Evidence for the mechanism of the irreversible inhibition of oestrone sulphatase (ES) by aminosulphonate based compounds

In our search for the mechanism of the enzyme oestrone sulphatase (ES) we have synthesised and evaluated a number of compounds that were predicted to possess some inhibitory activity. Some of these compounds were indeed found to be inhibitors of ES, whilst other compounds were not. From a consideration of the structure–activity relationship (SAR) of the inhibitors and non-inhibitors of this enzyme, we discovered a factor which we now believe is the main inhibitory moiety within the aminosulphonated inhibitors. We therefore report the results of our study into a series of phenyl and alkyl sulphamated compounds as inhibitors of ES. The results of the study show that the substituted phenyl sulphamates are potent inhibitors, whereas the alkyl compounds are, in general, non-inhibitors. Using the results of our SAR study, we postulate the probable mechanism for the irreversible and reversible inhibition of ES, and rationalise the role of the different physicochemical factors in the inhibition of this crucial enzyme.     

Common action of certain viruses,toxins, and activated complement: pore formation and its prevention by extracellular Ca2+

Haemolysis by Sendal virus, -toxin, and activated complement is inhibited by high concentrations of divalent cations. In Daudi cells, sublytic amounts of these agents induce the following changes: collapse of surface membrane potential, uptake of Na⁺ and loss of K⁺ from cells, and leakage of phosphorylated metabo-tites from cells. The changes induced by Sendal virus and complement are sensitive to physiological concentrations of extracellular Ca²⁺. It is concluded that fluctuations in plasma Ca²⁺ concentration may affect the damaging action of certain pore-forming agents on susceptible cells.     

The molecular basis of increased glomerulosclerosis after blockade of the renin angiotensin system in growth hormone transgenic mice.

BACKGROUND: Angiotensin converting enzyme inhibitor (ACEi) therapy delays the onset of renal failure in diabetic nephropathy and inhibits or delays the onset of proteinuria in several animal models. MATERIALS AND METHODS: We examined this question using a transgenic model of chronic glomerulosclerosis caused by an excess production of growth hormone (GH) in which there is progressive glomerular scarring leading to uremia. In addition, since GH mice do not have systemic hypertension or an elevated glomerular filtration rate, we could address the question of whether ACEi or angiotensin II receptor antagonists (AII RA) had an effect on the development of glomerulosclerosis under these conditions. Since excess matrix accumulates in glomerulosclerosis because of alterations in the balance between its synthesis and degradation, we examined the effect of ACEi and AII RA on these parameters. RESULTS: Systemic blood pressure was unaffected by ACEi treatment, but the glomerular filtration rate decreased 85%. ACEi-treated mice had increased mesangial deposition of type I collagen and decreased 105 kD complex collagenase activity. In addition, ACEi-treated GH mice had increased glomerular alpha 1 type I collagen, alpha 1 type IV collagen, and alpha-smooth muscle cell actin mRNAs. No changes were noted in beta actin, or 72 kD metalloproteinase mRNAs. The result of these changes was a net increase in sclerosis. Surprisingly, GH mice treated with ACEi or AngII RA developed marked renal arteriolar lesions. CONCLUSIONS: In some forms of glomerulosclerosis, the lesions develop independently of angiotensin II. Pharmacological inhibition of angiotensin II, in this circumstance, may aggravate the lesions through disregulation of the levels and the balance between glomerular matrix synthesis and degradation.     

The precise radioimmunoassay of adenosine: minimization of sample collection artifacts and immunocrossreactivity.

Anti-adenosine antibodies were produced in rabbits immunized with N6-carboxymethyladenosine conjugated to methyl albumin. 125I-N6-Aminobenzyladenosine was synthesized and used as a high-specific-activity, high-affinity ligand. A radioimmunoassay (RIA) was developed that can detect 6.25 nM (312.5 fmol) of underivatized adenosine and cross-reacts less than 0.02% with adenine nucleotides and guanosine and not at all with 1 mM inosine. The sensitivity of the RIA can be increased to a detection limit of 0.125 nM (6.25 fmol) by derivitizing samples with benzyl bromide to form N6-benzyladenosine. The assay was adapted to an automated RIA procedure. Assay precision was increased by: (i) inhibiting slight adenosine deaminase activity present in anti-sera; (ii) treating buffers and albumin used in the RIA with charcoal to remove contaminating adenosine; and (iii) correcting for a small but variable component of immunoreactivity not attributable to adenosine. A second antibody prepared with a 2',3'-disuccinyladenosine-albumin conjugate was also found to detect some non-adenosine-mediated immunoreactivity in plasma samples. Immunointerference in human plasma was eliminated in samples treated with ZnSO4/Ba(OH)2 or partially purified over C18 Sep Paks to remove nucleotides and assayed after sample benzylation or succinylation. Human blood was mixed with a novel "stop" solution that was optimized to inhibit adenosine formation from AMP by greater than 99% and to inhibit adenosine uptake into red cells and degradation by greater than 94%. Human plasma/stop solution was assayed by RIA and HPLC with equivalent results.     

Antitumor activity of Noscapine in combination with Doxorubicin in triple negative breast cancer

Background

The aim of this study was to investigate the anticancer activity and mechanism of action of Noscapine alone and in combination with Doxorubicin against triple negative breast cancer (TNBC).

Methods

TNBC cells were pretreated with Noscapine or Doxorubicin or combination and combination index values were calculated using isobolographic method. Apoptosis was assessed by TUNEL staining. Female athymic Nu/nu mice were xenografted with MDA-MB-231 cells and the efficacy of Noscapine, Doxorubicin and combination was determined. Protein expression, immunohistochemical staining were evaluated in harvested tumor tissues.

Results

Noscapine inhibited growth of MDA-MB-231 and MDA-MB-468 cells with the IC₅₀ values of 36.16±3.76 and 42.7±4.3 µM respectively. The CI values (<0.59) were suggestive of strong synergistic interaction between Noscapine and Doxorubicin and combination treatment showed significant increase in apoptotic cells. Noscapine showed dose dependent reduction in the tumor volumes at a dose of 150–550 mg/kg/day compared to controls. Noscapine (300 mg/kg), Doxorubicin (1.5 mg/kg) and combination treatment reduced tumor volume by 39.4±5.8, 34.2±5.7 and 82.9±4.5 percent respectively and showed decreased expression of NF-KB pathway proteins, VEGF, cell survival, and increased expression of apoptotic and growth inhibitory proteins compared to single-agent treatment and control groups.

Conclusions

Noscapine potentiated the anticancer activity of Doxorubicin in a synergistic manner against TNBC tumors via inactivation of NF-KB and anti-angiogenic pathways while stimulating apoptosis. These findings suggest potential benefit for use of oral Noscapine and Doxorubicin combination therapy for treatment of more aggressive TNBC.     

The primase active site is on the outside of the hexameric bacteriophage T7 gene 4 helicase-primase ring

Gene 4 of bacteriophage T7 encodes a protein (gp4) that can translocate along single-stranded DNA, couple the unwinding of duplex DNA with the hydrolysis of dTTP, and catalyze the synthesis of short RNA oligoribonucleotides for use as primers by T7 DNA polymerase. Electron microscopic studies have shown that gp4 forms hexameric rings, and X-ray crystal structures of the gp4 helicase domain and of the highly homologous RNA polymerase domain of Escherichia coli DnaG have been determined. Earlier biochemical studies have shown that when single-stranded DNA is bound to the hexameric ring, the primase domain remains accessible to free DNA. Given these results, a model was suggested in which the primase active site in the gp4 hexamer is located on the outside of the hexameric ring. We have used electron microscopy and single-particle image analysis to examine T7 gp4, and have determined that the primase active site is located on the outside of the hexameric ring, and therefore provide direct structural support for this model.     

Development and validation of high-throughput liquid chromatography-tandem mass spectrometric method for simultaneous quantification of loratadine and desloratadine in human plasma

As a continuation of effort to improve our high flow on-line bioanalytical approach for high-throughput quantification of drugs and metabolites in plasma by high-throughput liquid chromatography tandem mass spectrometry (HTLC-MS/MS), we have developed a simple, sensitive and reliable method for simultaneous quantification of loratadine and desloratadine in human plasma. We have performed on-line coupling of extraction with Cyclone P 50 mm x 0.5 mm 50 microm HTLC column and chromatographic separation is performed with Zorbax XDB C18 50 mm x 2.1 mm 5 microm, followed by quantification with mass detector. The method is validated and showed good performances in terms of linearity, sensitivity, precision, accuracy and stability. A marked improvement in sample throughput efficiency is realized with this method and the proposed method will be useful for pharmacokinetic and/or bioequivalence studies.     

A257T linker region mutant of T7 helicase-primase protein is defective in DNA loading and rescued by T7 DNA polymerase

The helicase and primase activities of the hexameric ring-shaped T7 gp4 protein reside in two separate domains connected by a linker region. This linker region is part of the subunit interface between monomers, and point mutations in this region have deleterious effects on the helicase functions. One such linker region mutant, A257T, is analogous to the A359T mutant of the homologous human mitochondrial DNA helicase Twinkle, which is linked to diseases such as progressive external opthalmoplegia. Electron microscopy studies show that A257T gp4 is normal in forming rings with dTTP, but the rings do not assemble efficiently on the DNA. Therefore, A257T, unlike the WT gp4, does not preassemble on the unwinding DNA substrate with dTTP without Mg(II), and its DNA unwinding activity in ensemble assays is slow and limited by the DNA loading rate. Single molecule assays measured a 45 times slower rate of A257T loading on DNA compared with WT gp4. Interestingly, once loaded, A257T has almost WT-like translocation and DNA unwinding activities. Strikingly, A257T preassembles stably on the DNA in the presence of T7 DNA polymerase, which restores the ensemble unwinding activity of A257T to ～75% of WT, and the rescue does not require DNA synthesis. The DNA loading rate of A257T, however, remains slow even in the presence of the polymerase, which explains why A257T does not support T7 phage growth. Similar types of defects in the related human mitochondrial DNA helicase may be responsible for inefficient DNA replication leading to the disease states.     

4-(2-pyridyl)piperazine-1-carboxamides: potent vanilloid receptor 1 antagonists

A series of 4-(2-pyridyl)piperazine-1-carboxamide analogues based on the lead compound 1 was synthesized and evaluated for VR1 antagonist activity in capsaicin-induced (CAP) and pH (5.5)-induced (pH) FLIPR assays in a rat VR1-expressing HEK293 cell line. Potent VR1 antagonists were identified through SAR studies. From these studies, 18 was found to be very potent in the in vitro assay [IC(50)=4.8 nM (pH) and 35 nM (CAP)] and orally available in rat (F%=15.1).