Molecular Pharmacology current issue

[MINIREVIEW] Diverse Actions and Target-Site Selectivity of Neonicotinoids: Structural Insights

Matsuda, K., Kanaoka, S., Akamatsu, M., Sattelle, D. B. — 2009-06-24

The nicotinic acetylcholine receptors (nAChRs) are targets for human and veterinary medicines as well as insecticides. Subtype-selectivity among the diverse nAChR family members is important for medicines targeting particular disorders, and pest-insect selectivity is essential for the development of safer, environmentally acceptable insecticides. Neonicotinoid insecticides selectively targeting insect nAChRs have important applications in crop protection and animal health. Members of this class exhibit strikingly diverse actions on their nAChR targets. Here we review the chemistry and diverse actions of neonicotinoids on insect and mammalian nAChRs. Electrophysiological studies on native nAChRs and on wild-type and mutagenized recombinant nAChRs have shown that basic residues particular to loop D of insect nAChRs are likely to interact electrostatically with the nitro group of neonicotinoids. In 2008, the crystal structures were published showing neonicotinoids docking into the acetylcholine binding site of molluscan acetylcholine binding proteins with homology to the ligand binding domain (LBD) of nAChRs. The crystal structures showed that 1) glutamine in loop D, corresponding to the basic residues of insect nAChRs, hydrogen bonds with the NO₂ group of imidacloprid and 2) neonicotinoid-unique stacking and CH- bonds at the LBD. A neonicotinoid-resistant strain obtained by laboratory-screening has been found to result from target site mutations, and possible reasons for this are also suggested by the crystal structures. The prospects of designing neonicotinoids that are safe not only for mammals but also for beneficial insects such as honey bees (Apis mellifera) are discussed in terms of interactions with non- nAChR subunits.

[MINIREVIEW] Inactivation and Biotransformation of the Endogenous Cannabinoids Anandamide and 2-Arachidonoylglycerol

Yates, M. L., Barker, E. L. — 2009-06-24

The cannabinoid field is currently an active research area. Anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are the most characterized endogenous cannabinoids (also known as endocannabinoids). These neuromodulators have been implicated in various physiologically relevant phenomena, including mood (Witkin et al., 2005), the immune response (Ashton, 2007), appetite (Kirkham and Tucci, 2006), reproduction (Wang et al., 2006), spasticity (Pertwee, 2002), and pain (Hohmann and Suplita, 2006). Pharmacological manipulation of AEA and 2-AG signaling should prove to have significant therapeutic applications in disorders linked to endocannabinoid signaling. One way to alter endocannabinoid signaling is to regulate the events responsible for termination of the endocannabinoid signal-cellular uptake and metabolism. However, to pharmacologically exploit AEA and/or 2-AG signaling in this way, we must first gain a better understanding of the proteins and mechanisms governing these processes. This review serves as an introduction to the endocannabinoid system with an emphasis on the proteins and events responsible for the termination of AEA and 2-AG signaling.

[ACCELERATED COMMUNICATION] Increased GABAB Receptor-Mediated Signaling Reduces the Susceptibility of Fragile X Knockout Mice to Audiogenic Seizures

Pacey, L. K. K., Heximer, S. P., Hampson, D. R. — 2009-06-24

Mice lacking the gene encoding fragile X mental retardation protein (FMR1) are susceptible to audiogenic seizures, and antagonists of the group I metabotropic glutamate receptors (mGluRs) have been shown to block seizures in FMR1 knockout mice. We investigated whether the G-protein-inhibitory activity of the regulator of G-protein signaling protein, RGS4, could also alter the susceptibility to audiogenic seizures in FMR1 mice. We were surprised to find that male FMR1/RGS4 double-knockout mice showed reduced susceptibility to audiogenic seizures compared with age-matched FMR1 mice. These data raised the intriguing possibility that loss of RGS4 increased signaling through another G-protein pathway that reduces seizure susceptibility in FMR1 mice. Indeed, administration of the GABA_B receptor agonist baclofen to FMR1 mice inhibited seizures, whereas the GABA_B receptor antagonist (3-aminopropyl)(cyclohexylmethyl)phosphinic acid (CGP 46381) increased seizure incidence in double-knockout mice but not in wild-type mice. Finally, audiogenic seizures could be induced in wild-type mice by coadministering CGP 46381 and the mGluR5-positive allosteric modulator 3-cyano-N-(1,2 diphenyl-1H-pyrazol-5-yl) benzamide. These data show for the first time that GABA_B receptor-mediated signaling antagonizes the seizure-promoting effects of the mGluRs in FMR1 knockout mice and point to the potential therapeutic benefit of GABA_B agonists for the treatment of fragile X syndrome.

[ARTICLES] Contribution of Binding Enthalpy and Entropy to Affinity of Antagonist and Agonist Binding at Human and Guinea Pig Histamine H1-Receptor

Wittmann, H.-J., Seifert, R., Strasser, A. — 2009-06-24

For several GPCRs, discrimination between agonism and antagonism is possible on the basis of thermodynamics parameters, such as binding enthalpy and entropy. In this study, we analyze whether agonists and antagonists can also be discriminated thermodynamically at the histamine H₁ receptor (H₁R). Because previous studies revealed species differences in pharmacology between human H₁R (hH₁R) and guinea pig H₁R (gpH₁R), we analyzed a broad spectrum of H₁R antagonists and agonists at hH₁R and gpH₁R. [³H]Mepyramine competition binding assay were performed at five different temperatures in a range from 283.15 to 303.15 K. In addition, we performed a temperature-dependent three-dimensional quantitative structure activity relationship study to predict binding enthalpy and entropy for histaprodifen derivatives, which can bind to H₁R in two different orientations. Our studies revealed significant species differences in binding enthalpy and entropy between hH₁R and gpH₁R for some antagonists and agonists. Furthermore, in some cases, we found changes in heat capacity of the binding process that were different from zero. Differences in flexibility of the ligands may be responsible for this observation. For most ligands, the binding process to hH₁R and gpH₁R is clearly entropy-driven. In contrast, for the endogenous ligand histamine, the binding process is significantly enthalpy-driven at both species isoforms. Thus, a definite discrimination between antagonism and agonism based on thermodynamic parameters is possible for neither hH₁R nor gpH₁R, but thermodynamic analysis of ligand-binding may be a novel approach to dissect agonist- and antagonist-specific receptor conformations.

[ARTICLES] Regulation of Renal Outer Medullary Potassium Channel and Renal K+ Excretion by Klotho

Cha, S.-K., Hu, M.-C., Kurosu, H., Kuro-o, M., Moe, O., Huang, C.-L. — 2009-06-24

Klotho is an aging-suppression protein predominantly expressed in kidney, parathyroid glands, and choroids plexus of the brain. The extracellular domain of Klotho, a type-1 membrane protein, is secreted into urine and blood and may function as an endocrine or paracrine hormone. The functional role of Klotho in the kidney remains largely unknown. Recent studies reported that treatment by the extracellular domain of Klotho (KLe) increases cell-surface abundance of transient receptor potential vanilloid type isoform 5, an epithelial Ca²⁺ channel critical for Ca²⁺ reabsorption in the kidney. Whether Klotho regulates surface expression of other channels in the kidney is not known. Here, we report that KLe treatment increases the cell-membrane abundance of the renal K⁺ channel renal outer medullary potassium channel 1 (ROMK1) by removing terminal sialic acids from N-glycan of the channel. Removal of sialic acids exposes underlying disaccharide galactose-N-acetylglucosamine, a ligand for a ubiquitous galactoside-binding lectin galectin-1. Binding to galectin-1 at the extracellular surface prevents clathrin-mediated endocytosis of ROMK1 and leads to accumulation of functional channel on the plasma membrane. Intravenous administration of KLe increases the level of Klotho in urine and increases urinary excretion of K⁺. These results suggest that Klotho may have a broader function in the regulation of ion transport in the kidney.

[ARTICLES] Thiazolidinediones Mimic Glucose Starvation in Facilitating Sp1 Degradation through the Up-Regulation of {beta}-Transducin Repeat-Containing Protein

2009-06-24

This study investigated the mechanism by which the transcription factor Sp1 is degraded in prostate cancer cells. We recently developed a thiazolidinedione derivative, (Z)-5-(4-hydroxy-3-trifluoromethylbenzylidene)-3-(1-methylcyclohexyl)-thiazolidine-2,4-dione (OSU-CG12), that induces Sp1 degradation in a manner paralleling that of glucose starvation. Based on our finding that thiazolidinediones suppress β-catenin and cyclin D1 by up-regulating the E3 ligase SCF^β-TrCP, we hypothesized that β-transducin repeat-containing protein (β-TrCP) targets Sp1 for proteasomal degradation in response to glucose starvation or OSU-CG12. Here we show that either treatment of LNCaP cells increased specific binding of Sp1 with β-TrCP. This direct binding was confirmed by in vitro pull-down analysis with bacterially expressed β-TrCP. Although ectopic expression of β-TrCP enhanced the ability of OSU-CG12 to facilitate Sp1 degradation, suppression of endogenous β-TrCP function by a dominant-negative mutant or small interfering RNA-mediated knockdown blocked OSU-CG12-facilitated Sp1 ubiquitination and/or degradation. Sp1 contains a C-terminal conventional DSG destruction box (⁷²⁷DSGAGS⁷³²) that mediates β-TrCP recognition and encompasses a glycogen synthase kinase 3β (GSK3β) phosphorylation motif (SXXXS). Pharmacological and molecular genetic approaches and mutational analyses indicate that extracellular signal-regulated kinase-mediated phosphorylation of Thr739 and GSK3β-mediated phosphorylation of Ser728 and Ser732 were critical for Sp1 degradation. The ability of OSU-CG12 to mimic glucose starvation to activate β-TrCP-mediated Sp1 degradation has translational potential to foster novel strategies for cancer therapy.

[ARTICLES] In Vivo and in Vitro Structure-Activity Relationships and Structural Conformation of Kisspeptin-10-Related Peptides

2009-06-24

Kisspeptins, the natural ligands of the G protein-coupled receptor KISS1R, comprise a family of related peptides derived from the proteolytic processing of a common precursor encoded by the KISS1 gene. Among those, Kisspeptin-10 (Kp-10) contains the basic residues to retain full functional activity and exhibits higher receptor affinity and biopotency than longer forms of the peptide. Although kisspeptins were first characterized by their ability to inhibit tumor metastasis, recent studies have revealed that the KISS1/KISS1R system plays an essential role in the neuroendocrine control of the reproductive axis. In this context, development and functional analysis of Kp-10 analogs may help in the search for new agonists and antagonists as valuable tools to manipulate the KISS1/KISS1R system and hence fertility. We report herein functional and structural analyses of a series of Ala-substituted rat kp-10 analogs, involving [Ca²⁺]_i responses in rat kiss1r-transfected Chinese hamster ovary cells, dynamic luteinizing hormone (LH) responses in vivo, and NMR structural studies. In vitro assays revealed that Ala substitutions in positions 6 or 10 of kp-10 resulted in a significant increase in EC₅₀ values (>6.46 x 10^-6 M versus 1.54 to 2.6 x 10^-8 M for rat and human Kp-10, respectively) and a substantial decrease in the proportion of responsive cells coupled to a marked increase in the time required to reach maximal response. In vivo assays showed that Ala⁶ substitution diminished and Ala¹⁰ substitution eliminated LH secretory responses, whereas coadministration of each analog failed to affect the LH-releasing ability of kp-10. Molecular modeling under NMR restraints revealed that kp-10 exhibits a helicoidal structure between the Asn⁴ and Tyr¹⁰ residues, with mixed - and 3₁₀-helix characteristics. Ala⁶ substitution induced limited destabilization of the helix around the position of the substitution. Ala¹⁰ substitution was found to totally disrupt the helical structure in the C-terminal region of the molecule. Taken together, our results indicate that positions 6 and 10 are critical for kp-10 action at kiss1r and suggest that modifications in these positions could lead to the generation of new kisspeptin agonists and/or antagonists with altered functional and perhaps binding properties. Furthermore, they emphasize the importance of using combined, multidisciplinary approaches, including in vivo studies, to reliably evaluate structure function properties of novel kisspeptin analogs.

[ARTICLES] Site Specificity of Agonist and Second Messenger-Activated Kinases for Somatostatin Receptor Subtype 2A (Sst2A) Phosphorylation

Liu, Q., Bee, M. S., Schonbrunn, A. — 2009-06-24

Somatostatin receptor subtype 2A (sst2A) mediates many of the endocrine and neuronal actions of somatostatin and is the target of somatostatin analogs in cancer therapy. As with many G-protein-coupled receptors, agonist stimulation causes sst2A receptor desensitization and internalization, events that require receptor phosphorylation. Furthermore, heterologous receptor activation of protein kinase C (PKC) also increases sst2A receptor phosphorylation and internalization. Here we analyzed a series of sst2A receptor mutants biochemically to identify residues in the receptor carboxyl terminus that were phosphorylated upon agonist stimulation, and we then generated four phosphorylation-sensitive antibodies to those residues. Once the selectivity of each antibody for its phosphorylated and nonphosphorylated target sequence was determined, the phospho-site-specific antibodies were used to demonstrate that somatostatin treatment of Chinese hamster ovary (CHO) cells expressing the wild type sst2A receptor increased phosphorylation on five residues in the receptor C terminus: Ser341, Ser343, Ser348, Thr353, and Thr354. Phorbol 12-myristate 13-acetate (PMA) increased receptor phosphorylation only on Ser343. Inhibition of PKC blocked PMA but not somatostatin stimulation, showing that different kinases catalyzed Ser343 phosphorylation. In contrast, somatostatin-stimulated sst2A receptor phosphorylation was inhibited by knockdown of G-protein coupled receptor kinase-2 with siRNA. Somatostatin increased sst2A receptor phosphorylation on the same five residues in GH4C1 pituitary cells as in CHO cells. However, PMA stimulated sst2A receptor phosphorylation on both Ser343 and Ser348 in GH4C1 cells. These results characterize the complex pattern of sst2A receptor phosphorylation by agonist and second messenger-activated kinases for the first time and indicate that cell type-specific regulation of sst2A receptor phosphorylation occurs.

[ARTICLES] Polyethylene Glycosylated Curcumin Conjugate Inhibits Pancreatic Cancer Cell Growth through Inactivation of Jab1

2009-06-24

Jab1 (Jun activation domain binding protein 1), integrated into COP9 signalosome complex (CSN), induces protein instability of many tumor suppressors and cell cycle regulators and is therefore a novel target in cancer therapy. Curcumin, an inhibitor of Jab1/CSN-associated kinase(s), has been reported to suppress tumor growth; however, curcumin is highly hydrophobic, and this feature prevents its usage as an antitumor drug. To increase the solubility and targeted delivery, we generated a water-soluble polyethylene glycol (PEG)-conjugated curcumin system, in which curcumin is covalently linked to PEG_35kD. PEGylated curcumin showed much greater reduction of cell growth than free curcumin in pancreatic cancer cells. Cells treated with PEGylated curcumin had increased arrest at the mitotic phase with the formation of abnormal multinucleated cells, indicating that this compound affects cell cycle progression, which may contribute to cell growth inhibition. The stabilities of Jab1 target proteins were also examined. PEGylated curcumin increased protein stability of these proteins in pancreatic cancer cells and directly inhibited the activity of Jab1/CSN-associated kinases. Moreover, the inhibitory effect of PEGylated curcumin on cell proliferation was blunted in pancreatic cancer cells with Jab1 knockdown. The results suggest that PEGylated curcumin inhibits cell proliferation through suppression of Jab1/CSN activity. More importantly, the new compound sensitized pancreatic cancer cells to gemcitabine-induced apoptosis and cell proliferation inhibitory effects. Collectively, the PEGylated curcumin conjugate has much more potent effects on pancreatic cancer cell growth inhibition than free curcumin. The current study provides a biologic rationale to treat patients with pancreatic adenocarcinoma with the nontoxic phytochemical conjugated to PEG for systemic delivery.

[ARTICLES] Apratoxin A Reversibly Inhibits the Secretory Pathway by Preventing Cotranslational Translocation

Liu, Y., Law, B. K., Luesch, H. — 2009-06-24

Apratoxin A is a potent cytotoxic marine natural product that rapidly inhibits signal transducer and activator of transcription (STAT) 3 phosphorylation by an undefined mechanism. We have used biochemical and proteomics approaches to illuminate upstream molecular events. Apratoxin A inhibits Janus kinase (JAK)/STAT signaling through rapid down-regulation of interleukin 6 signal transducer (gp130). Apratoxin A also depletes cancer cells of several cancer-associated receptor tyrosine kinases by preventing their N-glycosylation, leading to their rapid proteasomal degradation. A proteomics approach revealed that several proteins in the endoplasmic reticulum, the site of N-glycoprotein synthesis, are down-regulated upon apratoxin A exposure. Using in vitro cell free systems, we demonstrated that apratoxin A prevents cotranslational translocation of proteins destined for the secretory pathway. This process is reversible in living cells. Our study indicates that apratoxins are new tools to study the secretory pathway and raises the possibility that inhibition of cotranslational translocation may be exploited for anticancer drug development.

[ARTICLES] Inhibition of Aquaporin-1 and Aquaporin-4 Water Permeability by a Derivative of the Loop Diuretic Bumetanide Acting at an Internal Pore-Occluding Binding Site

2009-06-24

Aquaporin (AQP) water channels, essential for fluid homeostasis, are expressed in perivascular brain end-feet regions of astroglia (AQP4) and in choroid plexus (AQP1). At a high concentration, the loop diuretic bumetanide has been shown to reduce rat brain edema after ischemic stroke by blocking Na⁺-K⁺-2Cl^- cotransport. We hypothesized that an additional inhibition of AQP contributes to the protection. We show that osmotic water flux in AQP4-expressing Xenopus laevis oocytes is reduced by extracellular bumetanide (≥100 µM). The efficacy of block by bumetanide is increased by injection intracellularly. Forty-five synthesized bumetanide derivatives were tested on oocytes expressing human AQP1 and rat AQP4. Of these, one of the most effective was the 4-aminopyridine carboxamide analog, AqB013, which inhibits AQP1 and AQP4 (IC₅₀ ~20 µM, applied extracellularly). The efficacy of block was enhanced by mutagenesis of intracellular AQP4 valine-189 to alanine (V189A, IC₅₀ ~8 µM), confirming the aquaporin as the molecular target of block. In silico docking of AqB013 supported an intracellular candidate binding site in rat AQP4 and suggested that the block involves occlusion of the AQP water pore at the cytoplasmic side. AqB013 at 2 µM had no effect, and 20 µM caused 20% block of human Na⁺-K⁺-2Cl^- cotransporter activity, in contrast to >90% block of the transporter by bumetanide. AqB013 did not affect X. laevis oocyte Cl^- currents and did not alter rhythmic electrical conduction in an ex vivo gastric muscle preparation. The identification of AQP-selective pharmacological agents opens opportunities for breakthrough strategies in the treatment of edema and other fluid imbalance disorders.

[ARTICLES] The Spermostatic and Microbicidal Actions of Quinones and Maleimides: Toward a Dual-Purpose Contraceptive Agent

2009-06-24

There is an urgent need to develop safe, effective, dual-purpose contraceptive agents that combine the prevention of pregnancy with protection against sexually transmitted diseases. Here we report the identification of a group of compounds that on contact with human spermatozoa induce a state of "spermostasis," characterized by the extremely rapid inhibition of sperm movement without compromising cell viability. These spermostatic agents were more active and significantly less toxic than the reagent in current clinical use, nonoxynol 9, giving therapeutic indices (ratio of spermostatic to cytotoxic activity) that were orders of magnitude greater than this traditional spermicide. Although certain compounds could trigger reactive oxygen species generation by spermatozoa, this activity was not correlated with spermostasis. Rather, the latter was associated with alkylation of two major sperm tail proteins that were identified as A Kinase-Anchoring Proteins (AKAP3 and AKAP4) by mass spectrometry. As a consequence of disrupted AKAP function, the abilities of cAMP to drive protein kinase A-dependent activities in the sperm tail, such as the activation of SRC and the consequent stimulation of tyrosine phosphorylation, were suppressed. Furthermore, analysis of microbicidal activity using Chlamydia muridarum revealed powerful inhibitory effects at the same low micromolar doses that suppressed sperm movement. In this case, the microbicidal action was associated with alkylation of Major Outer Membrane Protein (MOMP), a major chlamydial membrane protein. Taken together, these results have identified for the first time a novel set of cellular targets and chemical principles capable of providing simultaneous defense against both fertility and the spread of sexually transmitted disease.

[ARTICLES] S3b Amino Acid Substitutions and Ancillary Subunits Alter the Affinity of Heteropoda venatoria Toxin 2 for Kv4.3

DeSimone, C. V., Lu, Y., Bondarenko, V. E., Morales, M. J. — 2009-06-24

Heteropoda venatoria toxin 2 (HpTx2) is an inhibitor cystine knot (ICK)-gating modifier toxin that selectively inhibits Kv4 channels. To characterize the molecular determinants of interaction, we performed alanine scanning of the Kv4.3 S3b region. HpTx2-Kv4.3 interaction had an apparent K_d value of 2.3 µM. Two alanine mutants in Kv4.3 increased K_d values to 6.4 µM for V276A and 25 µM for L275A. Simultaneous mutation of both amino acids to alanine nearly eliminated toxin interaction. Unlike Hanatoxin and other well characterized ICK toxins, HpTx2 binding does not require a charged amino acid for interaction. To determine whether the identity of the S3b binding site amino acids altered HpTx2 specificity, we constructed Kv4.3 [LV275IF]. This mutation decreased the K_d value to 0.54 µM, suggesting that the hydrophobic character of the putative binding site is the most important property for interaction with HpTx2. One mutant, N280A, caused stronger interaction of HpTx2 with Kv4.3; the K_d value for Kv4.3 [N280A] was 0.26 µM. To understand Kv4.3-based transient outward currents in native tissues, we tested the affinity of HpTx2 for Kv4.3 coexpressed with KChIP2b. The toxin's K_d value for Kv4.3 + KChIP2b was 0.95 µM. KChIP2b stabilizes the closed state of Kv4.3, suggesting that the increased toxin affinity is due to increased stabilization of the closed state. These data show that HpTx2 binding to Kv4.3 has aspects in common with other ICK gating modifier toxins but that the interventions that increase toxin affinity suggest flexibility toward channel binding that belies its unusual specificity for Kv4 channels.

[ARTICLES] {delta} Receptors Are Required for Full Inhibitory Coupling of {micro} Receptors to Voltage-Dependent Ca2+ Channels in Dorsal Root Ganglion Neurons

Walwyn, W., John, S., Maga, M., Evans, C. J., Hales, T. G. — 2009-06-24

Recombinant µ and opioid receptors expressed in cell lines can form heterodimers with distinctive properties and trafficking. However, a role for opioid receptor heterodimerization in neurons has yet to be identified. The inhibitory coupling of opioid receptors to voltage-dependent Ca²⁺ channels (VDCCs) is a relatively inefficient process and therefore provides a sensitive assay of altered opioid receptor function and expression. We examined µ-receptor coupling to VDCCs in dorsal root ganglion neurons of (+/+), (+/-), and (-/-) mice. Neurons deficient in receptors exhibited reduced inhibition of VDCCs by morphine and [d-Ala²,Phe⁴,Gly⁵-ol]-enkephalin (DAMGO). An absence of receptors caused reduced efficacy of DAMGO without affecting potency. An absence of receptors reduced neither the density of VDCCs nor their inhibition by either the GABA_B receptor agonist baclofen or intracellular guanosine 5'-O-(3-thio)triphosphate. Flow cytometry revealed a reduction in µ-receptor surface expression in (-/-) neurons without altered DAMGO-induced internalization. There was no change in µ-receptor mRNA levels. d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH₂-sensitive µ-receptor-coupling efficacy was fully restored to (+/+) levels in (-/-) neurons by expression of recombinant receptors. However, the dimerization-deficient -15 construct expressed in (-/-) neurons failed to fully restore the inhibitory coupling of µ receptors compared with that seen in (+/+) neurons, suggesting that, although not essential for µ-receptor function, µ- receptor dimerization contributes to full µ-agonist efficacy. Because DAMGO exhibited a similar potency in (+/+) and (-/-) neurons and caused similar levels of internalization, the role for heterodimerization is probably at the level of receptor biosynthesis.

[ARTICLES] {alpha}1-Adrenergic Receptor Stimulates Interleukin-6 Expression and Secretion through Both mRNA Stability and Transcriptional Regulation: Involvement of p38 Mitogen-Activated Protein Kinase and Nuclear Factor-{kappa}B

Perez, D. M., Papay, R. S., Shi, T. — 2009-06-24

Our previous studies have demonstrated that activation of ₁-adrenergic receptors (ARs) increased interleukin-6 (IL-6) mRNA expression and protein secretion, which is probably an important yet unknown mechanism contributing to the regulation of cardiac function. Using Rat-1 fibroblasts stably transfected with the _1A-AR subtype and primary mouse neonatal cardiomyocytes, we elucidated the basic molecular mechanisms responsible for the ₁-AR modulation of IL-6 expression. IL-6 mRNA production mediated by ₁-AR peaked at 1 to 2 h. Studies of the mRNA decay rate indicated that ₁-AR activation enhanced IL-6 mRNA stability. Analysis of IL-6 promoter activity using a series of deletion constructs indicated that ₁-ARs enhanced IL-6 transcription through several transcriptional elements, including nuclear factor B (NF-B). Inhibition of ₁-AR mediated IL-6 production and secretion by actinomycin D and the increase of intracellular IL-6 levels by ₁-AR activation suggest that ₁-AR mediated IL-6 secretion through de novo synthesis. Both IL-6 transcription and protein secretion mediated by ₁-ARs were significantly reduced by chemical inhibitors for p38 mitogen-activated protein kinase (MAPK) and NF-B and by a dominant-negative construct of p38 MAPK. Serum level of IL-6 was elevated in transgenic mice expressing a constitutively active mutant of the _1A-AR subtype but not in a similar mouse model expressing the _1B-AR subtype. Our results indicate that ₁-ARs stimulated IL-6 expression and secretion through regulating both mRNA transcription and stability, involving p38 MAPK and NF-B pathways.

[ARTICLES] KLYP956 Is a Non-Imidazole-Based Orally Active Inhibitor of Nitric-Oxide Synthase Dimerization

2009-06-24

Nitric-oxide synthases (NOS) generate nitric oxide (NO) through the oxidation of l-arginine. Inappropriate or excessive production of NO by NOS is associated with the pathophysiology of various disease states. Efforts to treat these disorders by developing arginine mimetic, substrate-competitive NOS inhibitors as drugs have met with little success. Small-molecule-mediated inhibition of NOS dimerization represents an intriguing alternative to substrate-competitive inhibition. An ultra-high-throughput cell-based screen of 880,000 small molecules identified a novel quinolinone with inducible NOS (iNOS) inhibitory activity. Exploratory chemistry based on this initial screening hit resulted in the synthesis of KLYP956, which inhibits iNOS at low nanomolar concentrations. The iNOS inhibitory potency of KLYP956 is insensitive to changes in concentrations of the substrate arginine, or the cofactor tetrahydrobiopterin. Mechanistic analysis suggests that KLYP956 binds the oxygenase domain in the vicinity of the active site heme and inhibits iNOS and neuronal NOS (nNOS) by preventing the formation of enzymatically active dimers. Oral administration of KLYP956 [N-(3-chlorophenyl)-N-((8-fluoro-2-oxo-1,2-dihydroquinolin-4-yl)methyl)-4-methylthiazole-5-carboxamide] inhibits iNOS activity in a murine model of endotoxemia and blocks pain behaviors in a formalin model of nociception. KLYP956 thus represents the first nonimidazole-based inhibitor of iNOS and nNOS dimerization and provides a novel pharmaceutical alternative to previously described substrate competitive inhibitors.

[ARTICLES] Potent Activity of Indolequinones against Human Pancreatic Cancer: Identification of Thioredoxin Reductase as a Potential Target

2009-06-24

The indolequinone ES936 {5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione} was previously developed in our lab as an antitumor agent against pancreatic cancer. The objective of this study was to identify indolequinones with improved potency against pancreatic cancer and to define their mechanisms of action. Pancreatic cancer cell lines PANC-1, MIA PaCa-2, and BxPC-3 were used in in vitro assays [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) and clonogenic assays]; indolequinones displayed potent cytotoxicity against all three cell lines, and two specific classes of indolequinone were particularly potent agents. These indolequinones induced caspase-dependent apoptosis but no redox cycling or oxidative stress in MIA PaCa-2 and BxPC-3 cells. Selected indolequinones were also screened against the NCI-60 cell line panel and were found to be particularly effective against colon, renal, and melanoma cancer cells. A potential target of these indolequinones was identified as thioredoxin reductase. Indolequinones were found to be potent inhibitors of thioredoxin reductase activity both in pancreatic cancer cells and in cell-free systems. The mechanism of action of the indolequinones was shown to involve metabolic reduction, loss of a leaving group to generate a reactive electrophile resulting in alkylation of the selenocysteine residue in the active site of thioredoxin reductase. In vivo efficacy of the indolequinones was also tested in the MIA PaCa-2 pancreatic tumor xenograft in nude mice, and lead indolequinones demonstrated high efficacy and low toxicity. Inhibition of thioredoxin reductase represents a potential novel target in pancreatic cancer and may provide a biomarker of effect of lead indolequinones in this type of cancer.

[ARTICLES] Heme-Oxygenase-1 Induction and Carbon Monoxide-Releasing Molecule Inhibit Lipopolysaccharide (LPS)-Induced High-Mobility Group Box 1 Release in Vitro and Improve Survival of Mice in LPS- and Cecal Ligation and Puncture-Induced Sepsis Model in Vivo

2009-06-24

We examined our hypothesis that heme-oxygenase-1 (HO-1)-derived carbon monoxide (CO) inhibits the release of high-mobility group box 1 (HMGB1) in RAW264.7 cells activated with lipopolysaccharide (LPS) in vitro and in LPS- or cecal ligation and puncture (CLP)-induced septic mice in vivo, so that HO-1 induction or CO improves survival of sepsis in rodents. We found that pretreatment with HO-1 inducers (hemin, cobalt protoporphyrin IX) or transfection of HO-1 significantly inhibited HMGB1 release, which was blocked by HO-1 small interfering RNA, in cells activated by LPS. Carbon monoxide-releasing molecule 2 (CORM-2) but not bilirubin or deferoxamine inhibited HMGB1 release in LPS-activated macrophages. Oxyhemoglobin reversed the effect of HO-1 inducers on HMGB1 release. Translocation of HMGB1 from nucleus to cytosol was significantly inhibited by HO-1 inducers, CORM-2, or HO-1 transfection. Neutralizing antibodies to tumor necrosis factor (TNF)-, interleukin (IL)-1β, interferon-β, and N-nitro-l-arginine methyl ester hydrochloride but not N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methane sulfonamide (NS-398) significantly inhibited HMGB1 release in LPS-activated cells. Production of TNF-, IL-1β, and IFN-β was significantly reduced by pretreatment of HO-1 inducers, CORM-2, or HO-1 transfection in LPS-activated cells. Plasma levels of HMGB1 in mice challenged with LPS or CLP were significantly reduced by the administration of HO-1 inducers or CORM-2, which was accompanied by either reduction (pretreatment) or no change (delayed administration) of serum TNF- and IL-1β levels. Regardless of pretreatment or delayed administration, CORM-2 and hemin rescued mice from lethal endotoxemia and sepsis induced by LPS or CLP. Taken together, we concluded that HO-1-derived CO reduces HMGB1 release in LPS-activated cells and LPS- or CLP-induced animal model of sepsis.

[ARTICLES] Mesalamine Suppresses the Expression of TC22, a Novel Tropomyosin Isoform Associated with Colonic Neoplasia

Das, K. K., Bajpai, M., Kong, Y., Liu, J., Geng, X., Das, K. M. — 2009-06-24

Although a protective role for mesalamine against colon cancer in ulcerative colitis has been shown epidemiologically, its molecular mechanism is unknown. We cloned and sequenced a novel human tropomyosin (hTM) isoform, TC22, which is an alternatively spliced variant of normal epithelial hTM isoform 5 (hTM5), identical apart from 25 C-terminal amino acids. TC22 is expressed in 100% of colorectal carcinoma but is not expressed in normal colon epithelial cells. To explore a molecular mechanism of chemoprevention, we examined the effect of mesalamine on TC22 expression using LS180 colon cancer cells. Expression of hTM5 and TC22 was investigated at the protein and gene levels by fluorescence-activated cell sorting and real-time reverse transcription-polymerase chain reaction. Small interference RNA (siRNA) against the TC22 variant were transfected into LS180 colon cancer cells, reducing protein and transcript levels by 45 to 50%. Mesalamine or sulfasalazine (2 mM), but not sulfapyridine, significantly (p < 0.02-0.006) reduced the expression of the TC22 transcript and significantly (p < 0.05 to <0.0002) reduced the expression of TC22 protein in a dose-dependent and reversible manner. Rosiglitazone, a specific peroxisome proliferator-activated receptor- (PPAR) agonist, similarly and significantly (p < 0.002) reduced TC22 protein expression. A polymerase chain reaction array of 84 cancer-related genes performed on TC22 siRNA-transfected cells demonstrated a significant (more than two times) change in targets involved in apoptosis, adhesion, angiogenesis, and tissue remodeling. We conclude that mesalamine, sulfasalazine, and rosiglitazone significantly reduced the cellular expression of TC22, implicating PPAR in this modulation. Similar suppression of TC22 by siRNA produced gene level changes on several critical carcinogenic pathways. These findings suggest a novel antineoplastic molecular effect of mesalamine.

[ARTICLES] S-Adenosylmethionine and Methylthioadenosine Inhibit Cellular FLICE Inhibitory Protein Expression and Induce Apoptosis in Colon Cancer Cells

2009-06-24

S-Adenosylmethionine (SAMe) and its metabolite 5'-methylthioadenosine (MTA) inhibit mitogen-induced proliferative response in liver and colon cancer cells. SAMe and MTA are also proapoptotic in liver cancer cells by selectively inducing Bcl-x_S expression. The aims of this work were to assess whether these agents are proapoptotic in colon cancer cells, and if so, to elucidate the molecular mechanisms. We found that both SAMe and MTA are proapoptotic in HT-29 and RKO cells in a dose- and time-dependent manner. Gene microarray uncovered down-regulation of cellular FLICE inhibitory protein (cFLIP). SAMe and MTA treatment led to a decrease in the mRNA and protein levels of both the long and short cFLIP isoforms. This required de novo RNA synthesis and was associated with activation of procaspase-8, Bid cleavage, and release of cytochrome c from the mitochondria. Inhibiting caspase 8 activity or overexpression of cFLIP protected against apoptosis, whereas supplementing with polyamines did not. SAMe and MTA treatment sensitized RKO cells to tumor necrosis factor -related apoptosis-inducing ligand-induced apoptosis. Although SAMe and MTA are proapoptotic in colon cancer cells, they have no toxic effects in NCM460 cells, a normal colon epithelial cell line. In contrast to liver cancer cells, SAMe and MTA had no effect on Bcl-x_S expression in colon cancer cells. In conclusion, SAMe and MTA are proapoptotic in colon cancer cells but not normal colon epithelial cells. One molecular mechanism identified is the inhibition of cFLIP expression. SAMe and MTA may be attractive agents in the chemoprevention and treatment of colon cancer.

[ARTICLES] Activation Switch in the Transmembrane Domain of Metabotropic Glutamate Receptor

Yanagawa, M., Yamashita, T., Shichida, Y. — 2009-06-24

Metabotropic glutamate receptors (mGluRs), members of family 3 G protein-coupled receptors, play pivotal roles in the modulation of synaptic transmission and are important drug targets for various neurological diseases. The structures of the extracellular ligand-binding domain (ECD) of mGluRs and its changes upon ligand binding have been well studied by various techniques, including X-ray crystallography. In contrast, little is known about the structure and structural changes of the seven-transmembrane domain (TMD). Here we searched for constitutively active mutation (CAM) sites in the TMD of mGluR8 to get insight into the epicenter of the structural changes in TMD, a potential target for allosteric ligands. Mutational analyses based on the knowledge of activating mutations of calcium-sensing receptor showed the presence of several CAM sites in the TMD of mGluR8. Among them, the site at position 764 in helix V, where threonine is present, is unique in that any substitution resulted in elevation of the basal activity, and some substitutions caused a loss of responsiveness to agonist. Further comprehensive mutational analyses indicated that the additional mutation of the CAM site at position 663 in helix III, where a tyrosine residue is present, generated a revertant phenotype. Mutations at these sites also increased the agonist binding affinity, although these sites are far from the ECD. These results indicated that the specific pair of amino acids at these CAM sites forms an activation switch that stabilizes the inactive state of mGluR8 and mediates the signal flow from the ECD to the cytoplasmic G protein-interacting site.

[ARTICLES] Cisplatin-Induced DNA Damage Activates Replication Checkpoint Signaling Components that Differentially Affect Tumor Cell Survival

Wagner, J. M., Karnitz, L. M. — 2009-06-24

Cisplatin and other platinating agents are some of the most widely used chemotherapy agents. These drugs exert their antiproliferative effects by creating intrastrand and interstrand DNA cross-links, which block DNA replication. The cross-links mobilize signaling and repair pathways, including the Rad9-Hus1-Rad1-ATR-Chk1 pathway, a pathway that helps tumor cells survive the DNA damage inflicted by many chemotherapy agents. Here we show that Rad9 and ATR play critical roles in helping tumor cells survive cisplatin treatment. However, depleting Chk1 with small interfering RNA or inhibiting Chk1 with 3-(carbamoylamino)-5-(3-fluorophenyl)-N-(3-piperidyl)thiophene-2-carboxamide (AZD7762) did not sensitize these cells to cisplatin, oxaliplatin, or carboplatin. Moreover, when Rad18, Rad51, BRCA1, BRCA2, or FancD2 was disabled, Chk1 depletion did not further sensitize the cells to cisplatin. In fact, Chk1 depletion reversed the sensitivity seen when Rad18 was disabled. Collectively, these studies suggest that the pharmacological manipulation of Chk1 may not be an effective strategy to sensitize tumors to platinating agents.