Inhibitory mechanism of reveromycin A at the tRNA binding site of a class I synthetase.

The polyketide natural product reveromycin A (RM-A) exhibits antifungal, anticancer, anti-bone metastasis, anti-periodontitis and anti-osteoporosis activities by selectively inhibiting eukaryotic cytoplasmic isoleucyl-tRNA synthetase (IleRS). Herein, a co-crystal structure suggests that the RM-A molecule occupies the substrate tRNAIle binding site of Saccharomyces cerevisiae IleRS (ScIleRS), by partially mimicking the binding of tRNAIle. RM-A binding is facilitated by the copurified intermediate product isoleucyl-adenylate (Ile-AMP). The binding assays confirm that RM-A competes with tRNAIle while binding synergistically with L-isoleucine or intermediate analogue Ile-AMS to the aminoacylation pocket of ScIleRS. This study highlights that the vast tRNA binding site of the Rossmann-fold catalytic domain of class I aminoacyl-tRNA synthetases could be targeted by a small molecule. This finding will inform future rational drug design.

Glucose starvation-mediated inhibition of salinomycin induced autophagy amplifies cancer cell specific cell death.

Salinomycin has been used as treatment for malignant tumors in a small number of humans, causing far less side effects than standard chemotherapy. Several studies show that Salinomycin targets cancer-initiating cells (cancer stem cells, or CSC) resistant to conventional therapies. Numerous studies show that Salinomycin not only reduces tumor volume, but also decreases tumor recurrence when used as an adjuvant to standard treatments. In this study we show that starvation triggered different stress responses in cancer cells and primary normal cells, which further improved the preferential targeting of cancer cells by Salinomycin. Our in vitro studies further demonstrate that the combined use of 2-Fluoro 2-deoxy D-glucose, or 2-deoxy D-glucose with Salinomycin is lethal in cancer cells while the use of Oxamate does not improve cell death-inducing properties of Salinomycin. Furthermore, we show that treatment of cancer cells with Salinomycin under starvation conditions not only increases the apoptotic caspase activity, but also diminishes the protective autophagy normally triggered by the treatment with Salinomycin alone. Thus, this study underlines the potential use of Salinomycin as a cancer treatment, possibly in combination with short-term starvation or starvation-mimicking pharmacologic intervention.

Evaluation of zinc against salinomycin toxicity in broilers.

Salinomycin was studied for its toxicity and zinc (80 mg/kg) was assessed for prophylactic and therapeutic management in broiler chicks. Male broiler chicks were randomly divided into 7 groups consisting of 6 chicks in each. Group 1, 2 and 3 were maintained as control, therapeutic dose control (60 mg/kg feed) and toxic dose control (120 mg/kg feed), respectively. Group 4 was fed on feed containing salinomycin therapeutic dose and zinc. Group 5 received feed containing toxic dose of salinomycin. Group 6 and 7 were fed on feed containing toxic dose of salinomycin for the first 4 weeks for induction of ionophore toxicity and for the subsequent 2 weeks, group 6 received zinc and group 7 was fed on feed containing toxic dose of salinomycin along with zinc. Weekly body weights revealed a significant (P<0.01) decrease in toxic controls as compared to group 1, 2, 4 and 5. The activity of glutathione peroxidase, glutathione reductase and catalase, and the values of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total proteins, total cholesterol, triglycerides, low density lipoproteins (LDL), urea, creatinine and blood urea nitrogen (BUN) were significantly (P<0.01) elevated in toxic controls, whereas glutathione (GSH) and high density lipoproteins (HDL) were significantly (P<0.01) lowered as compared to group 1, 2, 4 and 5. Following toxicity, zinc supplementation in group 6 and 7, all serobiochemical parameters were revived to normal. Thus, it is enunciated that salinomycin toxicity is due to oxidative damage and use of zinc in feed tends to cure and avoid any accidental toxicity.

Metabolism of territrem a by liver microsomes of Wistar rats: identification of the metabolites and their metabolic sequence.

The metabolism of territrem A (TRA) was investigated in liver microsomes of male Wistar rats. The results indicated that three metabolites were produced from TRA and these metabolic reactions were inhibited by metyrapone, an inhibitor of cytochrome P-450. Based on analysis by high-performance liquid chromatography (HPLC), mass, and nuclear magnetic resonance (NMR) spectroscopic techniques, the structure of these metabolites were identified as 4beta-hydroxymethyl-4beta-demethylterritrem A (MA1), 4beta-oxo-4beta-demethylterritrem A (MAX), and 2-dihydro-4beta-demethylterritrem A (MA2). It was proposed that reactions proceeded by three sequential oxidative reactions in the pyran moiety of TRA: first, hydroxylation at the 4beta-C methyl group of TRA to form MA1; second, oxidation at the 4beta hydroxyl group of MA, to form MAX; and third, decarbonylation at the 4beta-C oxo group of MAX to form MA2.

Cardioprotective actions of potassium channel openers.

The potential cardioprotective effect of two pure potassium channel openers, bimakalim (EMD 52692) and aprikalim (RP 52891), on myocardial ischaemia/reperfusion injury was investigated in barbital-anaesthetized dogs. In a model of reversible ischaemia/reperfusion injury, administration of bimakalim as an intravenous bolus prior to ischaemia or administration of a non-hypotensive dose of aprikalim as a constant intravenous infusion resulted in a reduction in reperfusion contractile dysfunction (myocardial 'stunning') produced by a single 15-min coronary artery occlusion. Administration of aprikalim only during the reperfusion period had no beneficial effect. Similarly, in a model of irreversible ischaemia/reperfusion injury (90 min of coronary artery occlusion followed by 5 h of reperfusion), intravenous infusion of bimakalim at a dose which reduced aortic blood pressure approximately 15-20 mmHg or infusion of aprikalim at a non-hypotensive dose throughout the entire experiment produced a significant reduction in myocardial infarct size. A protective effect of bimakalim was not observed when it was administered during the reperfusion period only. In both the stunned myocardium model as well as the infarcted myocardium model, the beneficial effects of the potassium channel openers could not be attributed to differences in the traditional determinants of the extent of ischaemia/reperfusion injury; area at risk size, oxygen consumption, or collateral blood flow. Furthermore, the anti-ischaemic actions of the potassium channel openers were blocked by pre-treatment with the ATP-dependent potassium (KATP) channel antagonist, glibenclamide.(ABSTRACT TRUNCATED AT 250 WORDS)

Vascular pharmacology of ATP-sensitive K+ channels: interactions between glyburide and K+ channel openers.

This study in isolated rabbit superior artery (RMA) investigated the interactions between glyburide, a known blocker of vascular ATP-sensitive K+ channels (KATP), and several chemically diverse potassium channel openers (PCOs): minoxidil sulfate (MNXS; 5 microM), pinacidil (1 microM), cromakalim (0.5 microM) and RP-49356 (1 microM; a PCO from Rhône Poulenc). Relaxation time courses for these PCOs were obtained in norepinephrine (NE; 5 microM)-precontracted RMA, and the concentrations of PCOs found to be equipotent to each other in terms of the degree of maximum relaxation (about 80%) and the time course of relaxation (within 15 min) were chosen for further study. This was taken as a functional indicator of a similar degree as well as similar kinetics of K+ channel opening by these PCOs. Pretreatment with glyburide (10-500 nM) produced a dose-dependent inhibition of the PCO relaxation time course. The glyburide IC50s against pinacidil, MNXS and RP-49356 were statistically similar and ranged from 72-79 nM. The glyburide IC50 against cromakalim was a modest 2-fold higher, at 148 nM. In contrast, pretreatment with charybdotoxin (200 nM) produced no significant inhibition of the maximum relaxation produced by these PCOs. Furthermore, glipizide, a sulfonylurea that is 10- to 25-fold less potent than glyburide for insulin secretion, was found to be 20- to 30-fold less potent than glyburide as a vascular KATP antagonist. These data suggest a mechanistic model in which these structurally diverse PCOs share a common critical step in the sequence of events leading to the KATP opening, and that glyburide interferes with this common critical step to produce a similar type of blockade against all four PCOs. Interaction studies with glyburide and pinacidil demonstrated 15 min to be the optimal pretreatment time for glyburide to produce maximal inhibition. Glyburide also reversed existing pinacidil relaxation regardless of the degree of pre-existing relaxation. These data suggest that glyburide is able to produce its blockade regardless of the state of K+ channel activation. Studies on the effect of pH (6.4 vs. 7.3) showed that at acidic pH, pinacidil became less effective and the effectiveness of glyburide was significantly enhanced, whereas the actions of D600 remained unchanged. These data suggest the effects of both openers and blockers of the KATP are strongly pH dependent.

Characterization of sulfonylurea receptors and the action of potassium channel openers on cholinergic neurotransmission in guinea pig isolated small intestine.

Specific binding sites for [3H]glibenclamide, a potent ATP-sensitive K+ channel blocker, have been characterized in the isolated guinea pig longitudinal muscle-myenteric plexus preparation. The Scatchard plot of the saturation isotherm was curvilinear and revealed two binding sites, one of high affinity (Kd = 0.42 nM; maximum binding site = 156 fmol/mg of protein), the other of low affinity (Kd = 83 nM; maximum binding site = 3100 fmol/mg of protein). Displacement experiments in the presence of various sulfonylureas showed the same order of potency for the two binding sites (glibenclamide greater than gliquidone greater than glipizide glibornuride greater than chlorpropamide greater than tolbutamide). The K+ channel opener RP 49356 (but not diazoxide or cromakalim) displaced the [3H] glibenclamide with an IC50 of 4.8 microM. The effects of the K+ channel openers diazoxide, RP 49356, cromakalim and its two optical isomers BRL 38226 and BRL 38227 were also studied on the electrically induced contractions of isolated guinea pig small intestine. These compounds produce an inhibition of neurally evoked twitch height with pD2 values of 4.5 to 6.2 (BRL 38227 greater than cromakalim greater than RP 49356 greater than diazoxide, whereas BRL 38226 was practically ineffective). The effects of cromakalim and RP 49356 were antagonized competitively by glibenclamide (pA2 = 7.2) and other sulfonylureas, suggesting they act on ATP-sensitive K+ channels. Affinities of the sulfonylureas obtained from concentration-response curves to cromakalim on electrically induced contractions are better correlated with the IC50 value corresponding to the low affinity binding site than to the high affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of thiol compounds versus the cytotoxicity of valepotriates on cultured hepatoma cells.

The antagonistic activity of various thiol compounds versus the cytotoxic effects of valtrate and didrovaltrate has been evaluated on cultured hepatoma cells. Compounds with free SH groups like cysteine, mercaptoethanol, dithioerythritol, and glutathione were able to suppress the cytotoxicity of the valepotriates in a dose-dependent way, whereas compounds with blocked SH groups did not antagonize these toxic effects. The possible interactions between the valepotriates and thiol compounds are discussed.

Glibenclamide is a competitive antagonist of cromakalim, pinacidil and RP 49356 in guinea-pig pulmonary artery.

The relaxant effect of cromakalim (BRL 34915), pinacidil and RP 49356 (N-methyl-2-(3-pyridyl)-tetrahydro-thiopyran-2-carbothioamide-1-ox ide) on the sustained contractions induced by 20 mM KCl were compared with the effects of nicorandil. The preparation used was vascular smooth muscle of phenoxybenzamine-treated pulmonary artery rings from reserpinized guinea-pigs. Cromakalim, pinacidil, RP 49356 and nicorandil relaxed the tissues with -log EC50 values of 6.78, 6.12, 6.02 and 5.46, respectively. The inhibitory effect of cromakalim, pinacidil and RP 49356, but not of nicorandil, was competitively antagonized by glibenclamide (10(-7)-3 X 10(-6) M), yielding uniform pA2 values of 7.17-7.22 against all three relaxant drugs. The order of potency of other K+ channel blocking agents for the inhibition of vasorelaxation by cromakalim, pinacidil and RP 49356 was procaine greater than 4-aminopyridine greater than tetraethylammonium. The mainly competitive type of inhibition of the RP 49356-induced response was more comparable to that with pinacidil than with cromakalim. The relaxation caused by nicorandil was only attenuated by a high concentration of 4-aminopyridine or tetraethylammonium but was markedly antagonized by methylene blue (3 X 10(-6)-10(-5) M) and potentiated by M & B 22948 (3 X 10(-6)-10(-5) M). These results suggest that the vascular relaxation caused in guinea-pig pulmonary artery by cromakalim, pinacidil and RP 49356 is mediated through the same glibenclamide-sensitive K+ channel whereas the major mechanism for the effect of nicorandil seems to involve stimulation of guanylate cyclase.

Prostaglandin and thromboxane biosynthesis inhibitors.

All acidic non-steroidal anti-inflammatory drugs suppress the activity of microsomal cyclo-oxygenase of arachidonic acid; therefore, these drugs are equipotent inhibitors of prostaglandin and thromboxane generation. A non-acidic anti-inflammatory agent, 1'-(isopropyl-2-indolyl)-3-pyridyl-3-ketone (L 8027), selectively inhibits biosynthesis of thromboxane A2 in blood platelets and in lungs.

Stability of patulin to sulfur dioxide and to yeast fermentation.

The affinity of patulin for sulfur dioxide (SO2) is much less than was previously reported and is of little significance at the SO2 concentrations (below 200 ppm) used in the processing of apple juice and cider. However, at concentrations of 2000 ppm SO2 and 15 ppm patulin, combination was 90% complete in 2 days. Removal of SO2 liberated only part of the patulin, which suggests that 2 mechanisms are involved: one reversible (opening the hemiacetal ring) and one irreversible (SO2 addition at the double bond). Test with 2 yeasts used in English commercial cider making confirmed that patulin is effectively removed during yeast fermentation.