Design,green synthesis and pharmacological evaluation of novel 5,6-diaryl-1,2,4-triazines bearing 3-morpholinoethylamine moiety as potential antithrombotic agents*

The aim of this research work was to investigate a series of novel 5,6-diaryl-1,2,4-triazines (3a–3q) containing 3-morpholinoethylamine side chain, and to address their antiplatelet activity by in vitro, ex vivo and in vivo methods. All compounds were synthesized by environment benign route and their structures were unambiguously confirmed by spectral data. Compounds (3l) and (3m) were confirmed by their single crystal X-ray structures. Out of all the synthesized compounds, 10 were found to be more potent in vitro than aspirin; six of them were found to be prominent in ex vivo assays and one compound (3d) was found to have the most promising antithrombotic profile in vivo. Moreover, compound (3d) demonstrated less ulcerogenicity in rats as compared to aspirin. The selectivity of the most promising compound (3d) for COX-1 and COX-2 enzymes was determined with the help of molecular docking studies and the results were correlated with the biological activity.     

Folding of the natural hammerhead ribozyme is enhanced by interaction of auxiliary elements

It has been shown that the activity of the hammerhead ribozyme at microM magnesium ion concentrations is markedly increased by the inclusion of loops in helices I and II. We have studied the effect of such loops on the magnesium ion-induced folding of the ribozyme, using fluorescence resonance energy transfer. We find that with the loops in place, folding into the active conformation occurs in a single step, in the microM range of magnesium ion concentration. Disruption of the loop-loop interaction leads to a reversion to two-step folding, with the second stage requiring mM concentrations of magnesium ion. Sodium ions also promote the folding of the natural form of the ribozyme at high concentrations, but the folding occurs as a two-stage process. The loops clearly act as important auxiliary elements in the function of the ribozyme, permitting folding to occur efficiently under physiological conditions.     

Artificial tertiary motifs stabilize trans-cleaving hammerhead ribozymes under conditions of submillimolar divalent ions and high temperatures

Tertiary stabilizing motifs (TSMs) between terminal loops or internal bulges facilitate folding of natural hammerhead ribozymes (hRz) under physiological conditions. However, both substrate and enzyme strands contribute nucleotides to the TSMs of trans-cleaving hRz, complicating the design of hRz that exploit TSMs to target specific mRNA. To overcome this limitation, we used SELEX to identify new, artificial TSMs that are less sensitive to sequence context. Nucleotides in loop II or in a bulge within the ribozyme strand of stem I were randomized, while the interaction partner was held constant. All nucleotides of the substrate pair with the ribozyme, minimizing their possible recruitment into the TSM, as such recruitment could constrain choice of candidate target sequences. Six cycles of selection identified cis-acting ribozymes that were active in 100 microM MgCl2. The selected motifs partially recapitulate TSMs found in natural hRz, suggesting that the natural motifs are close to optimal for their respective contexts. Ribozyme "RzB" showed enhanced thermal stability by retaining trans-cleavage activity at 80 degrees C in 10 mM MgCl2 and at 70 degrees C in 2 mM MgCl2. A variant of ribozyme "RzB" with a continuously paired stem 1 rapidly lost activity as temperature was increased. The selected motifs are modular, in that they permit trans-cleavage of several substrates in submillimolar MgCl2, including two substrates derived from the U5 genomic region of HIV-1. The new, artificial tertiary stabilized hRz are thus nearly independent of sequence context and enable for the first time the use of highly active hRz targeting almost any mRNA at physiologically relevant magnesium concentrations.     

Functional Coupling of ATP-binding Cassette Transporter Abcb6 to Cytochrome P450 Expression and Activity in Liver

Although endogenous mechanisms that negatively regulate cytochrome P450 (P450) monooxygenases in response to physiological and pathophysiological signals are not well understood, they are thought to result from alterations in the level of endogenous metabolites, involved in maintaining homeostasis. Here we show that homeostatic changes in hepatic metabolite profile in Abcb6 (mitochondrial ATP-binding cassette transporter B6) deficiency results in suppression of a specific subset of hepatic P450 activity. Abcb6 null mice are more susceptible to pentobarbital-induced sleep and zoxazolamine-induced paralysis, secondary to decreased expression and activity of Cyp3a11 and Cyp2b10. The knock-out mice also show decrease in both basal and xeno-inducible expression and activity of a subset of hepatic P450s that appear to be related to changes in hepatic metabolite profile. These data, together with the observation that liver extracts from Abcb6-deficient mice suppress P450 expression in human primary hepatocytes, suggest that this mouse model may provide an opportunity to understand the physiological signals and the mechanisms involved in negative regulation of P450s.     

Synthesis of a new 4-aza-2,3-didehydropodophyllotoxin analogues as potent cytotoxic and antimitotic agents

A series of novel conjugates of 4-aza-2,3-didehydropodophyllotoxins (11a-w) were synthesized by a straightforward one-step multicomponent synthesis that demonstrated cytotoxicity against five human cancer cell lines (breast, oral, colon, lung and ovarian). All the twenty three compounds (11a-w) have been examined for the inhibition of tubulin polymerization. Among these compounds, 11a, 11k and 11p exhibited inhibition of polymerization tubulin comparable to podophyllotoxin apart from disruption of microtubule organization within the cells. Flow cytometric analysis showed that these compounds (11a, 11k and 11p) arrested the cell cycle in the G2/M phase of cell cycle leading to caspase-3 dependent apoptotic cell death.     

Reduction of 8-iso-prostaglandin F2α in the first week after Roux-en-Y gastric bypass surgery

Obesity is associated with increased markers of oxidative stress. We examined whether oxidative stress is reduced within the first week after Roux‐en‐Y gastric bypass (RYGB) surgery and could be related to changes in adipose tissue depots. The reactive oxygen species (ROS) marker 8‐iso‐prostaglandin F2α (8‐iso‐PGF2α) and activity of antioxidant glutathione peroxidases (GPX) in plasma were compared before and ～1 week after RYGB. The effects of RYGB on subcutaneous adipose tissue and interstitial fluid 8‐iso‐PGF2α levels and subcutaneous adipose tissue expression of GPX‐3 were also assessed. Levels of 8‐iso‐PGF2α in subcutaneous and visceral adipose tissue were determined. Plasma 8‐iso‐PGF2α levels decreased (122 ± 75 to 56 ± 15 pg/ml, P = 0.001) and GPX activity increased (84 ± 18 to 108 ± 25 nmol/min/ml, P = 0.003) in the first week post‐RYGB. RYGB also resulted in reductions of 8‐iso‐PGF2α in subcutaneous adipose tissue (1,742 ± 931 to 1,132 ± 420 pg/g fat, P = 0.046) and interstitial fluid (348 ± 118 to 221 ± 83 pg/ml, P = 0.046) that were comparable to plasma (26–33%, P = 0.74). Adipose GPX‐3 expression was increased (6.7 ± 4.7‐fold, P = 0.004) in the first postoperative week. The improvements in oxidative stress occurred with minimal weight loss (2.4 ± 3.4%, P = 0.031) and elevations in plasma interleukin‐6 (18.0 ± 46.8 to 28.0 ± 58.9 pg/ml, P = 0.004). Subcutaneous and visceral adipose tissues express comparable 8‐iso‐PGF2α levels (1,204 ± 470 and 1,331 ± 264 pg/g fat, respectively; P = 0.34). These data suggest that RYGB affects adipose tissue leading to the restoration of adipose redox balance within the first postoperative week and that plasma 8‐iso‐PGF2α is primarily derived from subcutaneous adipose tissue.