The antiproliferative and apoptotic effects of apigenin on glioblastoma cells.

OBJECTIVES: Glioblastoma (GBM) is highly proliferative, infiltrative, malignant and the most deadly form of brain tumour. The epidermal growth factor receptor (EGFR) is overexpressed, amplified and mutated in GBM and has been shown to play key and important roles in the proliferation, growth and survival of this tumour. The goal of our study was to investigate the antiproliferative, apoptotic and molecular effects of apigenin in GBM. METHODS: Proliferation and viability tests were carried out using the trypan blue exclusion, MTT and lactate dehydrogenase (LDH) assays. Flow cytometry was used to examine the effects of apigenin on the cell cycle check-points. In addition, we determined the effects of apigenin on EGFR-mediated signalling pathways by Western blot analyses. KEY FINDINGS: Our results showed that apigenin reduced cell viability and proliferation in a dose- and time-dependent manner while increasing cytotoxicity in GBM cells. Treatment with apigenin-induced is poly ADP-ribose polymerase (PARP) cleavage and caused cell cycle arrest at the G2M checkpoint. Furthermore, our data revealed that apigenin inhibited EGFR-mediated phosphorylation of mitogen-activated protein kinase (MAPK), AKT and mammalian target of rapamycin (mTOR) signalling pathways and attenuated the expression of Bcl-xL. CONCLUSION: Our results demonstrated that apigenin has potent inhibitory effects on pathways involved in GBM proliferation and survival and could potentially be used as a therapeutic agent for GBM.

A Pan-GTPase Inhibitor as a Molecular Probe.

Overactive GTPases have often been linked to human diseases. The available inhibitors are limited and have not progressed far in clinical trials. We report here a first-in-class small molecule pan-GTPase inhibitor discovered from a high throughput screening campaign. The compound CID1067700 inhibits multiple GTPases in biochemical, cellular protein and protein interaction, as well as cellular functional assays. In the biochemical and protein interaction assays, representative GTPases from Rho, Ras, and Rab, the three most generic subfamilies of the GTPases, were probed, while in the functional assays, physiological processes regulated by each of the three subfamilies of the GTPases were examined. The chemical functionalities essential for the activity of the compound were identified through structural derivatization. The compound is validated as a useful molecular probe upon which GTPase-targeting inhibitors with drug potentials might be developed.

Optimization of potent and selective quinazolinediones: inhibitors of respiratory syncytial virus that block RNA-dependent RNA-polymerase complex activity.

A quinazolinedione-derived screening hit 2 was discovered with cellular antiviral activity against respiratory syncytial virus (CPE EC50 = 2.1 µM), moderate efficacy in reducing viral progeny (4.2 log at 10 µM), and marginal cytotoxic liability (selectivity index, SI ∼ 24). Scaffold optimization delivered analogs with improved potency and selectivity profiles. Most notable were compounds 15 and 19 (EC50 = 300-500 nM, CC50 > 50 µM, SI > 100), which significantly reduced viral titer (>400,000-fold), and several analogs were shown to block the activity of the RNA-dependent RNA-polymerase complex of RSV.

Ebselen inhibits hepatitis C virus NS3 helicase binding to nucleic acid and prevents viral replication.

The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is both a protease, which cleaves viral and host proteins, and a helicase that separates nucleic acid strands, using ATP hydrolysis to fuel the reaction. Many antiviral drugs, and compounds in clinical trials, target the NS3 protease, but few helicase inhibitors that function as antivirals have been reported. This study focuses on the analysis of the mechanism by which ebselen (2-phenyl-1,2-benzisoselenazol-3-one), a compound previously shown to be a HCV antiviral agent, inhibits the NS3 helicase. Ebselen inhibited the abilities of NS3 to unwind nucleic acids, to bind nucleic acids, and to hydrolyze ATP, and about 1 µM ebselen was sufficient to inhibit each of these activities by 50%. However, ebselen had no effect on the activity of the NS3 protease, even at 100 times higher ebselen concentrations. At concentrations below 10 µM, the ability of ebselen to inhibit HCV helicase was reversible, but prolonged incubation of HCV helicase with higher ebselen concentrations led to irreversible inhibition and the formation of covalent adducts between ebselen and all 14 cysteines present in HCV helicase. Ebselen analogues with sulfur replacing the selenium were just as potent HCV helicase inhibitors as ebselen, but the length of the linker between the phenyl and benzisoselenazol rings was critical. Modifications of the phenyl ring also affected compound potency over 30-fold, and ebselen was a far more potent helicase inhibitor than other, structurally unrelated, thiol-modifying agents. Ebselen analogues were also more effective antiviral agents, and they were less toxic to hepatocytes than ebselen. Although the above structure-activity relationship studies suggest that ebselen targets a specific site on NS3, we were unable to confirm binding to either the NS3 ATP binding site or nucleic acid binding cleft by examining the effects of ebselen on NS3 proteins lacking key cysteines.

Potent and selective inhibitors of the TASK-1 potassium channel through chemical optimization of a bis-amide scaffold.

TASK-1 is a two-pore domain potassium channel that is important to modulating cell excitability, most notably in the context of neuronal pathways. In order to leverage TASK-1 for therapeutic benefit, its physiological role needs better characterization; however, designing selective inhibitors that avoid the closely related TASK-3 channel has been challenging. In this study, a series of bis-amide derived compounds were found to demonstrate improved TASK-1 selectivity over TASK-3 compared to reported inhibitors. Optimization of a marginally selective hit led to analog 35 which displays a TASK-1 IC50=16 nM with 62-fold selectivity over TASK-3 in an orthogonal electrophysiology assay.

A selective ATP-binding cassette subfamily G member 2 efflux inhibitor revealed via high-throughput flow cytometry.

Chemotherapeutics tumor resistance is a principal reason for treatment failure, and clinical and experimental data indicate that multidrug transporters such as ATP-binding cassette (ABC) B1 and ABCG2 play a leading role by preventing cytotoxic intracellular drug concentrations. Functional efflux inhibition of existing chemotherapeutics by these pumps continues to present a promising approach for treatment. A contributing factor to the failure of existing inhibitors in clinical applications is limited understanding of specific substrate/inhibitor/pump interactions. We have identified selective efflux inhibitors by profiling multiple ABC transporters against a library of small molecules to find molecular probes to further explore such interactions. In our primary screening protocol using JC-1 as a dual-pump fluorescent reporter substrate, we identified a piperazine-substituted pyrazolo[1,5-a]pyrimidine substructure with promise for selective efflux inhibition. As a result of a focused structure-activity relationship (SAR)-driven chemistry effort, we describe compound 1 (CID44640177), an efflux inhibitor with selectivity toward ABCG2 over ABCB1. Compound 1 is also shown to potentiate the activity of mitoxantrone in vitro as well as preliminarily in vivo in an ABCG2-overexpressing tumor model. At least two analogues significantly reduce tumor size in combination with the chemotherapeutic topotecan. To our knowledge, low nanomolar chemoreversal activity coupled with direct evidence of efflux inhibition for ABCG2 is unprecedented.

A competitive nucleotide binding inhibitor: in vitro characterization of Rab7 GTPase inhibition.

Mapping the functionality of GTPases through small molecule inhibitors represents an underexplored area in large part due to the lack of suitable compounds. Here we report on the small chemical molecule 2-(benzoylcarbamothioylamino)-5,5-dimethyl-4,7-dihydrothieno[2,3-c]pyran-3-carboxylic acid (PubChem CID 1067700) as an inhibitor of nucleotide binding by Ras-related GTPases. The mechanism of action of this pan-GTPase inhibitor was characterized in the context of the Rab7 GTPase as there are no known inhibitors of Rab GTPases. Bead-based flow cytometry established that CID 1067700 has significant inhibitory potency on Rab7 nucleotide binding with nanomolar inhibitor (K(i)) values and an inhibitory response of ≥97% for BODIPY-GTP and BODIPY-GDP binding. Other tested GTPases exhibited significantly lower responses. The compound behaves as a competitive inhibitor of Rab7 nucleotide binding based on both equilibrium binding and dissociation assays. Molecular docking analyses are compatible with CID 1067700 fitting into the nucleotide binding pocket of the GTP-conformer of Rab7. On the GDP-conformer, the molecule has greater solvent exposure and significantly less protein interaction relative to GDP, offering a molecular rationale for the experimental results. Structural features pertinent to CID 1067700 inhibitory activity have been identified through initial structure-activity analyses and identified a molecular scaffold that may serve in the generation of more selective probes for Rab7 and other GTPases. Taken together, our study has identified the first competitive GTPase inhibitor and demonstrated the potential utility of the compound for dissecting the enzymology of the Rab7 GTPase, as well as serving as a model for other small molecular weight GTPase inhibitors.

Antinociceptive effects of herkinorin, a MOP receptor agonist derived from salvinorin A in the formalin test in rats: new concepts in mu opioid receptor pharmacology: from a symposium on new concepts in mu-opioid pharmacology.

Herkinorin is the first µ opioid (MOP) selective agonist derived from salvinorin A, a hallucinogenic natural product. Previous work has shown that, unlike other opioids, herkinorin does not promote the recruitment of ß-arrestin-2 to the MOP receptor and does not lead to receptor internalization. This paper presents the first in vivo evaluation of herkinorin's antinociceptive effects in rats, using the formalin test as a model of tonic inflammatory pain. Herkinorin was found to produce a dose-dependent decrease in the number of flinches evoked by formalin. These antinociceptive effects were substantially blocked by pretreatment with the nonselective antagonist naloxone, indicating that the antinociception is mediated by opioid receptors. Contralateral administration of herkinorin did not attenuate the number of flinches evoked by formalin, indicating that its effects are peripherally restricted to the site of injection. Following chronic administration (5-day), herkinorin maintained antinociceptive efficacy in both phases of the formalin test. Furthermore, unlike morphine, herkinorin was still able to inhibit flinching in both phases of the formalin test in animals made tolerant to chronic systemic morphine treatment. Collectively, these results suggest that herkinorin may produce peripheral antinociception with decreased tolerance liability and thereby represents a promising template for the development of agents for the treatment of a variety of pain states.

Potential Drug Abuse Therapeutics Derived from the Hallucinogenic Natural Product Salvinorin A.

Previous structure-activity relationship studies of salvinorin A have shown that modification of the acetate functionality off the C-2 position to a methoxy methyl or methoxy ethyl ether moiety leads to increased potency at KOP receptors. However, the reason for this increase remains unclear. Here we report our efforts towards the synthesis and evaluation of C-2 constrained analogs of salvinorin A. These analogs were evaluated at opioid receptors in radioligand binding experiments as well as in the GTP-γ-S functional assay. One compound, 5, was found to have affinity and potency at κ opioid (KOP) receptors comparable to salvinorin A. In further studies, 5 was found to attenuate cocaine-induced drug seeking behavior in rats comparably to salvinorin A. This finding represents the first example of a salvinorin A analog that has demonstrated anti-addictive capabilities.

Tetranortriterpenoids from Spathelia sorbifolia (Rutaceae).

Two new tetranortriterpenoids were obtained by chromatography of the acetone extract of the wood of Spathelia sorbifolia L.; these compounds were characterized as their p-bromobenzoyl derivatives (2, 3). This extract also yielded a known tetranortriterpenoid and the chromones allopteroxylin, spatheliabischromene and anhydrosorbifolin.

Identification of a novel "almost neutral" micro-opioid receptor antagonist in CHO cells expressing the cloned human mu-opioid receptor.

The basal (constitutive) activity of G protein-coupled receptors allows for the measurement of inverse agonist activity. Some competitive antagonists turn into inverse agonists under conditions where receptors are constitutively active. In contrast, neutral antagonists have no inverse agonist activity, and they block both agonist and inverse agonist activity. The mu-opioid receptor (MOR) demonstrates detectable constitutive activity only after a state of dependence is produced by chronic treatment with a MOR agonist. We therefore sought to identify novel MOR inverse agonists and novel neutral MOR antagonists in both untreated and agonist-treated MOR cells. CHO cells expressing the cloned human mu receptor (hMOR-CHO cells) were incubated for 20 h with medium (control) or 10 microM (2S,4aR,6aR,7R,9S,10aS,10bR)-9-(benzoyloxy)-2-(3-furanyl)dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naphtho-[2,1-c]pyran-7-carboxylic acid methyl ester (herkinorin, HERK). HERK treatment generates a high degree of basal signaling and enhances the ability to detect inverse agonists. [(35)S]-GTP-gamma-S assays were conducted using established methods. We screened 21 MOR "antagonists" using membranes prepared from HERK-treated hMOR-CHO cells. All antagonists, including CTAP and 6beta-naltrexol, were inverse agonists. However, LTC-274 ((-)-3-cyclopropylmethyl-2,3,4,4alpha,5,6,7,7alpha-octahydro-1H-benzofuro[3,2-e]isoquinolin-9-ol)) showed the lowest efficacy as an inverse agonist, and, at concentrations less than 5 nM, had minimal effects on basal [(35)S]-GTP-gamma-S binding. Other efforts in this study identified KC-2-009 ((+)-3-((1R,5S)-2-((Z)-3-phenylallyl)-2-azabicyclo[3.3.1]nonan-5-yl)phenol hydrochloride) as an inverse agonist at untreated MOR cells. In HERK-treated cells, KC-2-009 had the highest efficacy as an inverse agonist. In summary, we identified a novel and selective MOR inverse agonist (KC-2-009) and a novel MOR antagonist (LTC-274) that shows the least inverse agonist activity among 21 MOR antagonists. LTC-274 is a promising lead compound for developing a true MOR neutral antagonist.

Synthetic studies of neoclerodane diterpenes from Salvia divinorum: role of the furan in affinity for opioid receptors.

Further synthetic modification of the furan ring of salvinorin A (1), the major active component of Salvia divinorum, has resulted in novel neoclerodane diterpenes with opioid receptor affinity and activity. A computational study has predicted 1 to be a reproductive toxicant in mammals and is suggestive that use of 1 may be associated with adverse effects. We report in this study that piperidine 21 and thiomorpholine 23 have been identified as selective partial agonists at kappa opioid receptors. This indicates that additional structural modifications of 1 may provide ligands with good selectivity for opioid receptors but with reduced potential for toxicity.

Utilizing nature as a source of new probes for opioid pharmacology.

BACKGROUND: Traditional and current opioid pharmacology is fundamentally based on interactions between opioid receptors and compounds isolated from natural sources. Adverse effects associated with opioids have led to the search for compounds with diminished side effects. DISCUSSION: Recent discoveries of non-nitrogenous and structurally diverse alkaloids as novel opioid ligands have led to renewed interest in the development of novel chemotypes for opioid receptors. CONCLUSION: The strong history of natural products as opioid receptor ligands suggests that nature is one of the most promising for the identification of novel opioids. This review highlights the vast potential of investigating natural products as novel probes of opioid receptors.

Differential effects of opioid agonists on G protein expression in CHO cells expressing cloned human opioid receptors.

Recent evidence indicates that agonist ligands of G protein coupled receptors (GPCR) can activate different signaling systems. Such "agonist-directed" signaling also occurs with opioid receptors. Previous work from our laboratory showed that chronic morphine, but not DAMGO, up-regulates the expression of Galpha12 and that both morphine and DAMGO decreased Galphai3 expression in CHO cells expressing the cloned human mu opioid receptor. In this study, we tested the hypothesis that chronic opioid regulation of G protein expression is agonist-directed. Following a 20h treatment of CHO cells expressing the cloned human mu (hMOR-CHO), delta (hDOR-CHO) or kappa (hKOR-CHO) opioid receptors with various opioid agonists, we determined the expression level of Galpha12 and Galphai3 by Western blots. Among five mu agonists (morphine, etorphine, DADLE, DAMGO, herkinorin) tested with hMOR-CHO cells, only chronic morphine and etorphine up-regulated Galpha12 expression. All five mu agonists decreased Galphai3 expression. Among six delta agonists (SNC80, DPDPE, deltorphin-1, morphine, DADLE, etorphine) tested with hDOR-CHO cells, all six agonists down-regulated Galphai3 expression or moderately up-regulated Galpha12 expression. Among five kappa agonists, ((-)-ethylketocyclazocine, salvinorin A, U69,593, etorphine, (-)-U50,488) tested with hKOR-CHO cells, only chronic (-)-U50,488 and (-)-EKC up-regulated Galpha12 expression. All kappa agonists decreased Galphai3 expression. These data demonstrate that chronic opioid agonist regulation of G protein expression depends not only on the agonist tested, but also on the type of opioid receptor expressed in a common cellular host, providing additional evidence for agonist-directed signaling.

The effects of herkinorin, the first mu-selective ligand from a salvinorin A-derived scaffold, in a neuroendocrine biomarker assay in nonhuman primates.

Herkinorin is the first mu-opioid receptor-selective ligand from the salvinorin A diterpenoid scaffold. Herkinorin has relative mu > kappa > delta binding selectivity, and it can act as an agonist at both mu- and kappa-receptors, in vitro. These studies were the first in vivo evaluation of the effects of herkinorin in nonhuman primates, using prolactin release, a neuroendocrine biomarker assay that is responsive to both mu- and kappa-agonists, as well as to compounds with limited ability to cross the blood-brain barrier. In cumulative dosing studies (0.01-0.32 mg/kg i.v.), herkinorin produced only small effects in gonadally intact males (n = 4), but a more robust effect in females (n = 4). Time course studies with herkinorin (0.32 mg/kg) confirmed this greater effectiveness in females and revealed a fast onset after i.v. administration (e.g., by 5-15 min). Antagonism experiments with different doses of nalmefene (0.01 and 0.1 mg/kg) caused dose-dependent and complete prevention of the effect of herkinorin in females. This is consistent with a principal mu-agonist effect of herkinorin, with likely partial contribution by kappa-agonist effects. The peripherally selective antagonist quaternary naltrexone (1 mg/kg s.c.) caused approximately 70% reduction in the peak effect of herkinorin (0.32 mg/kg) in females, indicating that this effect of herkinorin is prominently mediated outside the blood-brain barrier.

Synthetic studies of neoclerodane diterpenes from Salvia divinorum: preparation and opioid receptor activity of salvinicin analogues.

Further modification of salvinorin A (1a), the major active component of Salvia divinorum, has resulted in the synthesis of novel neoclerodane diterpenes with opioid receptor affinity and activity. We report in this study that oxadiazole 11a and salvidivin A (12a), a photooxygenation product of 1a, have been identified as the first neoclerodane diterpenes with kappa antagonist activity. This indicates that additional structural modifications of 1a may lead to analogues with higher potency and utility as drug abuse medications.