Synthesis and evaluation of general mechanism-based inhibitors of sulfatases based on (difluoro)methyl phenyl sulfate and cyclic phenyl sulfamate motifs.

Several model mechanism-based inhibitors (MbIs) were designed and evaluated for their ability to inhibit sulfatases. The MbI motifs were based on simple aromatic sulfates, which are known to be commonly accepted substrates across this highly conserved enzyme class, so that they might be generally useful for sulfatase labeling studies. (Difluoro)methyl phenol sulfate analogs, constructed to release a reactive quinone methide trap, were not capable of irreversibly inactivating the sulfatase active site. On the other hand, the cyclic sulfamates (CySAs) demonstrated inhibition profiles consistent with an active site-directed mode of action. These molecules represent a novel scaffold for labeling sulfatases and for probing their catalytic mechanism.

Evidence for the Cd(2+) activation of the aryl sulfatase from helix pomatia.

Often used to remove sulfate groups from carbohydrates, the regulatory properties of the aryl sulfatase from Helix pomatia remain little characterized. As many hydrolytic enzymes utilize exogenous metal ions in catalysis, the effect of various divalent metal ions on the sulfatase was investigated. Evidence for metal ion activation was collected, with Cd(2+) being notable for effective activation. The enzyme was inhibited by Cu(2+). The response of other common hydrolases to divalent metal ions was characterized. Activation by Cd(2+) was not observed for chymotrypsin, rabbit liver esterase, or beta-galactosidase. Instead, Cd was found to inhibit both the esterase and the galactosidase. Inhibition by Cu(2+) and Zn(2+) was also observed for some of these hydrolases.

The glutathione peroxidase homologous gene from Chlamydomonas reinhardtii is transcriptionally up-regulated by singlet oxygen.

The glutathione peroxidase homologous gene (Gpxh gene) in Chlamydomonas reinhardtii is up-regulated under oxidative stress conditions. The Gpxh gene showed a remarkably strong and fast induction by the singlet oxygen-generating photosensitizers neutral red, methylene blue and rose Bengal. The Gpxh mRNA levels strongly increased, albeit much more slowly, upon exposure to the organic hydroperoxides tert-butyl hydroperoxide (t-BOOH) and cumene hydroperoxide. In contrast, the Gpxh mRNA levels were only weakly induced by exposure to the superoxide-generating compound paraquat and by hydrogen peroxide. A comparison of the Gpxh mRNA levels with those of the heat shock protein HSP70A and the iron superoxide dismutase gene showed qualitative and quantitative differences for the three genes under oxidative stress conditions tested. The Gpxh gene is specifically induced by singlet-oxygen photosensitizers and the relative induction by other compounds is much weaker for Gpxh than for the other genes investigated. Using Gpxh promoter fusions with the arylsulfatase reporter gene, we have shown that the Gpxh was transcriptionally up-regulated by singlet-oxygen photosensitizers. It is also shown that the Gpxh promoter contains a region between 104 and 179 bp upstream of the transcription start that is responsible for the mRNA up-regulation upon exposure to 1O2 but not t-BOOH. Within this region a regulatory sequence homologous to the mammalian cAMP response element (CRE) and activator protein 1 (AP-1) binding site was identified within a 16 bp palindrome.

Influence of quercetin on B16 melanotic melanoma growth in C57BL/6 mice and on activity of some acid hydrolases in melanoma tissue.

Quercetin (QC) (5, 7, 3', 4' -tetra oxyflavonolol) is an ubiquitous flavonoid in many plants. The influence of QC on the growth of B16 melanotic melanoma in C57BL/6 mice and activity of some acid hydrolases in the tumor homogenates were investigated. Two series of experiments were carried out: In the first experimental group mice were inoculated s.c. with 10(6) of tumor cells (TC) suspended in 1 ml of saline. TC were obtained from the current serial passages. In the second series of experimental group mice were inoculated with melanoma cells preincubated 15 min. in different concentrations of QC. Mice of both series were divided into three subgroups. Mice of the first series were treated with QC i.p. every second day in a dose of 0.1 mg, 0.5 mg or 1.0 mg (total dose of 1.0 mg, 5.0 mg or 10.0 mg per mice). Animals of the second series did not obtain any treatment. After the nineteenth day of experiment the mice were killed, tumors excised and weighed. Tumor tissue pieces were homogenized for enzyme activity determination. Fragments of tumor tissue were taken for electron microscopy (EM) investigation. In mice injected i.p. with QC mean tumor weight was significantly higher than in control I. The mean tumor weight in the first experimental group was higher than in control from 170% to 196% and in the second experimental group from 69% to 147%. Enzymes activity was also higher in both experimental groups as compared to controls. Arylsulphatase activity in the first group was higher from 102% to 144% and in the second one - from 97% to 115% than in control I. Acid phosphatase activity was higher from 100% to 155% in the first experimental group and from 56% to 161% in the second one. Cathepsin D activity was greater from 133% to 333% and from 113% to 300%, respectively. EM studies revealed the presence of greater number of Golgi structures and primary lysosomes in experimental groups of tumors (mice treated with QC and mice with melanoma preincubated in QC). These results clearly indicate that QC significantly enhances melanotic melanoma growth and increases acid phosphatase and cathepsin D activity in these tumors. The mechanism of QC action on the melanotic melanoma is not fully understood and remains to be defined.

Steroidal and nonsteroidal sulfamates as potent inhibitors of steroid sulfatase.

Synthetic routes to potent steroidal and nonsteroidal sulfamate-based active site-directed inhibitors of the enzyme steroid sulfatase, a topical target in the treatment of postmenopausal women with hormone-dependent breast cancer, are described. Novel compounds were examined for estrone sulfatase (E1-STS) inhibition in intact MCF-7 breast cancer cells and placental microsomes. Reaction of the sodium salt of estrone with sulfamoyl chloride gave estrone 3-O-sulfamate (EMATE, 2) which inhibits E1-STS activity potently (> 99% at 0.1 microM in intact MCF-7 cells, IC50 = 65 pM) in a time- and concentration-dependent manner, suggesting that EMATE is an active site-directed inhibitor. EMATE is also active in vivo orally. 5,6,7,8-Tetrahydronaphthalene 2-O-sulfamate (7) and its N-methylated derivatives (8 and 9) were synthesized, and 7 inhibits the E1-STS activity in intact MCF-7 cells by 79% at 10 microM. 4-Methylcoumarin 7-O-sulfamate (COUMATE) and its derivatives (14, 16, and 18) were prepared to extend this series of nonsteroidal inhibitors, and COUMATE reduces the E1-STS activity in placental microsomes by > 90% at 10 microM. Although the orally active COUMATE is less potent than EMATE as an active site-directed inhibitor, it has the important advantage of being nonestrogenic. Analogues (20, 22, 24, 26, 27, 31, 33, 39, and 44) of COUMATE were synthesized to study its structure-activity relationships, and sulfamates of tetralones (46 and 48) and indanones (49, 51, and 53) were also prepared. While most of these compounds were found to inhibit E1-STS activity less effectively than COUMATE, one analogue, 3,4-dimethylcoumarin 3-O-sulfamate (24), was found to be some 12-fold more potent than COUMATE as an E1-STS inhibitor in intact MCF-7 cells (IC50 = 30 nM for 24, cf. 380 nM for COUMATE). Hence, highly potent sulfamate-based inhibitors of steroid sulfatase, such as EMATE, COUMATE, and 24, possess therapeutic potential and will allow the importance of estrogen formation in breast tumors via the E1-STS pathway to be assessed. A pharmacophore for active site-directed sulfatase inhibition is proposed.

Multiple sulfatase deficiency: catalytically inactive sulfatases are expressed from retrovirally introduced sulfatase cDNAs.

Multiple sulfatase deficiency (MSD) is an inherited lysosomal storage disease characterized by the deficiency of at least seven sulfatases. The basic defect in MSD is thought to be in a post-translational modification common to all sulfatases. In accordance with this concept, RNAs of normal size and amount were detected in MSD fibroblasts for three sulfatases tested. cDNAs encoding arylsulfatase A, arylsulfatase B, or steroid sulfatase were introduced into MSD fibroblasts and fibroblasts with a single sulfatase deficiency by retroviral gene transfer. Infected fibroblasts overexpressed the respective sulfatase polypeptides. While in single-sulfatase-deficiency fibroblasts a concomitant increase of sulfatase activities was observed, MSD fibroblasts expressed sulfatase polypeptides with a severely diminished catalytic activity. From these results we conclude that the mutation in MSD severely decreases the capacity of a co- or post-translational process that renders sulfatases enzymatically active or prevents their premature inactivation.

Temporary changes of arylsulphatase activity in liver after exposure of AET or MEA pre-treated mice to 60Co gamma-irradiation.

The adult male Swiss mice were either whole-body 60Co gamma-irradiated with a single dose of 10 Gy at 1900, or treated intraperitoneally with AET (2-aminoethylisothiouronium bromide hydrobromide) or MEA (cysteamine hydrochloride), in a dose of 400 mg/kg body weight, 15 minutes before irradiation. The measurements of the arylsulphatase (E.C. 3.1.6.1) activity in crude homogenate of liver were done every four hours during 24-hour period, starting at 2000. The enzyme activity towards the artificial substrate p-nitrocatechol sulphate was related to 1 mg of protein, 1 g of fresh tissue and to the whole organ weight. No fluctuations in the activity of arylsulphatase in the control mice were found. There were temporary changes in the enzyme activity expressed in nkat per 1 mg of protein and 1 g of fresh tissue in the irradiated only males, in that related to 1 g of fresh tissue and the whole organ weight in mice injected with AET before irradiation, as well as that calculated in nkat per 1 mg of protein in animals pre-treated with MEA to exposure of gamma rays. Between the particular groups of males, differences in the arylsulphatase activity related to 1 g of fresh tissue and the whole liver weight at 0400 were only observed. 60Co gamma-irradiation influenced the activity of arylsulphatase in liver. AET and MEA modified the effect of exposure of mice to gamma rays on the enzyme activity in a different way.