Carbonic anhydrase inhibitors: purification and inhibition studies of pigeon (Columba livia var. domestica) red blood cell carbonic anhydrase with sulfonamides

A carbonic anhydrase (CA, EC 4.2.1.1) from red blood cells of pigeons (Columba livia var. domestica), clCA, was purified to homogeneity. Its kinetic parameters for the CO(2) hydration reaction were measured. With a k(cat)/K(m) of 1.1?×?10(8) M(-1) s(-1), and a k(cat) of 1.3?×?10(6) s(-1), clCA has a high activity, similar to that of the human isoform hCA II. A group of 25 aromatic/heterocyclic sulfonamides incorporating the sulfanilamide, homosulfanilamide, benzene-1,3-disulfonamide, and acetazolamide scaffolds showed variable inhibitory activity against the pigeon enzyme, with K(I)s in the range of 1.9-3460?nM. Red blood cells of pigeons, like those of ostriches, contain thus just one CA isoform, unlike the blood of mammals, which normally contain two isoforms, one of low (CA I-like) and one of very high activity (CA II-like). However, from the sulfonamide inhibition viewpoint, the pigeon enzyme was more similar to hCA II than to the ostrich enzyme.     

Inhibition studies of a β-carbonic anhydrase from Brucella suis with a series of water soluble glycosyl sulfanilamides

A β-carbonic anhydrase (CA, EC 4.2.1.1) from the bacterial pathogen Brucella suis, bsCA 1, has been cloned, purified characterized kinetically and for inhibition with a series of water soluble glycosylated sulfanilamides. bsCA 1 has appreciable activity as catalyst for the hydration of CO₂ to bicarbonate, with a k_cat of 6.4 × 10⁵ s^?1 and k_cat/K_m of 3.9 × 10⁷ M^?1 s^?1. All types of inhibitory activities have been detected, with K_Is in the range of 8.9–110 nM. The best bsCA 1 inhibitor were the galactose and ribose sulfanilamides, with inhibition constants of 8.9–9.2 nM. Small structural changes in the sugar moiety led to dramatic differences of enzyme inhibitory activity for this series of compounds. One of the tested glycosylsulfonamides and acetazolamide significantly inhibited the growth of the bacteria in cell cultures.     

Carbonic anhydrase activators: X-ray crystal structure of the adduct of human isozyme II with L-histidine as a platform for the design of stronger activators

Activation of the carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA I, II, and IV with l-histidine and some of its derivatives has been investigated by kinetic and X-ray crystallographic methods. l-His was a potent activator of isozymes I and IV (activation constants in the range of 4-33microM), and a moderate hCA II activator (activation constant of 113microM). Both carboxy- as well as amino-substituted l-His derivatives, such as the methyl ester or the dipeptide carnosine (beta-Ala-His), acted as more efficient activators as compared to l-His. The X-ray crystallographic structure of the hCA II-l-His adduct showed the activator to be anchored at the entrance of the active site cavity, participating in an extended network of hydrogen bonds with the amino acid residues His64, Asn67, and Gln92 and, with three water molecules connecting it to the zinc-bound water. Although the binding site of l-His is similar to that of histamine, the first CA activator for which the X-ray crystal structure has been reported in complex with hCA II (Briganti, F.; Mangani, S.; Orioli, P.; Scozzafava, A.; Vernaglione, G.; Supuran, C. T. Biochemistry1997, 36, 10384) there are important differences of binding between the two structurally related activators, since histamine interacts among others with Asn67 and Gln92 (similarly to l-His), but also with Asn62 and not His64, whereas the number of water molecules connecting them to the zinc-bound water is different (two for histamine, three for l-His). Furthermore, the imidazole moieties of the two activators adopt different conformations when bound to the enzyme active site. Since neither the amino- nor carboxy moieties of l-His participate in interactions with amino acid moieties of the active site, they can be derivatized for obtaining more potent activators, with pharmacological applications for the enhancement of synaptic efficacy. This may constitute a novel approach for the treatment of Alzheimer's disease, aging, and other conditions in need of achieving spatial learning and memory therapy.     

Carbonic anhydrase inhibitors: Inhibition studies of a coral secretory isoform with inorganic anions

The inhibition of a coral carbonic anhydrase (CA, EC 4.2.1.1) has been investigated with a series of inorganic anions such as halogenides, pseudohalogenides, bicarbonate, carbonate, nitrate, nitrite, hydrogen sulfide, bisulfite, perchlorate, sulfate. The full-length scleractinian coral Stylophora pistillata CA, STPCA, has a significant catalytic activity for the physiological reaction of CO₂ hydration to bicarbonate, similarly to the ubiquitous human isoforms hCA I (cytosolic) and hCA VI (secreted). The best STPCA anion inhibitors were bromide, iodide, carbonate, and sulfamate, with inhibition constants of 9.0–10.0 μM.     

Dexamethasone and GLP-2 administered to rat dams during pregnancy and lactation have late effects on intestinal sugar transport in their postweaning offspring

Intestinal function in young animals is influenced by maternal factors, such as alterations in the maternal diet. Glucagon-like peptide 2 (GLP-2) enhances intestinal growth and absorption in mature animals. Glucocorticosteroids induce intestinal maturation in neonates and increase sugar uptake in adult animals. It is not known if maternally administered GLP-2 or glucocorticosteroids have persistent effects on intestinal transport in the offspring. This study was undertaken to determine (1) the influence of maternal GLP-2, dexamethasone (DEX) and GLP-2+DEX on intestinal sugar uptake in postweaning offspring and (2) if alterations in uptake are due to variations in intestinal morphology, sugar transporter abundance or the abundance of selected signals. Nursing rat dams were treated during pregnancy and lactation with GLP-2 (0.1 mug/g per day sc), DEX (0.128 microg/g per day sc), GLP-2+DEX or placebo. The offspring were sacrificed 4 weeks after weaning, and glucose and fructose uptake was determined using an in vitro intestinal ring uptake technique. sodium-dependent glucose transporter, glucose transporter (GLUT) 5, GLUT2, sodium potassium adenosine triphosphatase and selected signals were assessed by immunohistochemistry. The treatments did not affect body weights or intestinal morphology. GLP-2 and GLP-2+DEX increased jejunal fructose uptake, and GLP-2+DEX increased the jejunal and ileal maximal transport rate for glucose uptake. Protein kinase B and mammalian target of rapamycin abundance were also increased, while transporter abundance was unchanged. We speculate that these alterations in sugar uptake may be due to changes in the intrinsic activity of the transporters mediated by the phosphatidylinositol-3-kinase pathway. These alterations in uptake may have nutritional implications for the offspring of mothers who may be treated with GLP-2 or glucocorticosteroids.     

Carbonic anhydrase IX from cancer-associated fibroblasts drives epithelial-mesenchymal transition in prostate carcinoma cells

Extracellular acidification, a mandatory feature of several malignancies, has been mainly correlated with metabolic reprogramming of tumor cells toward Warburg metabolism, as well as to the expression of carbonic anydrases or proton pumps by malignant tumor cells. We report herein that for aggressive prostate carcinoma, acknowledged to be reprogrammed toward an anabolic phenotype and to upload lactate to drive proliferation, extracellular acidification is mainly mediated by stromal cells engaged in a molecular cross-talk circuitry with cancer cells. Indeed, cancer-associated fibroblasts, upon their activation by cancer delivered soluble factors, rapidly express carbonic anhydrase IX (CA IX). While expression of CAIX in cancer cells has already been correlated with poor prognosis in various human tumors, the novelty of our findings is the upregulation of CAIX in stromal cells upon activation. The de novo expression of CA IX, which is not addicted to hypoxic conditions, is driven by redox-based stabilization of hypoxia-inducible factor-1. Extracellular acidification due to carbonic anhydrase IX is mandatory to elicit activation of stromal fibroblasts delivered metalloprotease-2 and -9, driving in cancer cells the epithelial-mesenchymal transition epigenetic program, a key event associated with increased motility, survival and stemness. Both genetic silencing and pharmacological inhibition of CA IX (with sulfonamide/sulfamides potent inhibitors) or metalloprotease-9 are sufficient to impede epithelial-mesenchymal transition and invasiveness of prostate cancer cells induced by contact with cancer-associated fibroblasts. We also confirmed in vivo the upstream hierarchical role of stromal CA IX to drive successful metastatic spread of prostate carcinoma cells. These data include stromal cells, as cancer-associated fibroblasts as ideal targets for carbonic anhydrase IX-directed anticancer therapies.     

Paraoxon, 4-nitrophenyl phosphate and acetate are substrates of α- but not of β-, γ- and ζ-carbonic anhydrases

Carbonic anhydrases (CAs, EC 4.2.1.1) belonging to α-, β-, γ- and ζ-classes and from various organisms, ranging from the bacteria, archaea to eukarya domains, were investigated for their esterase/phosphatase activity with 4-nitrophenyl acetate, 4-nitrophenyl phosphate and paraoxon as substrates. Only α-CAs showed esterase/phosphatase activity, whereas enzymes belonging to the β-, γ- and ζ-classes were completely devoid of such activity. Paraoxon, the metabolite of the organophosphorus insecticide parathione, was a much better substrate for several human/murine α-CA isoforms (CA I, II and XIII), with k_cat/K_M in the range of 2681.6–4474.9 M^?1 s^?1, compared to 4-nitrophenyl phosphate (k_cat/K_M of 14.9–1374.4 M^?1 s^?1).     

Carbonic anhydrase inhibitors. Inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, IX, and XII with Schiff's bases incorporating chromone and aromatic sulfonamide moieties, and their zinc complexes

A series of Schiff's bases was prepared by reaction of 3-formyl-chromone or 6-methyl-3-formyl-chromone with aromatic sulfonamides, such as sulfanilamide, homosulfanilamide, 4-aminoethyl-benzenesulfonamide, a pyrimidinyl-substituted sulfanilamide derivative, sulfaguanidine and 4-amino-6-trifluoromethyl-benzene-1,3-disulfonamide. The zinc complexes of these sulfonamides have also been obtained. The new derivatives and their Zn(II) complexes were investigated for the inhibition of four physiologically relevant isozymes of carbonic anhydrase (CA, EC 4.2.1.1): the cytosolic isoforms I and II, as well as the tumor-associated, transmembrane isozymes CA IX and XII. Except for the sulfaguanidine-derived compounds which were devoid of activity against all isozymes, the other sulfonamides and their metal complexes showed interesting inhibitory activity. Against isozyme CA I, the inhibition constants were in the range of 13-100 nM, against isozyme CA II in the range of 1.9-102 nM, against isozyme CA IX in the range of 6.3-48nM, and against CA XII in the range of 5.9-50nM. Generally, the formyl-chromone derived compounds were better CA inhibitors as compared to the corresponding 6-methyl-chromone derivatives, and for the simple, benzenesulfonamide derivatives activity increased with an increase of the spacer from sulfanilamide to homosulfanilamide and 4-aminoethylbenzenesulfonamide derivatives, respectively. Some of these compounds may show applications for the development of therapies targeting hypoxic tumors in which CA IX and XII are often highly overexpressed.     

In-vitro antibacterial and cytotoxic activity of cobalt (ii), copper (ii), nickel (ii) and zinc (ii) complexes of the antibiotic drug cephalothin (Keflin)

Keflin (kefl) interacts with Co(II), Cu(II), Ni(II) and Zn(II) metal ions leading to complexes of the type M(kefl)2Cl2 and M(kefl)Cl2, which have been characterized by physicochemical and spectroscopic methods. Magnetic moment, IR, electronic spectral and elemental analyses data suggest that keflin behaves tridentately forming octahedral or trigonal bipyramidal complexes with the metal ions mentioned above. The new compounds have been screened in-vitro for antibacterial and cytotoxic activity against Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhi, Shigella dysentriae, Bacillus cereus, Corynebacterium diphtheriae, Staphylococcus aureus and Streptococcus pyogenes bacterial strains. Compounds, 4 and 8 showed promising activity (90%) against seven, compound 6 showed significant activity (52%) against four and, compounds 1 and 5 showed activity (40%) against three test bacterial strains at concentration of 10 microM.