Selective inhibition of HIV-1 reverse transcriptase-associated ribonuclease H activity by hydroxylated tropolones. (9/37)

High-throughput screening of a National Cancer Institute library of pure natural products identified the hydroxylated tropolone derivatives beta-thujaplicinol (2,7-dihydroxy-4-1(methylethyl)-2,4,6-cycloheptatrien-1-one) and manicol (1,2,3,4-tetrahydro-5-7-dihydroxy-9-methyl-2-(1-methylethenyl)-6H-benzocyclohepte n-6-one) as potent and selective inhibitors of the ribonuclease H (RNase H) activity of human immunodeficiency virus-type 1 reverse transcriptase (HIV-1 RT). beta-Thujaplicinol inhibited HIV-1 RNase H in vitro with an IC50 of 0.2 microM, while the IC50 for Escherichia coli and human RNases H was 50 microM and 5.7 microM, respectively. In contrast, the related tropolone analog beta-thujaplicin (2-hydroxy-4-(methylethyl)-2,4,6-cycloheptatrien-1-one), which lacks the 7-OH group of the heptatriene ring, was inactive, while manicol, which possesses a 7-OH group, inhibited HIV-1 and E.coli RNases H with IC50 = 1.5 microM and 40 microM, respectively. Such a result highlights the importance of the 2,7-dihydroxy function of these tropolone analogs, possibly through a role in metal chelation at the RNase H active site. Inhibition of HIV-2 RT-associated RNase H indirectly indicates that these compounds do not occupy the nonnucleoside inhibitor-binding pocket in the vicinity of the DNA polymerase domain. Both beta-thujaplicinol and manicol failed to inhibit DNA-dependent DNA polymerase activity of HIV-1 RT at a concentration of 50 microM, suggesting that they are specific for the C-terminal RNase H domain, while surface plasmon resonance studies indicated that the inhibition was not due to intercalation of the analog into the nucleic acid substrate. Finally, we have demonstrated synergy between beta-thujaplicinol and calanolide A, a nonnucleoside inhibitor of HIV-1 RT, raising the possibility that both enzymatic activities of HIV-1 RT can be simultaneously targeted.  (+info)

Discriminative stimulus effects of gamma-hydroxybutyrate (GHB) in rats discriminating GHB from baclofen and diazepam. (10/37)

Gamma-hydroxybutyrate (GHB) is a drug of abuse with actions at GHB and GABA receptors. This study tried to increase the selectivity of the discriminative stimulus effects of GHB by training animals to discriminate GHB from compounds that share pharmacological mechanisms with GHB. In comparison with a previous GHB versus saline discrimination (group 1), rats were trained to discriminate GHB (200 mg/kg) either from saline and the GABA(B) agonist baclofen (3.2 mg/kg) (group 2) or from saline, baclofen, and the positive GABA(A) modulator diazepam (1 mg/kg) (group 3). In all groups, GHB produced more than 80% GHB-appropriate responding. Baclofen produced 84% GHB-appropriate responding in group 1 but less than 30% in groups 2 and 3. Diazepam produced 68% GHB-appropriate responding in group 1, 30% in group 2, and only 5% in group 3. The GABA(B) receptor antagonists CGP35348 [3-[aminopropyl(diethoxymethyl)phosphinic acid] and CGP52432 [3-[[[((3,4-dichlorophenyl)methyl]amino]propyl]diethoxymethyl)phosphinic acid] attenuated the discriminative stimulus effects of GHB; CGP35348 did so with similar potency in all groups, but CGP52432 was significantly less potent in groups 2 and 3 than in group 1. In all groups, the GHB antagonist NCS-382 [(2E)-(5-hydroxy-5,7,8,9-tetrahydro-6H-benzo[a][7]annulen-6-ylidene ethanoic acid] partially attenuated the discriminative stimulus effects of GHB. The selective GHB receptor ligand UMB86 (4-hydroxy-4-napthylbutanoic acid sodium) tended to attenuate the discriminative stimulus effects of GHB more in group 3 than in the other groups. The finding that animals can discriminate GHB from baclofen is further evidence that the effects of GHB and baclofen are not identical. Effects that GHB does not share with baclofen may involve GHB receptors or differential interactions with GABA(B) receptors.  (+info)

Novel cyclic gamma-hydroxybutyrate (GHB) analogs with high affinity and stereoselectivity of binding to GHB sites in rat brain. (11/37)

Gamma-hydroxybutyrate (GHB) is a psychotropic compound endogenous to the brain. Despite its potentially great physiological significance, its exact molecular mechanism of action is unknown. GHB is a weak agonist at GABA(B) receptors, but there is also evidence of specific GHB receptor sites, the molecular cloning of which remains a challenge. Ligands with high affinity and specificity for the reported GHB binding site are needed for pharmacological dissection of the GHB and GABA(B) effects and for mapping the structural requirements of the GHB receptor-ligand interactions. For this purpose, we have synthesized and assayed three conformationally restricted GHB analogs for binding against the GHB-specific ligand [3H]NCS-382 [(E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene-)acetic acid] in rat brain homogenate. The cyclohexene and cyclopentene analogs, 3-hydroxycyclohex-1-enecarboxylic acid [(RS)-HOCHCA] and 3-hydroxycyclopent-1-enecarboxylic acid [(RS)-HOCPCA], were found to be high-affinity GHB ligands, with IC50 values in the nanomolar range, and had 9 and 27 times, respectively, higher affinity than GHB. The stereo-selectively synthesized R,R-isomer of the trans-cyclopropyl GHB analog, HOCPrCA, proved to have 10-fold higher affinity than its enantiomer. Likewise, the R-enantiomers of HOCHCA and HOCPCA selectively inhibited [3H]NCS-382 binding. The best inhibitor of these, (R)-HOCPCA, has an affinity 39 times higher than GHB and is thus among the best GHB ligands reported to date. Neither of the cycloalkenes showed any affinity (IC50 > 1 mM) for GABA(A) or GABA(B) receptors. These compounds show excellent potential as lead structures and novel tools for studying specific GHB receptor-mediated pharmacology.  (+info)

Blockade of alpha v beta3 and alpha v beta5 integrins by RGD mimetics induces anoikis and not integrin-mediated death in human endothelial cells. (12/37)

Alpha v integrins are thought to play an important role in tumor angiogenesis. However, discrepancies between findings with Arg-Gly-Asp (RGD) mimetics, which block angiogenesis in animal models, and knockout mice, in which loss of some alpha v integrins enhances tumor angiogenesis, raise questions concerning the function of these integrins and the precise role of alpha v substrate mimetics in antiangiogenic therapies. We have examined the effects of a novel non-peptide RGD mimetic, S 36578-2, on human endothelial cells to elucidate its antagonist activity and to identify possible agonist functions. S 36578-2 is highly selective for alpha v beta3 and alpha v beta5 integrins and induces detachment, caspase-8 activation, and apoptosis in human umbilical endothelial cells (HUVECs) plated on vitronectin. Importantly, the compound has no effect on the morphology or survival of cells plated on interstitial matrix components such as fibronectin, and it does not potentiate the apoptotic process in suspended cells. Identical results were obtained with a cyclic RGD peptide with similar target specificity. In microvascular endothelial cells, S 36578-2-induced death was also linked to its antiadhesive effect, with established lines markedly more resistant than primary cultures to the antiadhesive and proapoptotic effects. Altogether, these findings have important implications for the development of this class of antiangiogenics.  (+info)

Gamma-aminobutyric acidB (GABAB)-receptor mediation of different in vivo effects of gamma-butyrolactone. (13/37)

The endogenous brain constituent, gamma-hydroxybutyric acid (GHB), as well as its prodrug, gamma-butyrolactone (GBL), have recently gained interest in the drug addiction field due to their abuse potential and fatalities caused by overdose. It is known that GHB has two sites of actions: the gamma-aminobutyric acid(B) (GABA(B)) receptor and a specific-GHB binding site. The present study was designed to extend to GBL the investigations on the contribution of the GABA(B) receptor and the specific-GHB binding site to its in vivo effects. To this aim, DBA mice were pretreated either with GABA(B)-receptor antagonists, (3-aminopropyl)(diethoxymethyl)phosphinic acid (CGP 35348) and (2S)(+)-5,5-dimethyl-2-morpholineacetic acid (SCH 50911), or a putative antagonist of the specific-GHB binding site, 6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylideneacetic acid (NCS-382), prior to the administration of doses of GBL that induced hypothermia, motor-incoordination (measured as motor-impairment at the Rota-Rod task), and sedation/hypnosis. The capability of SCH 50911 and NCS-382 to protect against GBL-induced lethality was also investigated. Pretreatment with either GABA(B)-receptor antagonist completely prevented GBL-induced hypothermia, motor-incoordination, and sedation /hypnosis. SCH 50911 also provided complete protection against GBL-associated lethality. Vice versa, NCS-382 failed to exert any antagonistic or protective effect. These results suggest that the in vivo GBL effects tested in the present study are mediated by activation of the GABA(B) receptor.  (+info)

Inhibition of LPS-stimulated NO production in mouse macrophage-like cells by benzocycloheptoxazines. (14/37)

Twenty-six benzocycloheptoxazine derivatives were investigated for their effect on nitric oxide (NO) production by lipopolysaccharide (LPS)-stimulated mouse macrophage-like RAW 264.7 cells. Benzo[b]cyclohepta[e][1,4]thiazine most effectively inhibited the LPS-stimulated NO production at noncytotoxic concentrations. 6H-Benzo[b]cyclohepta[e][1,4]-diazine cation, and benzo[b]cyclohepta[e][1,4]oxazine and its 6-bromo derivative also efficiently inhibited the LPS-stimulated NO production. Another sixteen benzo[b]cyclohepta[e]-[1,4]oxazine derivatives, 14H-[1,4]benzoxazino[3',2' :3,4]-cyclohepta[1,2-b][1,4]benzoxazine and its 7-bromo- and 7-isopropyl derivatives were slightly less active (selectivity index (SI)=83-66). Bromination of benzo[b]cyclohepta[e][1,4]-thiazine, benzo[b]cyclohepta[e][1,4]oxazine and 2-methylbenzo[b]cyclohepta[e][1,4]oxazine at C-6, C-8 or C-10 positions resulted in the significant reduction of the inhibitory activity. The observed inhibitory activity of benzo[b]cyclohepta-[e][1,4]thiazine and its 6,8-dibromo derivatives were not due to the reduction of the intracellular level of inducible NO synthase protein (based on Western blot analysis), nor to NO scavenging activity (based on ESR spectroscopy). These results suggest the possible anti-inflammatory action of benzocyclo-heptoxazines via inhibition of LPS-activated macrophages.  (+info)

Agonizing integrin antagonists? (15/37)


Inhibition of NO production in LPS-stimulated mouse macrophage-like cells by benzo[b]cyclohept[e] [1,4]oxazine and 2-aminotropone derivatives. (16/37)

The aim of this study was to investigate whether a total of twenty benzo[b]cyclohept[e][1,4]oxazines and their S-analogs, and 2-aminotropone derivatives affect the function of activated macrophages. These compounds inhibited the production of pro-inflammatory substances such as nitric oxide (NO) by lipopolysaccharide (LPS)-activated mouse macrophage-like RAW264.7 cells to different extents. Among them, benzo[b]cyclohept[e][1,4]oxazin-6(11H)-one [5] and 7-bromo-2-(4-hydroxyanilino)tropone [16] showed the highest inhibitory effects at concentrations that did not affect cellular viability (selectivity index=74.89 and 54.15, respectively). Western blot and RT-PCR analyses showed that [16] inhibited the expression of both inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 at both protein and mRNA levels, whereas [5] inhibited only iNOS protein expression. Electron-spin resonance (ESR) spectroscopy revealed that both [5] and [16] scavenged nitric oxide (generated from NOC-7) and superoxide anion (generated by HX-XOD reaction) only at much higher concentration. These data suggest that [16] but not [5] exerts its anti-inflammatory action against macrophages via the inhibition of iNOS and COX-2 protein expressions.  (+info)