and of 9.9 microM for carbamoyl phosphate. Foscarnet was the best inhibitor of this isozyme (K(I) of 0.82 mM) highly abundant in the kidneys, which may explain some of the renal side effects of the drug. The mitochondrial isozyme hCA V was weakly inhibited by all phosphates/phosphonates, except carbamoyl phosphate, which showed a K(I) of 8.5 microM. Thus, CA V cannot be the isozyme involved in the carbamoyl phosphate synthetase I biosynthetic reaction, as hypothesized earlier. Furthermore, the relative resistance of CA V to inhibition by inorganic phosphates suggests an evolutionary adaptation of this mitochondrial isozyme to the presence of high concentrations of such anions in these energy-converting organelles, where high amounts of ATP are produced by ATP synthetase, from ADP and inorganic phosphates. The transmembrane, tumor-associated isozyme hCA IX was on the other hand slightly inhibited by all these anions.     

Carbonic anhydrase inhibitors: inhibition of human cytosolic isozyme II and mitochondrial isozyme V with a series of benzene sulfonamide derivatives

Among the 14 human isozymes of carbonic anhydrase (CA, EC 4.2.1.1) presently known, the cytosolic hCA II is the most active and plays a host of physiological functions, whereas the mitochondrial hCA V is unique due to its role in several biosynthetic reactions. An inhibition study of these isozymes with a series of sulfonamides is reported here, with the scope to detect lead molecules for the design of isozyme-specific CA inhibitors (CAIs) targeting the mitochondrial isoform. Indeed, recently it has been shown that CA V is a novel target for the drug design of anti-obesity agents among others. Compounds included in this study were mainly ortho-, meta-, and para-substituted-benzenesulfonamides, together with several halogeno-substituted sulfanilamides and disubstituted-benzene-1,3-disulfonamide derivatives. Isozyme V showed an inhibition profile with these sulfonamides different of that of hCA II. Thus, IC(50) values in the range of 80 nM to 74 microM against hCA II, and 0.78-63.7 microM against hCA V with these derivatives have been obtained. Only one compound, 2-carboxymethyl-benzenesulfonamide, was more active against hCA V over hCA II (selectivity ratio of 1.39), whereas all other derivatives investigated here were much better hCA II inhibitors (selectivity ratios CA II/CA V in the range of 0.0008-0.73) than hCA V inhibitors.     

Carbonic anhydrase inhibitors. Inhibition of cytosolic isozyme XIII with aromatic and heterocyclic sulfonamides: a novel target for the drug design

The inhibition of the newly discovered cytosolic carbonic anhydrase isozyme XIII (CA XIII) has been investigated with a series of aromatic and heterocyclic sulfonamides, including some of the clinically used derivatives, such as acetazolamide, methazolamide, dichlorophenamide, dorzolamide, and valdecoxib. Inhibition data for the physiologically relevant isozymes I and II (cytosolic forms) and the tumor associated isozyme IX (transmembrane) were also provided for comparison. A very interesting and unusual inhibition profile against CA XIII with these sulfonamides has been observed. The clinically used compounds (except valdecoxib, which was a weak CA XIII inhibitor) potently inhibit CA XIII, with Ki's in the range of 17-23 nM, whereas sulfanilamide, halogenated sulfanilamides, homosulfanilamide, 4-aminoethylbenzenesulfonamide, and orthanilamide were slightly less effective, with Ki's in the range of 32-56 nM. Several low nanomolar (Ki's in the range of 1.3-2.4 nM) CA XIII inhibitors have also been detected, all of them belonging to the sulfanilyl-sulfonamide type of inhibitors, of which aminobenzolamide is the best known representative. Because CA XIII is an active isozyme predominantly expressed in salivary glands, kidney, brain, lung, gut, uterus, and testis, where it probably plays an important role in pH regulation, its inhibition by sulfonamides may lead to novel therapeutic applications for this class of pharmacological agents.     

Redox state and carbonic anhydrase isozyme IX expression in human renal cell carcinoma: biochemical and morphological investigations

Clear renal cell carcinomas (RCC) frequently express carbonic anydrase IX (CA IX) because of non-functional mutation of von Hippel Lindau (VHL) tumor suppressor gene. CA IX is a tumor-associated transmembrane antigen, which catalyzes the extracellular, reversible hydration of carbon dioxide to bicarbonate and proton and thereby contributes to acidification of extracellular milieu. Extracellular acidic pH facilitates tumor growth and progression. CA IX expression is upregulated by Hypoxia Inducible Factor-1 (HIF-1), which is negatively controlled by oxygen via wild type VHL protein and is also regulated by the cell redox state. We investigated the immunohistochemical pattern of distribution of CA IX in a small series (14 cases) of RCCs. CA IX expression was matched with the redox state of RCC, stratifying our series in relation to clinical and histopathological parameters, such as Fuhrman grade, staging, proliferation markers expression, and particularly, the presence of necrosis. Our results show for the first time the existence of a perivascular pattern of CA IX distribution in RCC. We also found a significant relationship between CA IX expression and the presence of necrosis. Tumors with higher CA IX expression exhibited higher degree of necrosis (p < 0.05). Notably, an almost significant relationship between the redox state and CA IX expression was detected in RCC patients with 5 years disease-free survival, most of them showing organ-confined disease. Tumors with lower redox state showed an algebraically higher degree of CA IX expression. On the contrary, tumors with higher redox state exhibited an algebraically lower CA IX expression (p = 0.057). The observed relationship of CA IX expression and necrosis suggests a role for CA IX in RCC. Further investigations are necessary to further establish the role of the redox state in regulation of CA IX expression in RCC.