Plasmatic carbonic anhydrase IX as a diagnostic marker for clear cell renal cell carcinoma

Carbonic anhydrase (CA, EC 4.2.1.1) IX is regarded as a tumour hypoxia marker and CA inhibitors have been proposed as a new class of antitumor agents, with one such agent in Phase II clinical trials. The expression of some CAs, in particular the isoforms CA IX and CA XII, has been correlated with tumour aggressiveness and progression in several cancers. The aim of this study was to evaluate the possibility that CA IX could represent a marker related to clear cell Renal Cell Carcinoma (ccRCC). Bcl-2 and Bax, and the activity of caspase-3, evaluated in tissue biopsies from patients, were congruent with resistance to apoptosis in ccRCCs with respect to healthy controls, respectively. In the same samples, the CA IX and pro-angiogenic factor VEGF expressions revealed that both these hypoxia responsive proteins were strongly increased in ccRCC with respect to controls. CA IX plasma concentration and CA activity were assessed in healthy volunteers and patients with benign kidney tumours and ccRCCs. CA IX expression levels were found strongly increased only in plasma from ccRCC subjects, whereas, CA activity was found similarly increased both in plasma from ccRCC and benign tumour patients, compared to healthy volunteers. These results show that the plasmatic level of CA IX, but not the CA total activity, can be considered a diagnostic marker of ccRCCs. Furthermore, as many reports exist relating CA IX inhibition to a better outcome to anticancer therapy in ccRCC, plasma levels of CA IX could be also predictive for response to therapy.     

Carbonic anhydrase inhibition with a series of novel benzenesulfonamide-triazole conjugates

We report the synthesis and characterisation of a novel series of triazole benzenesulfonamide derivatives, which incorporate the general pharmacophore associated with carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. The synthesised compounds were tested in vitro against four human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes, hCA I, hCA II, hCA IV and hCA IX. The obtained results showed that the tumour-associated hCA IX was the most sensitive to inhibition with the synthesised derivatives, with the triazolo-pyridine benzenesulfonamides 14, 16 and 17 being the most effective inhibitors. Some selected compounds were chosen for a single dose anti-proliferative activity testing against a panel of 57 human tumour cell lines and show some anti-proliferative activity ex vivo.     

Photodynamics of Porphyric Insecticides

The discovery of porphyric insecticides was a direct fallout of the discovery and development of photodynamic herbicides. Tetrapyrrole-dependent photodynamic herbicides are compounds that force green plants to accumulate undesirable amounts of metabolic intermediates of the chlorophyll and heme metabolic pathways, namely, tetrapyrroles. In light, the accumulated tetrapyrroles photosensitize the formation of singlet oxygen that kills treated plants by oxidation of their cellular membranes. Demonstration of the potential for tetrapyrrole accumulation in insects was achieved by spraying T. ni larvae with δ-aminolevulinic acid (ALA) and 2,2-dipyridyl (Dpy). Treated larvae were placed overnight in darkness at 28°C in order to allow for tetrapyrrole accumulation. Extraction of treated, dark-incubated larvae with ammoniacal acetone, followed by spectrofluorometric examination of the larval extract, revealed the accumulation of massive amounts of protoporphyrin IX (Proto). A high degree of correlation was observed between Proto accumulation in darkness and larval death in the light. A few hours after exposure to light, the larvae became sluggish and flaccid due to loss of body fluids. Death was accompanied by extensive desiccation. Because control of insects by ingestion is as viable an option as control by spraying, and offers certain advantages under household conditions, studies were conducted to determine whether combinations of ALA and porphyric insecticide modulators would be effective if ingested with the food. The effect of ALA and 1,10-phenanthroline (Oph) were determined by incorporating them into the diet of T. ni larvae. After exposure to light, following 17 h of dark incubation, larvae underwent violent convulsions and vomiting and died within 20 to 40 s. Tetrapyrrole analysis of the treated larvae immediately after dark incubation revealed significant amounts of Proto and Zn-Proto accumulation. Correlation between tetrapyrrole accumulation and larval death was significant. Similar results were obtained when ALA and Dpy were administered to the larvae with the diet. The above results indicated that in addition to contact via spraying, porphyric insecticides had the potential to be very potent when ingested. For a more thorough understanding of the mode of action of porphyric insecticides, the phenomenology of tissue, cellular, and subcellular sites of tetrapyrrole accumulation in representative insect species was investigated. In T. ni larvae, on a unit protein basis, about 59% of the accumulated Proto was observed in the hemolymph, 35% in the gut, and 6% in the integument. Further understanding of the response of insect organs and tissues to porphyric insecticide treatment was obtained by investigating the response of isolated organs and tissues to incubation with ALA + Dpy or ALA + Oph in adult Blattella germanica (German cockroach), adult Anthonomus grandis (cotton boll weevil), fifth instar larvae of Heliothus zea (corn earworm), and fifth instar larvae of T. ni (cabbage looper). In T. ni, and H. zea, significant Proto accumulation was observed in incubated midgut and fat bodies. Proto accumulation occurred when tissues were incubated with Dpy, ALA + Dpy, Oph, and ALA + Oph (2). No response to treatment with ALA alone was observed. In cockroaches, more of the Proto appeared to accumulate in the male and female guts than in their abdomen. As in T. ni and H. zea, the response was elicited by each of the treatments that included Dpy or Oph. Cotton boll weevil abdomens appeared to be less responsive than the abdomens of the other three species. To determine whether Proto accumulation resulted in photodynamic damage of incubated tissues, T. ni midguts were incubated in darkness either in buffer, with ALA, or with Oph + ALA. Oxygen consumption of the tissue was monitored before and after exposure to 2-h of illumination. A 30% decrease in O₂ consumption was observed in midguts treated with Oph or with ALA + Oph after 2 h in the light. The decrease in oxygen consumption observed in isolated T. ni midguts was shown to be caused by photodynamic damage to mitochondrial enzymes. Finally, structure-function photodynamic insecticidal studies led to the identification of 36 compounds belonging to 10 different chemical families that were effective (>70% mortality) against at least one insect species. Of the 36 modulators, 10 exhibited potent activity toward cockroaches.     

Increment of hFIX expression with endogenous intron 1 in Vitro

This paper probes into the feasibility of increasing expression level of hFIX gene with endogenous intron 1 sequence.hFIX minigene was obtained with middle sequence truncated intron 1 inserted into the relative site of hFIX cDNA,and plasmid vector pKG5i‘IX,retroviral vector G1NaCi‘IX were constructed.These vectors were transduced into target cells of PA317,C2C12,primary rabbit skin fibroblasts (RSF) and primary human skin fibroblasts (HSF).The expression level of mixed colonies are PA317/pKG5i‘IX,151 ng/10^6 cells/24h;PA317/G1NaCi‘IX,308 ng/10^6 cells/24 h;C2C12/G1NaCi‘IX,186 ng/10^6 cells/24 h;RSF/G1NaCi‘IX,1929 ng/10^6 cells/24 h;HSF/G1NaCi‘IX,1646 ng/10^6 cells/24 h.These results indicated that hFIX minigene with intron l is able to increase the expression level to about 3 times of that of hFIX cDNA.Meanwhile,in order to study the application of hFIX minigene in the retroviral-mediated gene transfer system and refrain from intron splicing during viral production,a retroviral vector G1NaCi‘IXR with reversely inserted hFIX minigene expression cassette was constructed.The expression level of reverse constructor in PA317 cells was 390 ng/10^6 cells/24 h with 79% of bioactivity.PCR detection of HT/G1NaCi‘IXR cells infected with PA317/G1NaCi‘IXR supernatant confirmed the existence of intron 1 sequence.These results suggested that expression vector with forward-inserted intronl-carrying hFIX expression cassette can be used in directed gene transfer,but when using the retroviral-mediated gene transfer system,reversely-inserted intronl-carrying hFIX expression cassette should be considered.     

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.     

Mercaptide-anion as a trans-ligand in oxycytochrome P450

Oxygenation of heme-mercaptide as well as spectroscopic characteristics of the dioxygen complex formed have been studied. Absorption and magnetic circular dichroism spectra of the O₂ complex support the retention of mercaptide in the heme fifth position. A release of $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ in the decomposition of the oxygenated complex and an independent formation of the latter from hemine-dimercaptide and $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ together with electron paramagnetic resonance and Mössbauer data support the O₂ presence in the heme coordination sphere. The similarity of optical and magnetic circular dichroism spectra and the closeness of the ratio for oxy-heme-mercaptide and oxycytochrome P450 unequivocally confirm the presence of an axial cystein mercaptide ligand in oxycytochrome P450.     

SLC-0111 enaminone analogs, 3/4-(3-aryl-3-oxopropenyl) aminobenzenesulfonamides,as novel selective subnanomolar inhibitors of the tumor-associated carbonic anhydrase isoform IX

SLC-0111, an ureido substituted benzenesulfonamide, is a selective carbonic anhydrase (CA, EC 4.2.1.1) IX inhibitor that is currently in Phase I/II clinical trials for the treatment of advanced hypoxic tumors complicated with metastases. Herein we report the synthesis of two series of 3/4-(3-aryl-3-oxopropenyl) aminobenzenesulfonamides 5a–i and 6a–j as SLC-0111 enaminone congeners. The prepared enaminones were in vitro investigated as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA I, II, IV and IX, using a stopped-flow CO₂ hydrase assay. All these isoforms were inhibited by the enaminones reported here in variable degrees. The target tumor-associated isoform hCA IX was undeniably the most affected one (K_Is: 0.21–7.1 nM), with 6- to 21-fold enhanced activity than SLC-0111 (K_I = 45 nM). All the prepared enaminones displayed interesting selectivity towards hCA IX over hCA I (SI: 32 – >35714), hCA II (SI: 2 – 1689) and hCA IV (SI: 11 – >45454). Of particular interest, bioisosteric replacement of phenyl tail with the bulkier 2-naphthyl tail, sulfonamide 6h, achieved the higher II/IX selectivity herein reported with SI of 1689.