Natural Products Discovered in a High-Throughput Screen Identified as Inhibitors of RGS17 and as Cytostatic and Cytotoxic Agents for Lung and Prostate Cancer Cell Lines.

Regulator of G Protein Signaling (RGS) 17 is an overexpressed promoter of cancer survival in lung and prostate tumors, the knockdown of which results in decreased tumor cell proliferation in vitro. Identification of drug-like molecules inhibiting this protein could ameliorate the RGS17's pro-tumorigenic effect. Using high-throughput screening, a chemical library containing natural products was interrogated for inhibition of the RGS17-Gαo interaction. Initial hits were verified in control and counter screens. Leads were characterized via biochemical, mass spectrometric, Western blot, microscopic, and cytotoxicity measures. Four known compounds (1-4) were identified with IC50 values ranging from high nanomolar to low micromolar. Three compounds were extensively characterized biologically, demonstrating cellular activity determined by confocal microscopy, and two compounds were assessed via ITC exhibiting high nanomolar to low micromolar dissociation constants. The compounds were found to have a cysteine-dependent mechanism of binding, verified through site-directed mutagenesis and cysteine reactivity assessment. Two compounds, sanguinarine (1) and celastrol (2), were found to be cytostatic against lung and prostate cancer cell lines and cytotoxic against prostate cancer cell lines in vitro, although the dependence of RGS17 on these phenomena remains elusive, a result that is perhaps not surprising given the multimodal cytostatic and cytotoxic activities of many natural products.

Functional selectivity at the µ-opioid receptor: implications for understanding opioid analgesia and tolerance.

Opioids are the most effective analgesic drugs for the management of moderate or severe pain, yet their clinical use is often limited because of the onset of adverse side effects. Drugs in this class produce most of their physiological effects through activation of the µ opioid receptor; however, an increasing number of studies demonstrate that different opioids, while presumably acting at this single receptor, can activate distinct downstream responses, a phenomenon termed functional selectivity. Functional selectivity of receptor-mediated events can manifest as a function of the drug used, the cellular or neuronal environment examined, or the signaling or behavioral measure recorded. This review summarizes both in vitro and in vivo work demonstrating functional selectivity at the µ opioid receptor in terms of G protein coupling, receptor phosphorylation, interactions with ß-arrestins, receptor desensitization, internalization and signaling, and details on how these differences may relate to the progression of analgesic tolerance after their extended use.

Galpha(i2) inhibition of adenylate cyclase regulates presynaptic activity and unmasks cGMP-dependent long-term depression at Schaffer collateral-CA1 hippocampal synapses.

Cyclic AMP signaling plays a central role in regulating activity at a number of synapses in the brain. We showed previously that pairing activation of receptors that inhibit adenylate cyclase (AC) and reduce the concentration of cyclic AMP, with elevation of the concentration of cyclic GMP is sufficient to elicit a presynaptically expressed form of LTD at Schaffer collateral-CA1 synapses in the hippocampus. To directly test the role of AC inhibition and G-protein signaling in LTD at these synapses, we utilized transgenic mice that express a mutant, constitutively active inhibitory G protein, Galpha(i2), in principal neurons of the forebrain. Transgene expression of Galpha(i2) markedly enhanced LTD and impaired late-phase LTP at Schaffer collateral synapses, with no associated differences in input/output relations, paired-pulse facilitation, or NMDA receptor-gated conductances. When paired with application of a type V phosphodiesterase inhibitor to elevate the concentration of intracellular cyclic GMP, constitutively active Galpha(i2) expression converted the transient depression normally caused by this treatment to an LTD that persisted after the drug was washed out. Moreover, this effect could be mimicked in control slices by pairing type V phosphodiesterase inhibitor application with application of a PKA inhibitor. Electrophysiological recordings of spontaneous excitatory postsynaptic currents and two-photon visualization of vesicular release using FM1-43 revealed that constitutively active Galpha(i2) tonically reduced basal release probability from the rapidly recycling vesicle pool of Schaffer collateral terminals. Our findings support the hypothesis that inhibitory G-protein signaling acts presynaptically to regulate release, and, when paired with elevations in the concentration of cyclic GMP, converts a transient cyclic GMP-induced depression into a long-lasting decrease in release.

High-content assays for ligand regulation of G-protein-coupled receptors.

High-content assays rely on the imaging of cellular events. They can be used to monitor the activation of G-protein-coupled receptors (or other receptors), their internalization into the cell, or alterations in their amount. In addition, multiplexed assays can provide further information about the characteristics of the receptor. Recent improvements in throughput using high-content screening platforms means that such assays are now an integral element of functional analysis in the drug discovery process.

Morphine-induced changes of gene expression in the brain.

Repeated opiate administration alters gene expression in different brain regions of rodents, an effect which may contribute to plastic changes associated with addictive behaviour. There is increasing evidence that multiple transcription factors are induced in morphine tolerance, sensitization and during morphine withdrawal. Whereas morphine treatment does not lead to major alterations in the expression of mu-opioid receptors (MOR), there is transcriptional regulation of proteins involved in MOR trafficking such as GRK2 or beta arrestin 2 as well as altered expression of other receptors such as dopamine receptors, NMDA receptors, GABA(A) receptor and alpha(2A) adrenoceptor. Recent gene expression profiling studies reveal additional clusters of morphine-responsive genes: whereas single dose administration has been shown to predominantly reduce expression of genes involved in metabolic function, ascending morphine doses leading to morphine tolerance revealed induction of genes which alter patterns of synaptic connectivity such as arc or ania-3. These genes remained elevated after precipitated withdrawal, which also triggered the expression of several transcriptional activators and repressors. In addition, morphine has been shown to be a strong inducer of heat shock protein 70, a cell protective protein which might counter-regulate opiate-induced neurotoxicity. Temporal expression profiles during a chronic morphine application schedule revealed discrete and fluctuating expression of gene clusters such as transcription factors, G-protein-coupled receptors and neuropeptides. Prolonged abstinence seems to be characterized by up-regulation of several transcription factors and persistent down-regulation of ligand gated ion channels such as glutamatergic and GABA-ergic receptor subunits. These long-term changes in receptor expression suggest a persistent alteration of synaptic signalling after morphine treatment.

Sphingosine-1-phosphate stimulates smooth muscle cell migration through galpha(i)- and pi3-kinase-dependent p38(MAPK) activation.

BACKGROUND: Sphingosine-1-phosphate (S-1-P) is an extracellular mediator released in response to vessel injury. S-1-P binds to G-protein-coupled receptors, which can be Galpha(i)-, Galpha(q)-, or G(12/13)-linked. This study examines the role of p38 mitogen-activated protein kinase (p38(MAPK)) in vascular smooth muscle cell migration after stimulation with S-1-P, and pathways leading to p38(MAPK) activation. S-1-P has previously been shown to stimulate migration of vascular smooth muscle cells (VSMCs) in vitro through ERK1/2 and G(i). We hypothesized that S-1-P-induced VSMC migration is also dependent on p38(MAPK) activation through a G(i)-coupled extracellular receptor and phosphoinositide 3-kinase (PI3-K). METHODS: VSMCs were cultured in vitro. A linear wound assay was performed in the presence of S-1-P and inhibitors of p38(MAPK) (SB203580) or epidermal growth factor (EGF) receptor kinase (AG1478). Chemotaxis stimulated by S-1-P was also assayed in a modified Boyden chamber with and without SB203580 pretreatment. Western blotting was performed to examine p38(MAPK) activation in response to S-1-P with and without SB203580, AG1478, or inhibitors of G(i) (pertussis toxin), PI3-K (Wortmannin and LY294002), or MEK1 (PD98059). Western blotting and immunoprecipitation for targets of p38(MAPK) (MAPKAP kinase-2) and PI3-K (Akt) were also performed.S-1-P stimulated migration of VSMCs in both wound and Boyden transwell assays. This migration was inhibited by SB203580 to the level of control, whereas AG478 had no effect. RESULTS: S-1-P stimulated activation of p38(MAPK) that peaked at 10 min, as well as activation of MAPKAP kinase-2. Activation of p38(MAPK) was significantly inhibited by SB203580, pertussis toxin, Wortmannin, and LY294002, but not by PD98059 or AG1478; MAPKAP kinase-2 activation was inhibited by SB203580. Akt was activated by S-1-P at 3 to 5 min; this response was inhibited by Wortmannin and LY294002, but not by SB203580 or pertussis toxin. CONCLUSIONS: S-1-P induced VSMC migration through a G(i)-linked and a PI3-K coupled, p38(MAPK)- dependent process. PI3-K appears to function upstream of p38(MAPK), but was not G(i)-dependent. S-1-P-stimulated activation of p38(MAPK) does not signal via transactivation of the EGF receptor. Understanding signal transduction will allow targeted molecular interventions to treat the response of a vessel to injury.