Design,synthesis & biological evaluation of ferulic acid-based small molecule inhibitors against tumor-associated carbonic anhydrase IX

Carbonic anhydrase IX (CAIX) is an emerging drug target for hypoxia associated cancers. To identify potent and selective inhibitors of CAIX, a small library of ferulic acid (FA) derivatives bearing triazole moiety has been designed, synthesized and evaluated against different human CA isoforms (CAII, CAVA & CAIX). Though most of the compounds showed CAIX inhibition in the micromolar range, compound 7i selectively inhibits CAIX in the nanomolar range (IC₅₀ = 24 nM). In silico analysis revealed binding of 7i with the catalytically important amino acid residues of CAIX. Further, cell-based studies indicate that 7i inhibits the activity of CAIX, decreases the epithelial to mesenchymal transitions, induces apoptosis, inhibits cell migration and colonization potential of cancer cells. Taken together, these results emphasized the use of 7i as a prospective pharmacological lead molecule in CAIX targeted anticancer therapeutics.     

Photodynamic Damage to Yeast Subcellular Organelles Induced by Elevated Levels of Endogenous Protoporphyrin IX

The 2,2"-dipyridyl-induced accumulation of protoporphyrin IX in Saccharomyces cerevisiae cells was shown to be accompanied by the photoinhibition of cell respiration and the enhancement of the photoinduced permeability of plasma membranes to the fluorescent dye primuline. The visible-light illumination (at 400–600 nm) of the mitochondria and plasma membranes isolated from yeast cells with a high level of endogenous protoporphyrin IX intensified lipid peroxidation in these subcellular organelles. Comparative studies showed that the rad 52 mutant cells, which are deficient in the postreplicative recombinational DNA repair system, are considerably more sensitive to the inactivating action of visible light than are the wild-type cells and the rad 3 mutant cells, which are deficient in the excision DNA repair system. The contribution of photodynamic damage to the yeast subcellular organelles to the lethal photodynamic effect is discussed.     

Characterization and quantification of class 1 integrons and associated gene cassettes in sewage treatment plants

Three hydrogen-based membrane biofilm reactors (MBfR) biologically reduced nitrate and perchlorate in a synthetic ion-exchange (IX) brine. Inocula from different natural saline environments were employed to initiate the three MBfRs. Under high-salinity (3%) conditions, bioreduction of nitrate and perchlorate occurred simultaneously, and the three MBfRs from the different inocula exhibited similar removal fluxes for nitrate and perchlorate. Clone libraries were generated from samples of the biofilms in the three MBfRs and compared to those of their inocula. When H₂ was the sole exogenous electron donor under high-salinity conditions, MBfR-driven community shifts were observed with a similar pattern regardless of inoculum. The following 16S rRNA gene phylogenetic analysis showed the presence of novel perchlorate-reducing bacteria in the salt-tolerant mBfR communities. These findings suggest that autohydrogenotrophic and high-salinity conditions provided strong selective pressure for novel perchlorate-reducing populations in the mBfRs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Intact intracellular tail is critical for proper functioning of the tumor-associated, hypoxia-regulated carbonic anhydrase IX

Carbonic anhydrase IX (CA IX) is a tumor-associated, hypoxia-induced enzyme involved in pH regulation and cell adhesion. Its catalytically active ectodomain (ECD) is linked to a transmembrane region and a short intracellular (IC) tail. Removal of the IC tail causes intracellular localization of CA IX. Mutations of basic amino acids within IC do not perturb the membrane position, but reduce shedding of the CA IX ectodomain as well as CA IX-mediated cell dissociation. Moreover, they abolish the CA IX capacity to acidify extracellular pH (pHe) and bind CA IX-selective sulfonamide inhibitor in hypoxia. These findings provide the first evidence for the critical contribution of the IC tail to the proper functioning of CA IX.

Structured summary

MINT-7293982: E-cadherin (uniprotkb:Q95LE0) and CA IX (genbank_protein_gi:223556027) colocalize (MI:0403) by fluorescence microscopy (MI:0416)     

Q-band electron paramagnetic resonance study of nitrosylprotoheme dimethyl ester complexes with N-,O-, and S-donor ligand as model systems for nitrosylhemoproteins

Electron paramagnetic resonance (epr) spectra (at X- and Q band frequency) of nitrosyl(proto-porphyrin IX dimethyl ester) iron( II) complexes with a trans axial ligand of nitrogen-, oxygen-, and sulfur-donor ligand, in the trozen glass state at 77°K, have been investigated in order to understand the epr spectra of nitrosylhemoproteins. The Q-band spectra resolved the spectral features more clearly than the X-band spectra and distinctly exhibited two groups of absorptions, which were attributable to two molecular species. Significant relations were found between two g values (e.g., g_x-g_z, g_x-g_y) and between the g value and the degree of the hyperfine splitting in central absorption. The epr parameters were not very sensitive to the π-bonding ability of the axial ligand, but registered the steric interaction of the axial ligand with porphynnato core. These findings can be utilized in the characterization of an axial ligand trans to the nitrosyl group in nitrosylhemoproteins.     

Tetrahydroquinazole-based secondary sulphonamides as carbonic anhydrase inhibitors: synthesis,biological evaluation against isoforms I,II, IV,and IX,and computational studies

A library of variously decorated N-phenyl secondary sulphonamides featuring the bicyclic tetrahydroquinazole scaffold was synthesised and biologically evaluated for their inhibitory activity against human carbonic anhydrase (hCA) I, II, IV, and IX. Of note, several compounds were identified showing submicromolar potency and excellent selectivity for the tumour-related hCA IX isoform. Structure–activity relationship data attained for various substitutions were rationalised by molecular modelling studies in terms of both inhibitory activity and selectivity.     

Synthesis,biological evaluation and in silico modelling studies of 1,3,5-trisubstituted pyrazoles carrying benzenesulfonamide as potential anticancer agents and selective cancer-associated hCA IX isoenzyme inhibitors

Inhibition of carbonic anhydrases (CAs, EC 4.2.1.1) has clinical importance for the treatment of several diseases. They participate in crucial regulatory mechanisms for balancing intracellular and extracellular pH of the cells. Among CA isoforms, selective inhibition of hCA IX has been linked to decreasing of cell growth for both primary tumors and metastases. The discovery of novel CA inhibitors as anticancer drug candidates is a current topic in medicinal chemistry. 1,3,5-Trisubstituted pyrazoles carrying benzenesulfonamide were evaluated against physiologically abundant cytosolic hCA I and hCA II and trans-membrane, tumor-associated hCA IX isoforms by a stopped-flow CO₂ hydrase method. Their in vitro cytotoxicities were screened against human oral squamous cell carcinoma (OSCC) cell lines (HSC-2) and human mesenchymal normal oral cells (HGF) via 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) test. Compounds 6, 8, 9, 11, and 12 showed low nanomolar hCA II inhibitory potency with Ki < 10 nM, whereas compounds 9 and 12 displayed Ki < 10 nM against hCA IX isoenzyme when compared with reference Acetazolamide (AZA). Compound 9, 4-(3-(hydrazinecarbonyl)-5-(4-nitrophenyl)-1H-pyrazol-1-yl)benzenesulfonamide, can be considered as the most selective hCA IX inhibitor over off-target cytosolic isoenzymes hCA I and hCA II with the lowest Ki value of 2.3 nM and selectivity ratios of 3217 (hCA I/hCA IX) and 3.9 (hCA II/hCA IX). Isoform selectivity profiles were also discussed using in silico modelling. Cytotoxicity results pointed out that compounds 5 (CC₅₀ = 37.7 μM) and 11 (CC₅₀ = 58.1 μM) can be considered as lead cytotoxic compounds since they were more cytotoxic than 5-Fluorouracil (5-FU) and Methotrexate (MTX).     

Methyl jasmonate-induced lateral root formation in rice: The role of heme oxygenase and calcium

Lateral roots (LRs) play important roles in increasing the absorptive capacity of roots as well as to anchor the plant in the soil. Therefore, understanding the regulation of LR development is of agronomic importance. In this study, we examined the effect of methyl jasmonate (MJ) on LR formation in rice. Treatment with MJ induced LR formation and heme oxygenase (HO) activity. As well, MJ could induce OsHO1 mRNA expression. Zinc protoporphyrin IX (the specific inhibitor of HO) and hemoglobin [the carbon monoxide/nitric oxide (NO) scavenger] reduced LR formation, HO activity and OsHO1 expression. LR formation and HO activity induced by MJ was reduced by the specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-oxide. The effects of Ca²⁺ chelators, Ca²⁺-channel inhibitors, and calmodulin (CaM) antagonists on LR formation induced by MJ were also examined. All these inhibitors were effective in reducing the action of MJ. However, Ca²⁺ chelators and Ca²⁺ channel inhibitors induced HO activity when combining with MJ further. It is concluded that Ca²⁺ may regulate MJ action mainly through CaM-dependent mechanism.     

Signalling through the platelet glycoprotein Ib-V-IX complex

The glycoprotein Ib-V–IX is one of the major adhesive receptors expressed on the surface of circulating platelets. It is composed of four different polypeptides—GPIb, GPIbβ, GPIX, and GPV—and represents a multifunctional receptor able to interact with a number of ligands, including the adhesive protein von Willebrand factor, the coagulation factors thrombin, factors XI and XII, and the membrane glycoproteins P-selectin and Mac-1. Interaction of GPIb-V–IX with the subendothelial von Willebrand factor is essential for primary haemostasis, as it initiates platelet adhesion to the subendothelial matrix at the sites of vascular injury even under high flow conditions. Upon interaction with von Willebrand factor, GPIb-V–IX initiates transmembrane signalling events for platelet activation, which eventually result in integrin _IIbβ₃ stimulation and platelet aggregation. The investigation of the biochemical mechanisms for platelet activation by GPIb-V–IX has attracted increasing attention during the last years. This review will describe and discuss recent findings that have provided new insights into the events underlying GPIb-V–IX transmembrane signalling. In particular, it will summarise basic concepts on the structure of this receptor, extracellular ligands, and intracellular interactors potentially involved in transmembrane signalling. The recently suggested role of membrane Fc receptors in GPIb-V–IX-initiated platelet activation will also be discussed, along with the involvement of lipid metabolising enzymes, tyrosine kinases, and the cytoskeleton in the crosstalk between GPIb-V–IX and integrin _IIbβ₃.     

Carbonic anhydrase IX reduces E-cadherin-mediated adhesion of MDCK cells via interaction with beta-catenin

Carbonic anhydrase IX (CA IX) is a cancer-associated transmembrane isoform of zinc metalloenzymes that catalyse interconversion between carbon dioxide and bicarbonate. CA IX is strongly induced by tumor hypoxia and has been proposed to participate in acidification of tumor microenvironment and in cell adhesion. To elucidate the cell adhesion-related role of CA IX, we investigated its subcellular localization and relationship to E-cadherin, a key adhesion molecule whose loss or destabilization is linked to tumor invasion. For this purpose, we generated MDCK cells with constitutive expression of human CA IX protein. During the monolayer formation, CA IX was localized to cell-cell contacts and its distribution in lateral membranes overlapped with E-cadherin. Calcium switch-triggered disruption and reconstitution of cell contacts resulted in relocalization of both CA IX and E-cadherin to cytoplasm and back to plasma membrane. A similar phenomenon was observed in hypoxia-treated and reoxygenated cells. Moreover, CA IX-expressing MDCK cells exhibited reduced cell adhesion capacity and lower levels of Triton-insoluble E-cadherin. Finally, CA IX was found to coprecipitate with beta-catenin. We conclude that CA IX has a capacity to modulate E-cadherin-mediated cell adhesion via interaction with beta-catenin, which could be of potential significance in hypoxia-induced tumor progression.