Alpha-hydroxytropolones are noncompetitive inhibitors of human RNase H1 that bind to the active site and modulate substrate binding.

The ribonucleases H (RNases H) of HIV and hepatitis B virus are type 1 RNases H that are promising drug targets because inhibiting their activity blocks viral replication. Eukaryotic ribonuclease H1 (RNase H1) is an essential protein and a probable off-target enzyme for viral RNase H inhibitors. α-hydroxytropolones (αHTs) are a class of anti-RNase H inhibitors that can inhibit the HIV, hepatitis B virus, and human RNases H1; however, it is unclear how these inhibitors could be developed to distinguish between these enzymes. To accelerate the development of selective RNase H inhibitors, we performed biochemical and kinetic studies on the human enzyme, which was recombinantly expressed in Escherichia coli. Size-exclusion chromatography showed that free RNase H1 is monomeric and forms a 2:1 complex with a substrate of 12 bp. FRET heteroduplex cleavage assays were used to test inhibition of RNase H1 in steady-state kinetics by two structurally diverse αHTs, 110 and 404. We determined that turnover rate was reduced, but inhibition was not competitive with substrate, despite inhibitor binding to the active site. Given the compounds' reversible binding to the active site, we concluded that traditional noncompetitive and mixed inhibition mechanisms are unlikely. Instead, we propose a model in which, by binding to the active site, αHTs stabilize an inactive enzyme-substrate-inhibitor complex. This new model clarifies the mechanism of action of αHTs against RNase H1 and will aid the development of RNase H inhibitors selective for the viral enzymes.

Potent and selective NOP receptor activation reduces cocaine self-administration in rats by lowering hedonic set point.

Developing new medications for the treatment of cocaine dependence continues to be a research priority. Compelling evidence indicates that mixed opioid receptor agonists, particularly bifunctional compounds that target nociceptin/orphanin FQ peptide (NOP) and mu opioid receptors, may be useful for the treatment of cocaine addiction. Here, we verify that potent and selective pharmacological activation of NOP receptors is sufficient to reduce relevant facets of cocaine addiction in animal models. Accordingly, we determined whether systemic injections of the small molecule AT-312 (0, 1, 3 mg/kg) could reduce operant cocaine self-administration, motivation for cocaine, and vulnerability to cocaine relapse in rats. Results indicate that a potent and selective NOP receptor agonist was equally efficacious in reducing the number of cocaine infusions in short (1-hour), as well as long (6-hour) access sessions. When tested on an economic-demand reinforcement schedule, AT-312 reduced Q0 , the parameter that describes the amount of drug consumed at zero price, while leaving the parameter α, a measure of motivation for drug consumption, unaltered. Furthermore, AT-312 successfully reduced conditioned reinstatement of cocaine seeking. In contrast, the NOP receptor agonist did not modify food self-administration. Blockade of the NOP receptor with the antagonist SB-612111 prevented the effect of AT-312 in decreasing cocaine-reinforced responding under a 2-hour fixed ratio 1 schedule, suggesting a NOP receptor-mediated mechanism. This work demonstrates that potent and selective activation of NOP receptors is sufficient to decrease cocaine taking and seeking behaviors in rats.

Inorganic polyphosphate is required for sustained free mitochondrial calcium elevation, following calcium uptake.

Mitochondrial free calcium is critically linked to the regulation of cellular metabolism. Free ionic calcium concentration within these organelles is determined by the interplay between two processes: exchange across the mitochondrial inner membrane and calcium-buffering within the matrix. During stimulated calcium uptake, calcium is primarily buffered by orthophosphate, preventing calcium toxicity while allowing for well-regulated yet elevated calcium loads. However, if limited to orthophosphates only, this buffering system is expected to lead to the irreversible formation of insoluble precipitates, which are not observed in living cells, under physiological conditions. Here, we demonstrate that the regulation of free mitochondrial calcium requires the presence of free inorganic polyphosphate (polyP) within the organelle. We found that the overexpression of a mitochondrial-targeted enzyme hydrolyzing polyP leads to the loss of the cellular ability to maintain elevated calcium concentrations within the organelle, following stimulated cytoplasmic signal. We hypothesize that the presence of polyP prevents the formation of calcium-phosphate insoluble clusters, allowing for the maintenance of elevated free calcium levels, during stimulated calcium uptake.

Nociceptin/orphanin FQ receptor activation attenuates antinociception induced by mixed nociceptin/orphanin FQ/mu-opioid receptor agonists.

Activation of brain nociceptin/orphanin FQ (NOP) receptors leads to attenuation of mu-opioid receptor (MOP receptor)-mediated antinociception. Buprenorphine, a high-affinity partial MOP receptor agonist also binds to NOP receptors with 80 nM affinity. The buprenorphine-induced inverted U-shaped dose-response curve for antinociception may be due to NOP receptor activation, given that, in the presence of the NOP receptor antagonist, 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (J113397), or in NOP receptor knockout mice, buprenorphine has a steeper dose-response curve and acts as a full agonist. To further explore the involvement of the direct activation of NOP receptors by buprenorphine and other compounds that activate both NOP and MOP receptors, the antinociceptive effects of 1-(1-(2,3,3alpha,4,5,6-hexahydro-1H-phenalen-1-yl)piperidin-4-yl)-indolin-2-one. (SR16435), 3-ethyl-1-(1-(4-isopropylcyclohexyl)piperidin-4-yl)-indolin-2-one (SR16507), buprenorphine, pentazocine, and morphine, compounds with varying levels of MOP and NOP receptor affinity and efficacy, were assessed in mice using the tail-flick assay. The ability of the selective NOP receptor antagonist (-)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB-612111) to potentiate antinociception induced by the above compounds was examined to investigate whether activation of NOP receptors leads to attenuation of MOP receptor-mediated antinociception. SB-612111 potentiated antinociception induced by buprenorphine and the other mixed NOP/MOP receptor agonists SR16435 and SR16507. However, SB-612111 had no effect on pentazocine or morphine antinociception, two compounds with no NOP receptor-binding affinity. These results further support the hypothesis that activation of NOP receptors can lead to attenuation of MOP receptor-mediated antinociception elicited by mixed NOP/MOP receptor compounds such as buprenorphine, SR16435, and SR16507 and that, although buprenorphine has low efficacy in vitro, it has significant NOP receptor agonist activity in vivo.

Antinociceptive profile of a selective metabotropic glutamate receptor 1 antagonist YM-230888 in chronic pain rodent models.

Metabotropic glutamate receptor 1 (mGlu(1) receptor) has been suggested to play an important role in pain transmission. In this study, the effects of a newly-synthesized mGlu(1) receptor antagonist, (R)-N-cycloheptyl-6-({[(tetrahydro-2-furyl)methyl]amino}methyl)thieno[2,3-d]pyrimidin-4-ylamine (YM-230888), were examined in a variety of rodent chronic pain models in order to characterize the potential analgesic profile of mGlu(1) receptor blockade. YM-230888 bound an allosteric site of mGlu(1) receptor with a K(i) value of 13+/-2.5 nM and inhibited mGlu(1)-mediated inositol phosphate production in rat cerebellar granule cells with an IC(50) value of 13+/-2.4 nM. It showed selectivity for mGlu(1) versus mGlu(2)-mGlu(7) subtypes and ionotropic glutamate receptors. YM-230888 recovered mechanical allodynia with an ED(50) value of 8.4 mg/kg p.o. in L5/L6 spinal nerve ligation models. It also showed antinociceptive response at doses of 10 and 30 mg/kg p.o. in streptozotocin-induced hyperalgesia models. In addition, it significantly reduced pain parameters at a dose of 30 mg/kg p.o. in complete Freund's adjuvant-induced arthritic pain models. Although YM-230888 showed no significant effect on rotarod performance time at doses of 10 or 30 mg/kg p.o., it significantly decreased it at a dose of 100 mg/kg p.o. On the other hand, YM-230888 showed no significant sedative effect in locomotor activity measurement up to 100 mg/kg p.o. These results suggest that the blockade of mGlu(1) receptors is an attractive target for analgesics. YM-230888 has potential as a new analgesic agent for the treatment of various chronic pain conditions. In addition, YM-230888 may be a useful tool for the investigation of mGlu(1) receptors.

Hemisynthesis, Antitumoral Effect, and Molecular Docking Studies of Ferutinin and Its Analogues.

The natural product ferutinin was shown to act as an agonist to estrogen receptor ERα and agonist/antagonist to ERß featuring a weak antiproliferative activity toward breast cancer cells. To enhance this activity, ferutinin analogues were synthesized by esterification of jaeschkenadiol with different acids. These compounds were assayed for their in vitro antiproliferative activity against estrogen-dependent (MCF-7) and estrogen-independent (MDA-MB-231) breast cancer cell lines. Among the compounds, 3c' exhibited a potent inhibitory selective activity against MCF-7 with IC50 value of 1 µM. Docking simulation of 3c' in the ligand binding domain of the ERs indicated a potential antagonism interaction with both ER subtypes. Functional assay showed that 3c' binds as an antagonist to ERα protein while ferutinin acts as an agonist.

In vitro functional characterization of novel nociceptin/orphanin FQ receptor agonists in recombinant and native preparations.

Nociceptin/Orphanin FQ (N/OFQ) regulates several biological functions via selective activation of the N/OFQ receptor (NOP). In this study novel nonpeptide NOP ligands were characterized in vitro in receptor binding and [35S]GTPγS stimulated binding in membranes of cells expressing human NOP and classical opioid receptors, calcium mobilization assay in cells coexpressing the receptors and chimeric G proteins, bioluminescence resonance energy transfer (BRET) based assay for studying NOP receptor interaction with G protein and arrestin, the electrically stimulated mouse vas deferens and the mouse colon bioassays. The action of the AT compounds were compared with standard NOP agonists (N/OFQ and Ro 65-6570) and the NOP selective antagonist SB-612111. AT compounds displayed high NOP affinity and behaved as NOP agonists in all the functional assays consistently showing the following rank order of potency AT-127≥AT-090≥AT-035>AT-004= AT-001. AT compounds behaved as NOP full agonists in the calcium mobilization and mouse colon assays and as partial agonists in the [35S]GTPγS and BRET assays. Interestingly AT-090 and AT-127, contrary to standard nonpeptide agonists that display G protein biased agonism, behaved as an unbiased agonists. AT-090 and AT-127 displayed higher NOP selectivity than Ro 65-6570 at native mouse receptors. AT-090 and AT-127 might be useful pharmacological tools for investigating the therapeutic potential of NOP partial agonists.

Synthesis of enantiopure termini-differentiated heptane stereotriads. Application to side chain-functionalized tetrahydrofurans of IKD-8344.

Enantiopure epoxy cycloheptenyl sulfones syn-7b and anti-7b are prepared in five high-yielding and stereospecific operations from 1, 3-cycloheptadiene. These substrates serve as effective precursors for cis- and trans-substituted tetrahydrofurans (12, 10) which are segments of the antineoplastic agent IKD-8344.

Asymmetric total synthesis of (+)-aphanamol I based on the transition metal catalyzed [5 + 2] cycloaddition of allenes and vinylcyclopropanes.

A concise asymmetric total synthesis of (+)-aphanamol I is described, based on the transition metal catalyzed [5 + 2] allenyl-vinylcyclopropane cycloaddition. The key cycloaddition precursor is convergently assembled from (R)-(+)-limonene and cyclopropane diester through a novel decarboxylative dehydration reaction. The metal-catalyzed [5 + 2] cycloaddition of this precursor proceeds with complete chemo, endo/exo, and diastereoselectivity in 93% yield, representing an effective general route to bicyclo[5.3.0]decane derivatives.

Butyl conformational reorganization as a possible explanation for the longitudinal flexibility of the binding site of bacteriorhodopsin. The azulene and C-22 retinoid analogs.

The UV-VIS absorption data of four bacteriorhodopsin (BR) analogs formed from azulene-retinals of varying polyene chain length show that the one-bond-shortened to one-bond-lengthened analogs possess comparable opsin shift values to that of BR. A two-bond-shortened analog exhibited a much smaller opsin shift. These data, combined with those reported for the C-22 retinal analog (Tokunaga et al., 1977, Biophys. J. 19, 191-198) were analyzed by molecular modelling and computer graphics in terms of a model where conformational flexibility of the appended butyl is the controlling factor in determining ease of pigment formation and protein/substrate interaction.

In vitro co-metabolism of ethanol and cyclic ketones.

The paper presents the results of studies concerning the effects of four cyclic ketones, i.e. cyclopentanone (Pen), cyclohexanone (Hex), cycloheptanone (Hep) and cyclooctanone (Oct) on metabolism of ethanol (EtOH) in vitro. The fraction S9 (supernatant with the removed mitochondria) was used obtained from homogenized rat livers. An increase in reduced nicotinamide adenine dinucleotide (NADH) was examined spectrophotometrically (at 340 nm) while the substrate disappearance and metabolite increase were determined using head-space gas chromatography. The addition of cyclic ketones statistically significantly affected a decrease in the A(340) level, particularly during co-metabolism of EtOH and Hex. The EtOH loss was significantly higher (than the loss observed during metabolism of EtOH alone) only in EtOH-Hex and EtOH-Hep systems, which may be explained by the fact that reoxidation of NADH to NAD+ is quicker in these systems than dissociation of the alcohol dehydrogenase (ADH)-NADH complex.

Neodictyoprolenol and dictyoprolenol, the possible biosynthetic intermediates of dictyopterenes, in the Japanese brown algae Dictyopteris.

Neodictyoprolenol [(-)-(3S)-(1,5Z,8Z)-undecatrien-3-ol] and dictyoprolenol [(-)-(3S)-(1,5Z,8Z)-undecadien-3-ol]), which had been proposed as possible biosynthetic intermediates of the sex pheromones of marine brown algae such as dictyopterene B [(-)-trans-1-((1'E,3'Z)-hexadienyl)-2-vinylcyclopropane], D' [(+)-6-((1'Z)-butenyl)-1,4-cycloheptadiene] and C' [(+)-6-butyl-1,4-cycloheptadiene], were again identified in the essential oils from Dictyopteris prolifera, D. latiscula, and in D. undulata, together with the C11-related volatile compounds such as neodictyoprolene, dictyoprolene and dictyopterenes. Incubation of D. prolifela preparation with racemic neodictyoprolenol and dictyoprolenol as substrates showed (S)-enantioselective decreases of the added substrates and increases in dictyopterenes. From these results, a possible pathway to form dictyopterenes is discussed.

Polyphenols in lahpet-so and two new catechin metabolites produced by anaerobic microbial fermentation of green tea.

The phenolic constituents of lahpet-so, a traditional postfermented tea of Myanmar produced under anaerobic conditions, were examined. The major polyphenols were identified to be pyrogallol and 4'-hydroxyphenyl-3-(2'',4'',6''-trihydroxyphenyl)-propan-2-ol, 3',4'-dihydroxyphenyl-3-(2'',4'',6''-trihydroxyphenyl)-propan-2-ol, and 3',4',5'-trihydroxyphenyl-3-(2'',4'',6''-trihydroxyphenyl)-propan-2-ol. The hydroxydiphenylpropan-2-ols were identical to the initial metabolites produced from green tea catechins by mammalian intestinal bacteria. In addition, an anaerobic mixed-fermentation experiment using lahpet-so and Japanese commercial green tea afforded two new catechin degradation products together with known compound bruguierol B and the above-mentioned catechin metabolites. Based on spectroscopic evidence, the structures of the new compounds were concluded to be 4-(2,5-dihydroxyhexyl)benzene-1,2-diol and (5S,8R)-6,7,8,9-tetrahydro-5-methyl-5·8-epoxy-5H-benzocycloheptene-2,3,4-triol. Interestingly, the production mechanism was deduced to be the inverse of the biosynthesis of the flavan-3-ol A ring.