o,p'-DDD in the mouse lung: selective uptake, covalent binding and effect on drug metabolism

By means of autoradiography a high and selective accumulation was observed in the lung alveolar region of C57Bl mice injected with o,p'-[14C]DDD. Exhaustive extraction of lung tissue showed that a large fraction of the radioactivity was covalently bound to protein. Covalent binding in liver was 20-30 times lower and represented a smaller fraction of the total radioactivity present in this tissue. Formation of a cytochrome P-450 catalysed reactive metabolite in lung and liver was indicated by a decreased covalent binding in these tissues in mice pretreated with metyrapone. Both beta-naphthoflavone (beta NF) and phenobarbital (PB) pretreatment decreased binding of o,p'-DDD in lung tissue, while binding in the liver was induced by PB but remained unaffected by beta NF. Pretreatment with high doses of o,p'-DDD and p,p'-DDT gave a significantly decreased binding of o,p'-[14C]DDD in lung, whereas binding in liver remained unchanged. Conjugation with glutathione does not appear to be a major inactivation pathway for the reactive lung metabolite, since a high dose of o,p'-DDD did not deplete non-protein thiols (NPSH) in lung tissue. Pretreatment with o,p'-DDD decreased the N-demethylation of [dimethyl-14C]aminopyrine in both lung and liver in a dose-dependent manner, suggesting that the drug-metabolizing enzyme system may be a target for o,p'-DDD in vivo.     

Kinetics of inhibition of leukocyte 12-lipoxygenase by the isoform-specific inhibitor 4-(2-oxapentadeca-4-yne)phenylpropanoic acid

Lipoxygenases (LOXs) are a ubiquitous family of enzymes that catalyze the dioxygenation of polyunsaturated fatty acids. Their role in a diverse range of biological processes has prompted the development of a large number of lipoxygenase inhibitors of possible therapeutic and probative value. The isoform-selective inhibitor 4-(2-oxapentadeca-4-yne)phenylpropanoic acid (OPP) was previously shown to inhibit leukocyte-type 12-LOX by a novel mechanism in which it binds to both the ferrous and ferric forms of the enzyme. The current study provides a detailed kinetic model of this inhibition. Nonlinear regression analysis of OPP's inhibition of arachidonic acid dioxygenation indicated mixed inhibition toward the ferric form of 12-LOX with apparent K(I) values in the low micromolar range: 2.0 +/- 0.2 microM for the free enzyme and 4.5 +/- 0.7 microM for the substrate-bound form of the enzyme. Rapid kinetic techniques allowed OPP's inhibition of the activation of the enzyme from the ferrous to the ferric form to be investigated. Titration of ferrous 12-LOX with OPP indicated that it bound to the ferrous form with an apparent K(I) value of 70 +/- 20 nM, suggesting a significantly higher affinity for the ferrous form than for the ferric form of the enzyme. Investigation of the LOX inhibitors nordihydroguaiaretic acid, N-(4-chlorophenyl)-N-hydroxy-N'-(3-chlorophenyl)urea, BWA137C, and eicosatetraynoic acid revealed that eicosatetraynoic acid also inhibited the activation of 12-LOX. These results demonstrate that LOX inhibitors are capable of binding to multiple forms of LOXs with high affinity and suggest that inhibition of enzyme activation may be an unrecognized mechanism of inhibition of additional LOX inhibitors.     

N-(4-chlorophenyl)-N-hydroxy-N'-(3-chlorophenyl)urea, a general reducing agent for 5-, 12-, and 15-lipoxygenases and a substrate for their pseudoperoxidase activities.

Lipoxygenases contain a nonheme iron that undergoes oxidation and reduction during the catalytic cycle. The conversion from the Fe3+ enzyme form to the Fe2+ form can be achieved using reducing inhibitors, a reaction that can be reversed with lipid hydroperoxides. The present study describes the properties of N-(4-chlorophenyl)-N-hydroxy-N'-(3-chlorophenyl)urea (CPHU), which functions as a reducing agent for various lipoxygenases and stimulates the degradation of lipid hydroperoxide catalyzed by these enzymes (pseudoperoxidase activity). CPHU was a substrate for the pseudoperoxidase reaction of purified soybean lipoxygenase-1 with apparent Km values for CPHU and 13-hydroperoxy-9,11-octadecadienoic acid (13-HpODE) of 14 and 15 microM, respectively. CPHU was converted during the pseudoperoxidase reaction to a mixture of products that can be resolved by reverse-phase high pressure liquid chromatography. By comparison with the chemical reaction of CPHU and potassium nitrosodisulfonate, the major enzymatic reaction product was tentatively identified as a one-electron oxidation product of CPHU. At low concentrations (50 microM), dithiothreitol completely protected against the degradation of hydroxyurea without inhibiting the pseudoperoxidase reaction. Under these conditions, the rate of the pseudoperoxidase reaction with CPHU as a substrate can be quantitated by the change in absorbance at 234 nm owing to the consumption of 13-HpODE. In addition to soybean lipoxygenase-1, CPHU was found to be a substrate for the pseudoperoxidase activities of purified recombinant human 5-lipoxygenase and porcine leukocyte 12-lipoxygenase. The results are consistent with CPHU reacting with lipoxygenase by a one-electron oxidation to generate the ferrous enzyme form and the nitroxide radical, which could be reduced back to CPHU by DTT.(ABSTRACT TRUNCATED AT 250 WORDS)     

The profile of urinary metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in rats is determined by its pulmonary metabolism.

Metabolism of the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in rats was compared to metabolism in primary lung and liver cells. Untreated rats and rats pretreated with phenobarbital, acetone or phenethyl isothiocyanate (PEITC) were used for all experiments. Also the influence of [-]-1-methyl-2-[3-pyridyl]-pyrrolidine (nicotine) administered concomitantly with NNK, or incubated with isolated cells, upon NNK metabolism was investigated and found to be only marginal upon alpha-hydroxylation and pyridine N-oxidation in vivo. In hepatocytes nicotine inhibited NNK pyridine N-oxidation, alpha-hydroxylation and glucuronidation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), whereas in lung cells the influence of nicotine was not as pronounced. In vivo phenobarbital induced alpha-hydroxylation and pyridine N-oxidation. In vitro the effects of the modulators were most pronounced upon hepatocytes, where phenobarbital greatly induced pyridine N-oxidation and PEITC inhibited alpha-hydroxylation. NNAL was conjugated to its beta-glucuronide in lung cells at four times higher rates than in hepatocytes. The ratios of the sum of N-oxides to the sum of alpha-hydroxylation products in vivo were similar to those in lung cells, especially at low NNK concentrations (1 microM), while in hepatocytes alpha-hydroxylation was more pronounced. The same correlation of metabolism in isolated lung cells with whole rats was observed if oxidative NNAL metabolism was related to oxidative NNK metabolism. Here hepatocytes showed a much higher formation of NNAL oxidation products than either lung cells formed, or rats excreted in urine. This was true despite a lower rate of metabolism in the lung than in liver if based on cell number, the rate based on mg protein was four times higher in lung than liver. Only after phenobarbital treatment was the contribution of hepatic metabolism to excreted metabolites important. In conclusion the lung which is also the target of NNK carcinogenesis, and not the liver, is the organ with the most important contribution to NNK and NNAL metabolism at concentrations relevant to human exposure.     

Identification of a butyrophenone analog as a potential atypical antipsychotic agent: 4-[4-(4-chlorophenyl)-1,4-diazepan-1-yl]-1-(4-fluorophenyl)butan-1-one

The synthesis and exploration of novel butyrophenones have led to the identification of a diazepane analogue of haloperidol, 4-[4-(4-chlorophenyl)-1,4-diazepan-1-yl]-1-(4-fluorophenyl)butan-1-one (compound 13) with an interesting multireceptor binding profile. Compound 13 was evaluated for its binding affinities at DA subtype receptors, 5HT subtype receptors, H-1, M-1 receptors and at NET, DAT, and SERT transporters. At each of these receptors, compound 13 was equipotent or better than several of the standards currently in use. In in vivo mouse and rat models to evaluate its efficacy and propensity to elicit catalepsy and hence EPS in humans, compound 13 showed similar efficacy as clozapine and did not produce catalepsy at five times its ED(50) value.     

Discovery of 2-(4-((1H-1,2,4-triazol-1-yl)methyl)-5-(4-bromophenyl)-1-(2-chlorophenyl)-1H-pyrazol-3-yl)-5-tert-butyl-1,3,4-thiadiazole (GCC2680) as a potent,selective and orally efficacious cannabinoid-1 receptor antagonist

Structure–activity relationship studies in a series of diarylpyrazolyl thiadiazoles identified cannabinoid-1 receptor antagonists with excellent potency and selectivity. Based on its exceptional in vivo efficacy in animal models and its favorable pharmacokinetic and toxicological profiles, 2-(4-((1H-1,2,4-triazol-1-yl)methyl)-5-(4-bromophenyl)-1-(2-chlorophenyl)-1H-pyrazol-3-yl)-5-tert-butyl-1,3,4-thiadiazole (GCC2680) was selected as a preclinical candidate for the treatment of obesity.     

O(2)-evoked regulation of HIF-1alpha and NF-kappaB in perinatal lung epithelium requires glutathione biosynthesis

To test the genetic capacity of the perinatal lung to respond to O(2) shifts that coincide with the first respiratory movements, rat fetal alveolar type II (fATII) epithelial cells were cultured at fetal distal lung PO(2) (23 Torr) and then exposed to postnatal (23 --> 76 Torr; mild hyperoxic shift), moderate (23 --> 152 Torr; moderate hyperoxic shift), or severe (23 --> 722 Torr; severe hyperoxic shift) oxygenation. Nuclear abundance and consensus binding characteristics of hypoxia-inducible factor (HIF)-1alpha and nuclear factor (NF)-kappaB (Rel A/p65) plus glutathione biosynthetic capacity were determined. Maximal HIF-1alpha activation at 23 Torr was sustained over the postnatal shift in (Delta) PO(2) and was elevated in vivo throughout late gestation. NF-kappaB was activated by the acute postnatal DeltaPO(2) in fATII cells, becoming maximal with moderate and severe oxygenation in vitro and within 6 h of birth in vivo, declining thereafter. fATII cell and whole lung glutathione and GSH-to-GSSG ratio increased fourfold with a postnatal DeltaPO(2) and were matched by threefold activity increases in gamma-glutamylcysteine synthetase and glutathione synthase. GSH concentration depletion by L-buthionine-(S, R)-sulfoximine abrogated both HIF-1alpha and NF-kappaB activation, with HIF-1alpha showing a heightened sensitivity to GSH concentration. We conclude that O(2)-linked genetic regulation in perinatal lung epithelium is responsive to developmental changes in glutathione biosynthetic capacity.     

2(3)-tert-butyl-4-hydroxyanisole inhibits oxidative metabolism of aflatoxin B1 in isolated rat hepatocytes

Previous studies indicate that dietary administration of phenolic antioxidants, 2(3)-tert-butyl-4-hydroxyanisole (BHA) and 3,5-di-tert-butyl-4-hydroxytoluene, inhibits the carcinogenic effect of a number of chemical carcinogens including aflatoxin B1 (AFB1). Induction of hepatic enzymes, such as glutathione S-transferase, UDP-glucuronyltransferase, and epoxide hydrolase, has been shown to be responsible for the reduction of AFB1 cytotoxic and carcinogenic effects. The effect of BHA on AFB1 activation was examined in vitro utilizing isolated rat hepatocytes and liver microsomes. In hepatocytes, the total AFB1 content and bound form of AFB1 were 3.4 and 1.4 pmol/10(6) cells, respectively. In the cell-free microsomal activating system, 2.2 pmol were activated per mg of microsomal protein during 60 min of incubation. BHA (0.1-0.5 mM) inhibited AFB1 activation and binding in both systems in a dose-dependent manner; in hepatocytes, 90% inhibition was observed at 0.5 mM. Analyzing various AFB1 adducts, BHA (0.25 mM)-treated hepatocytes contained a significantly reduced amount of AFB1 macromolecular adducts. The antioxidant neither stimulated nor inhibited the cytosolic glutathione S-transferase and microsomal UDP-glucuronyltransferase activities. Analysis of various hydroxylated (aflatoxins M1 and Q1 (AFM1 and AFQ1] and demethylated (aflatoxin P1 (AFP1] metabolites of AFB1 in both the conjugated and unconjugated form indicated that there was a 30-50% reduction of unconjugated AFP1, AFQ1, and AFM1, whereas AFB1 was increased 3-fold. There was no significant change of conjugated metabolites. The effect of BHA on AFB1 activation in hepatocytes was compared with that of other cytochrome P-450 inhibitors; the ED50 values of SKF 525A, BHA, and metyrapone were 9 microM, 40 microM, and 280 microM, respectively. In the cell-free microsomal system, biotransformation of AFB1 to AFP1, AFM1, and AFQ1 was also inhibited. Kinetic analysis of p-nitroanisole O-demethylase activity of rat liver microsomes demonstrated that BHA inhibited noncompetitively with an apparent Ki of 90 microM. In the absence of enzyme induction, the phenolic antioxidant, BHA, blocks the oxidative biotransformation of AFB1 in isolated hepatocytes.     

Pesticide-induced changes in hepatic microsomal enzyme systems: further studies on the effects of 1,1,-di(p-chlorophenyl)-2-chloroethylene (DDMU) in the Japanese quail.

Changes in the liver resulting from the low level dietary administration of 1,1-di(p-chlorophenyl)-2-chloroethylene (DDMU),p,p'-DDT, o,p'-DDT, p,p'-DDD and p,p'-DDE to Japanese Quail have been monitored. DDMU was exceptional in causing substantial increases in relative liver wt. and hepatic glucose-6-phosphatase after feeding at 100 ppm for 28 days. The time course of liver enzyme induction by DDMU has also been studied in Japanese Quail after periods of dietary administration ranging from 1--28 days with particular reference to changes in hepatic cytochrome P-450 and relative liver wt. Structural changes in the liver have been followed by reference to protein and lipid components. The hepatic response to DDMU appears to be biphasic. Initially there are substantial increases in hepatic cytochrome P-450 and relative liver wt., but the latter is largely due to accumulation of triglycerides. After approximately 20 days the level of hepatic cytochrome P-450 remain at a high 'plateau' level. This secondary phase of liver induction probably involves cell proliferation. It is concluded that DDMU causes major changes in the avian liver and either directly or through a metabolite causes pronounced microsomal enzyme induction.     

Structural and thermodynamic consequences of 1-(4-chlorophenyl)imidazole binding to cytochrome P450 2B4

The crystal structure of P450 2B4 bound with 1-(4-chlorophenyl)imidazole (1-CPI) has been determined to delineate the structural basis for the observed differences in binding affinity and thermodynamics relative to 4-(4-chlorophenyl)imidazole (4-CPI). Compared with the previously reported 4-CPI complex, there is a shift in the 1-CPI complex of the protein backbone in helices F and I, repositioning the side chains of Phe-206, Phe-297, and Glu-301, and leading to significant reshaping of the active site. Phe-206 and Phe-297 exchange positions, with Phe-206 becoming a ligand-contact residue, while Glu-301, rather than hydrogen bonding to the ligand, flips away from the active site and interacts with His-172. As a result the active site volume expands from 200 A3 in the 4-CPI complex to 280 A3 in the 1-CPI complex. Based on the two structures, it was predicted that a Phe-206-->Ala substitution would alter 1-CPI but not 4-CPI binding. Isothermal titration calorimetry experiments indicated that this substitution had no effect on the thermodynamic signature of 4-CPI binding to 2B4. In contrast, relative to wild-type 1-CPI binding to F206A showed significantly less favorable entropy but more favorable enthalpy. This result is consistent with loss of the aromatic side chain and possible ordering of water molecules, now able to interact with Glu-301 and exposed residues in the I-helix. Hence, thermodynamic measurements support the active site rearrangement observed in the crystal structure of the 1-CPI complex and illustrate the malleability of the active site with the fine-tuning of residue orientations and thermodynamic signatures.     

Characterization of covalent binding of [14C]-2-chloro-4-acetotoluidide to microsomes of starling liver and kidney

In this study, we have characterized the covalent binding of [14C]-2-chloro-4-acetotoluidide (CAT) radioactivity to microsomes of starling liver and kidney. The maximal velocity (Vmax) of covalent binding and apparent Michaelis constant (Km) for both tissues were similar. The Vmax for liver and kidney were 52.8 and 68.9 pmol/min/mg protein, and the apparent Kms were 0.54 and 0.87 mM, respectively. The covalent binding of radioactivity to heat-denatured microsomes of liver and kidney was reduced by 62% and 15%, respectively. Incubation at 0 degrees C reduced the binding by 80% to liver and 70% to kidney microsomes. Absence of nicotinamide adenine dinucleotide phosphate (NADP) and molecular O2 reduced the binding to liver microsomes by 36 and 53%, as opposed to 28% increase and 26% decrease in binding to kidney microsomes, respectively. Inducers of cytochrome P450 monooxygenase (P450), phenobarbital, and 3-methylcholanthrene (3-MC), had opposite effects on the covalent binding of [14C]-CAT radioactivity to hepatic and renal microsomes. Phenobarbital increased the binding to hepatic microsomes by 100% and had no effect on binding to renal microsomes. 3-MC, on the other hand, increased the binding to kidney microsomes by threefold and had no effect on the binding to hepatic microsomes. SKF 525A, an inhibitor of P450, inhibited the binding to hepatic microsomes by 60% at 0.5 mM but failed to have any effect on binding to renal microsomes. alpha-Naphthoflavone, another inhibitor of P450, had no effect on the covalent binding of [14C]-CAT radioactivity to microsomes of either tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of species, MFO inducers and conjugation agents on the in vitro covalent binding of 14C-3-methylindole metabolite in liver and lung tissues

The degree of tissue covalent binding of 14C-3-methylindole metabolite in goat and rat pretreated with phenobarbital or 3-methylcholanthrene was compared. The effect of conjugating agents, i.e. glutathione (GSH), cysteine and sulfate, in reducing the degree of tissue covalent binding was measured. The degree of tissue covalent binding was significantly higher in the lung than the liver of goats. In rats, covalent binding was higher in the liver than the lung. Glutathione and cysteine were effective in decreasing the degree of in vitro covalent binding in both liver and lung tissues of goat and rat.     

Bioisosteric replacement of dihydropyrazole of 4S-(-)-3-(4-chlorophenyl)-N-methyl-N'-[(4-chlorophenyl)-sulfonyl]-4-phenyl-4,5-dihydro-1H-pyrazole-1-caboxamidine (SLV-319) a potent CB1 receptor antagonist by imidazole and oxazole

Design, synthesis and conformational analysis of few imidazole and oxazole as bioisosters of 4S-(-)-3-(4-chlorophenyl)-N-methyl-N'-[(4-chlorophenyl)-sulfonyl]-4-phenyl-4,5-dihydro-1H-pyrazole-1-caboxamidine (SLV-319) 2 is reported. Computer assisted conformational analysis gave a direct clue for the loss of CB1 antagonistic activity of the ligands without a fine docking simulation for the homology model.     

Organ-specific DNA damage of tris(2,3-dibromopropyl)-phosphate and its diester metabolite in the rat.

The organ specificity of tris(2,3-dibromopropyl)phosphate(Tris-BP)-induced DNA damage was investigated in the rat 2 h after a single i.p. injection of 350 mumol/kg. Extensive DNA damage, measured with the alkaline elution method, was found in the kidney, liver and small intestine. Less, but significant DNA damage was detected in the brain, lung, spleen, large intestine and testis. The role of different pathways in the activation of Tris-BP to DNA damaging products was studied in isolated liver and testicular cells. Concentrations as low as 2.5-5 microM Tris-BP caused DNA damage in the hepatocytes, whereas an approximately 10-fold higher concentration was needed in testicular cells to produce a similar amount of DNA damage. Depletion of GSH by diethyl maleate (DEM) did not affect the extent of DNA damage caused by Tris-BP in the liver cells, but blocked the genotoxic effect in testicular cells. Two specifically deuterated Tris-BP analogs, C3D2-Tris-BP and C2D1-Tris-BP, were significantly less potent in causing DNA damage than the protio compound in isolated liver cells and were somewhat less potent in testicular cells. The major urinary metabolite of Tris-BP, bis(2,3-dibromopropyl)phosphate (Bis-BP), was less potent than Tris-BP in causing kidney DNA damage after in vivo exposure. Furthermore, Bis-BP induced substantially less DNA damage in isolated liver and testicular cells. Similar to the effect of DEM on the DNA damage caused by Tris-BP, the DNA damage caused by Bis-BP could be decreased by DEM-pretreatment in testicular cells but not in liver cells. The present study shows that Tris-BP is a potent multiorgan genotoxic agent in vivo. The in vitro data indicate that P-450 mediated metabolism of Tris-BP is more important than activation by glutathione S-transferases of Tris-BP in liver cells, whereas the latter activation pathway seems to be most important in testicular cells.     

Glutathione S-transferase catalyzes the isomerization of (R)-2-hydroxymenthofuran to mintlactones.

(R)-(+)-Menthofuran is the proximate toxic metabolite of pulegone, the major constituent of the pennyroyal oil, that contributes significantly to the hepatotoxicity resulting from ingestion of this folklore abortifacient pennyroyal oil. Recently, menthofuran was shown to be metabolized by cytochrome P450 to form (R)-2-hydroxymenthofuran. In this paper it is demonstrated that glutathione S-transferase (GST) catalyzes the tautomerization of 2-hydroxymenthofuran to mintlactone and isomintlactone, apparently without the formation of stable glutathione (GSH) conjugates. The reaction strictly required GSH; S-methyl GSH, which binds to the active site and leaves the active site Tyr-9 partly ionized, did not support GST-catalyzed isomerization. It was also determined that the tautomerization reaction requires the active site tyrosine, Tyr-9. The rat GSTA1-1 mutant (Y9F), with the active site tyrosine replaced with phenylalanine, demonstrated no catalytic activity. Rat cytosolic GST A1-1, in the presence of GSH, tautomerized 2-hydroxymenthofuran with apparent K(M) and V(max) values of 110 microM and 190 nmol/min/nmol GST, respectively. However, the site-directed mutant (F220Y), in which Tyr-9 and GSH in the binary complex [GST. GSH] have lower pK(a)s, exhibited K(M) and V(max) values of 97 microM and 280 nmol/min/nmol GST, respectively. Similarly, human liver cytosol catalyzed the tautomerization of 2-hydroxymenthofuran in a GST-dependent reaction. The mechanism most consistent with the data is a general-base catalyzed isomerization with GS(-) serving to deprotonate the substrate to initiate the reaction.     

Metabolic activation of the herbicide dichlobenil in the olfactory mucosa of mice and rats

The metabolic activation of the herbicide dichlobenil (2,6-dichloro[ring-14C]benzonitrile) in the olfactory mucosa of C57BL mice and Sprague-Dawley rats was examined. In homogenates of the olfactory mucosa (mouse 1000 x g supernatants; rat microsomes), dichlobenil was metabolized and covalently bound to protein. The apparent Km, Vmax and V/K values showed that the olfactory mucosa had both a higher affinity for dichlobenil and a higher capacity/mg protein to activate dichlobenil in comparison to the liver. The covalent binding was dependent on NADPH and was inhibited by the addition of dithionite, metyrapone and glutathione indicating an oxidative cytochrome P-450 dependent activation of dichlobenil into an electrophilic intermediate. The covalent binding was also inhibited by the addition of superoxide dismutase whereas catalase, mannitol or dimethylsulfoxide had no effect indicating the involvement of O2- but not of H2O2 or OH. in the activation. In explants of the olfactory mucosa incubated with [14C]dichlobenil a preferential covalent binding was observed in the Bowman's glands suggesting an activation of dichlobenil in these structures. The highly efficient metabolic activation of dichlobenil to reactive intermediates in the olfactory mucosa is suggested to be of importance for the potent dichlobenil-induced toxicity in this tissue.     

Resolution, configurational assignment, and enantiopharmacology of 2-amino-3-[3-hydroxy-5-(2-methyl-2H- tetrazol-5-yl)isoxazol-4-yl]propionic acid, a potent GluR3- and GluR4-preferring AMPA receptor agonist

We have previously shown that (RS)-2-amino-3-[3-hydroxy-5-(2-methyl-2H-tetrazol-5-yl)isoxazol -4-yl] propionic acid (2-Me-Tet-AMPA) is a selective agonist at (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors, markedly more potent than AMPA itself, whereas the isomeric compound 1-Me-Tet-AMPA is essentially inactive. We here report the enantiopharmacology of 2-Me-Tet-AMPA in radioligand binding and cortical wedge electrophysiological assay systems, and using cloned AMPA (GluR1-4) and kainic acid (KA) (GluR5, 6, and KA2) receptor subtypes expressed in Xenopus oocytes. 2-Me-Tet-AMPA was resolved using preparative chiral HPLC. Zwitterion (-)-2-Me-Tet-AMPA was assigned the (R)-configuration based on an X-ray crystallographic analysis supported by the elution order of (-)- and (+)-2-Me-Tet-AMPA using four different chiral HPLC columns and by circular dichroism spectra. None of the compounds tested showed detectable affinity for N-methyl-D-aspartic acid (NMDA) receptor sites, and (R)-2-Me-Tet-AMPA was essentially inactive in all of the test systems used. Whereas (S)-2-Me-Tet-AMPA showed low affinity (IC(50) = 11 microM) in the [(3)H]KA binding assay, it was significantly more potent (IC(50) = 0.009 microM) than AMPA (IC(50) = 0.039 microM) in the [(3)H]AMPA binding assay, and in agreement with these findings, (S)-2-Me-Tet-AMPA (EC(50) = 0.11 microM) was markedly more potent than AMPA (EC(50) = 3.5 microM) in the electrophysiological cortical wedge model. In contrast to AMPA, which showed comparable potencies (EC(50) = 1.3-3.5 microM) at receptors formed by the AMPA receptor subunits (GluR1-4) in Xenopus oocytes, more potent effects and a substantially higher degree of subunit selectivity were observed for (S)-2-Me-Tet-AMPA: GluR1o (EC(50) = 0.16 microM), GluR1o/GluR2i (EC(50) = 0.12 microM), GluR3o (EC(50) = 0.014 microM) and GluR4o (EC(50) = 0.009 microM). At the KA-preferring receptors GluR5 and GluR6/KA2, (S)-2-Me-Tet-AMPA showed much weaker agonist effects (EC(50) = 8.7 and 15.3 microM, respectively). It is concluded that (S)-2-Me-Tet-AMPA is a subunit-selective and highly potent AMPA receptor agonist and a potentially useful tool for studies of physiological AMPA receptor subtypes.     

Triazole analog 1-(1-benzyl-5-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)-2-(4-bromophenylamino)-1-(4-chlorophenyl)ethanol induces reactive oxygen species and autophagy-dependent apoptosis in both in vitro and in vivo breast cancer models

Autophagy is considered as an important cell death mechanism that closely interacts with other common cell death programs like apoptosis. Critical role of autophagy in cell death makes it a promising, yet challenging therapeutic target for cancer. We identified a series of 1,2,3-triazole analogs having significant breast cancer inhibition property. Therefore, we attempted to study whether autophagy and apoptosis were involved in the process of cancer cell inhibition. The lead molecule, 1-(1-benzyl-5-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)-2-(4-bromophenylamino)-1-(4-chlorophenyl)ethanol (T-12) induced significant cell cycle arrest, mitochondrial membrane depolarization, apoptosis and autophagy in MCF-7 and MDA-MB-231 cells. T-12 increased reactive oxygen species and its inhibition by N-acetyl-l-cysteine protected breast cancer cells from autophagy and apoptosis. Autophagy inhibitor, 3-methyladenine abolished T-12 induced apoptosis, mitochondrial membrane depolarization and reactive oxygen species generation. This suggested that T-12 induced autophagy facilitated cell death rather than cell survival. Pan-caspase inhibition did not abrogate T-12 induced autophagy, suggesting that autophagy precedes apoptosis. In addition, T-12 inhibited cell survival pathway signaling proteins, Akt, mTOR and Erk1/2. T-12 also induced significant regression of tumor with oral dose of as low as 10 mg/kg bodyweight in rat mammary tumor model without any apparent toxicity. In presence of reactive oxygen species inhibitor (N-acetyl-l-cysteine) and autophagy inhibitor (chloroquine), T-12 induced tumor regression was significantly decreased. In conclusion, T-12 is a potent inducer of autophagy-dependent apoptosis in breast cancer cells both in vitro and in vivo and can serve as an important lead in development of new anti-tumor therapy.     

Intracellular aflatoxin B1-binding proteins in rat liver

Intracellular aflatoxin B1 binding in rat liver was studied under both in vitro and in vivo conditions. Binding in vivo appeared similar to that observed in vitro except that some covalent adduct formation was detected. Participation of previously described carcinogen-binding proteins such as the Ah receptor, h2-5S protein, 4-5S receptor for 3-methylcholanthrene and the Z-protein fraction was discounted on the grounds of competition binding studies and gel-permeation chromatography. The molecular weight of 45,000 was estimated for the major aflatoxin B1-binding component. Aflatoxin B1 co-eluted with the glutathione S-transferases during gel-permeation and separation of the various isozymes by cation-exchange chromatography indicated interactions with the YaYa and YaYc-forms. These proteins, however, account for less than 20% of the total intracellular aflatoxin binding. A protein of apparent monomeric structure appears to form the major in vitro/in vivo complex with aflatoxin B1.     

Allosteric inhibition of glycogen phosphorylase a by the potential antidiabetic drug 3-isopropyl 4-(2-chlorophenyl)-1,4-dihydro-1-ethyl-2-methyl-pyridine-3,5,6-tricarbo xylate.

The effect of the potential antidiabetic drug (-)(S)-3-isopropyl 4-(2-chlorophenyl)-1,4-dihydro-1-ethyl-2-methyl-pyridine-3,5,6-tricarbox ylate (W1807) on the catalytic and structural properties of glycogen phosphorylase a has been studied. Glycogen phosphorylase (GP) is an allosteric enzyme whose activity is primarily controlled by reversible phosphorylation of Ser14 of the dephosphorylated enzyme (GPb, less active, predominantly T-state) to form the phosphorylated enzyme (GPa, more active, predominantly R-state). Upon conversion of GPb to GPa, the N-terminal tail (residues 5-22), which carries the Ser14(P), changes its conformation into a distorted 3(10) helix and its contacts from intrasubunit to intersubunit. This alteration causes a series of tertiary and quaternary conformational changes that lead to activation of the enzyme through opening access to the catalytic site. As part of a screening process to identify compounds that might contribute to the regulation of glycogen metabolism in the noninsulin dependent diabetes diseased state, W1807 has been found as the most potent inhibitor of GPb (Ki = 1.6 nM) that binds at the allosteric site of T-state GPb and produces further conformational changes, characteristic of a T'-like state. Kinetics show W1807 is a potent competitive inhibitor of GPa (-AMP) (Ki = 10.8 nM) and of GPa (+1 mM AMP) (Ki = 19.4 microM) with respect to glucose 1-phosphate and acts in synergism with glucose. To elucidate the structural features that contribute to the binding, the structures of GPa in the T-state conformation in complex with glucose and in complex with both glucose and W1807 have been determined at 100 K to 2.0 A and 2.1 A resolution, and refined to crystallographic R-values of 0.179 (R(free) = 0.230) and 0.189 (R(free) = 0.263), respectively. W1807 binds tightly at the allosteric site and induces substantial conformational changes both in the vicinity of the allosteric site and the subunit interface. A disordering of the N-terminal tail occurs, while the loop of chain containing residues 192-196 and residues 43'-49' shift to accommodate the ligand. Structural comparisons show that the T-state GPa-glucose-W1807 structure is overall more similar to the T-state GPb-W1807 complex structure than to the GPa-glucose complex structure, indicating that W1807 is able to transform GPa to the T'-like state already observed with GPb. The structures provide a rational for the potency of the inhibitor and explain GPa allosteric inhibition of activity upon W1807 binding.