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Biochemical Pharmacology (v.84, #4)

Editorial Advisory Board (pp. iii).

Proactively maintaining publication standards (pp. 413-413).

Orthosteric versus allosteric GPCR activation: The great challenge of group-III mGluRs by Peter J. Flor; Francine C. Acher (pp. 414-424).

Group-III metabotropic glutamate receptors (mGluRs) comprise four structurally related brain and retinal G protein-coupled receptors (GPCRs), mGluR4, mGluR6, mGluR7 and mGluR8, which receive much attention as promising targets for nervous system drugs. In particular, activation of mGluR4 is a major focus for the development of new therapeutics in Parkinson's disease, while mGluR7 activation is considered a potential approach for future treatments of specific psychiatric conditions. The first generation group-III mGluR agonists, e.g.l-AP4 andl-SOP, are characterized by an essential phosphonate functional group, which became a major limitation for the development of systemically active, potent and receptor subtype-selective drugs. Recently however, two approaches emerged in parallel providing resolution to this constraint: in silico high-throughput screening of chemical libraries against a 3D-model of the mGluR4 extracellular domain identified a hit that was optimized into a series of potent and subtype-selective orthosteric agonists with drug-like properties and novel chemotype structures; secondly, high-throughput random screening of chemical libraries against recombinantly expressed group-III receptors identified diverse chemical sets of allosteric agonists and positive modulators, which are drug-like, display selectivity for mGluR4, mGluR7, or mGluR8 and act via novel pharmacological sites.Here, we illustrate new scientific insights obtained via the use of those strategies. Also, we compare advantages and disadvantages of both approaches to identify the desired group-III mGluR activators and we conclude with suggestions how to employ those discovery strategies with success for the identification, optimization, and development of clinical drug candidates; this may have important implications for the entire field of GPCR research.

Keywords: Abbreviations; ACPT-I; (1; S; ,3; R; ,4; S; )-1-aminocyclopentane-1,3,4-tricarboxylic acid; ADMET; absorption, distribution, metabolism, excretion, toxicity; ADX88178; 5-methyl-; N; -(4-methylpyrimidin-2-yl)-4-(1H-pyrazol-4-yl)thiazol-2-amine; AMN082; N; ,; N; ′-dibenzhydryl-ethane-1,2-diamine dihydrochloride; AMPA; 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid; AP4; 2-amino-4-phosphono-butyric acid; APCPr; 1-amino-2-(phosphonomethyl)cyclopropanecarboxylic acid; APDC; 4-Aminopyrrolidine-2,4-dicarboxylic acid; AZ12216052; 2-(4-bromobenzylthio)-; N; -(4-sec-butylphenyl)acetamide; BBB; blood-brain barrier; CNS; central nervous system; CPCCOEt; 7-(hydroxyimino)cyclopropa[; b; ]chromen-1a-carboxylate ethyl ester; CPPG; α; -cyclopropyl-4-phosphonophenylglycine; l; -DOPA; l; -3,4-dihydroxyphenylalanine; EC; ₅₀; half maximal effective concentration; GPCR; G protein-coupled receptor; HTS; high-throughput screening; iGluR; ionotropic glutamate receptor; LSP1-2111; [((3; S; )-3-Amino-3-carboxy)propyl][(4-hydroxy-5-methoxy-3-nitrophenyl)hydroxymethyl]phosphinic acid; LSP1-3081; [(3; S; )-3-(3-amino-3-carboxypropyl(hydroxy)phosphinyl)-hydroxymethyl]-5-nitrothiophene; LSP4-2022; [((3; S; )-3-Amino-3-carboxy)propyl][(4-(carboxymethoxy)phenyl)hydroxymethyl]phosphinic acid; mGluR; metabotropic glutamate receptor; Lu AF21934; (1; S; ,2; R; )-; N; 1-(3,4-dichlorophenyl)-cyclohexane-1,2-dicarboxamide); LY2140023; N; -methionine amide of LY404039; LY354740; (1; S; ,2; S; ,5; R; ,6; S; )-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid; LY404039; (1; R; ,4; S; ,5; S; ,6; S; )-4-amino-2-sulfonylbicyclo[3.1.0]hexane-4,6-dicarboxylic acid; MAP4; 2-amino-2-methyl-4-phosphonobutanoic acid; MMPIP; 6-(4-methoxyphenyl)-5-methyl-3-(4-pyridinyl)-isoxazolo[4,5-; c; ]pyridin-4(5; H; )-one; MPEP; 2-methyl-6-(phenylethynyl)pyridine; MPPG; α-methyl-4-phosphonophenylglycine; MSOP; α-methylserine-O-phosphate; NAM; negative allosteric modulator; NMDA; N; -methyl-D-aspartate; PAM; positive allosteric modulator; PCEP; 3-amino-3-carboxypropyl-2′-carboxyethyl phosphinic acid; PEPT1; peptide transporter 1; PHCCC; N; -phenyl-7-(hydroxylimino)cyclopropa[b]chromen-1a-carboxyamide; PK; pharmacokinetics; PPG; 4-phosphonophenylglycine; PTSD; post-traumatic stress disorder; siRNA; small interfering RNA; SOP; serine-O-phosphate; TM; transmembrane; VFT; Venus flytrap; VU0155041; cis; -2-(3,5-dichlorphenylcarbamoyl)cyclohexanecarboxylic acid; VU0364770; N; -(3-chlorophenyl)picolinamideGPCR; Metabotropic glutamate; LSP1-2111; LSP4-2022; PHCCC; AMN082

Cyclosporin A suppresses prostate cancer cell growth through CaMKKβ/AMPK-mediated inhibition of mTORC1 signaling by Chae Ryun Lee; Jung Nyeo Chun; Sang-Yeob Kim; Soonbum Park; Su-Hwa Kim; Eun-Jung Park; In-San Kim; Nam-Hyuk Cho; In-Gyu Kim; Insuk So; Tae Woo Kim; Ju-Hong Jeon (pp. 425-431).

Cyclosporin A (CsA) has antitumor effects on various cancers including prostate cancer. However, its antitumor mechanism is poorly understood. In this study, we showed that AMP-activated protein kinase (AMPK) mediates the antitumor effect of CsA on prostate cancer cells. CsA attenuated cell growth by inducing a G1 arrest through the inhibition of mTOR complex 1 (mTORC1) signaling. In this context, Akt was paradoxically activated downstream of the EGF receptor (EGFR)-mediated increase in phosphatidylinositol 3,4,5-trisphosphate (PIP₃) production. However, CsA also caused a Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ)-dependent activation of AMPK, which inhibits mTORC1 signaling; this led to ineffective Akt signaling. An EGFR or Akt inhibitor increased the growth suppressive activity of CsA, whereas the combination of an AMPK inhibitor and CsA markedly rescued cells from the G1 arrest and increased cell growth. These results provide novel insights into the molecular mechanisms of CsA on cancer signaling pathways.

Keywords: Cyclosporin A; AMPK; Akt; mTOR; Prostate cancer

Epothilone B inhibits migration of glioblastoma cells by inducing microtubule catastrophes and affecting EB1 accumulation at microtubule plus ends by Alessandra Pagano; Stéphane Honoré; Renu Mohan; Raphael Berges; Anna Akhmanova; Diane Braguer (pp. 432-443).

Invasion of normal brain tissue by tumor cells is a major contributing factor to the recurrence of glioblastoma and its resistance to therapy. Here, we have assessed the efficacy of the microtubule (MT) targeting agent Epothilone B (patupilone) on glioblastoma cell migration, a prerequisite for invasive tumor cell behavior. At non-cytotoxic concentrations, patupilone inhibited glioblastoma cell movement, as shown by transwell cell migration, random motility and spheroid assays. This anti-migratory effect was associated with a reduced accumulation of EB1 and other MT plus end tracking proteins at MT ends and with the induction of MT catastrophes, while the MT growth rate and other MT dynamic instability parameters remained unaltered. An increase in MT catastrophes led to the reduction of the number of MTs reaching the leading edge. Analysis of the effect of patupilone on MT dynamics in a reconstituted in vitro system demonstrated that the induction of MT catastrophes and an alteration of EB1 accumulation at MT plus end are intrinsic properties of patupilone activity. We have thus demonstrated that patupilone antagonizes glioblastoma cell migration by a novel mechanism, which is distinct from suppression of MT dynamic instability. Taken together, our results suggest that EB proteins may represent a new potential target for anti-cancer therapy in highly invasive tumors.

Keywords: Microtubule; EB proteins; Glioblastoma; Cell migration; Epothilone B

Interaction of pseudolaric acid B with the colchicine site of tubulin by Taradas Sarkar; Tam Luong Nguyen; Zhi-Wei Su; Jun Hao; Ruoli Bai; Rick Gussio; Samuel X. Qiu; Ernest Hamel (pp. 444-450).

We purified pseudolaric acid B (PAB) from the root and stem bark of Pseudolarix kaempferi (Lindl.) Gorden. Confirming previous findings, we found that the compound had high nanomolar IC₅₀ antiproliferative effects in several cultured cell lines, causing mitotic arrest and the disappearance of intracellular microtubules. PAB strongly inhibited tubulin assembly (IC₅₀, 1.1μM) but weakly inhibited the binding of colchicine to tubulin, as demonstrated by fluorescence and with [³H]colchicine. Kinetic analysis demonstrated that the mechanism of inhibition was competitive, with an apparent K_i of 12–15μM. Indirect studies demonstrated that PAB bound rapidly to tubulin and dissociated more rapidly from tubulin than the colchicine analog 2-methoxy-5-(2′,3′,4′-trimethoxyphenyl)tropone, whose complex with tubulin is known to have a half-life of 17s at 37°C. We modeled PAB into the colchicine site of tubulin, using the crystal structure 1SA0 that contains two αβ-tubulin heterodimers, both bound to a colchicinoid and to a stathmin fragment. The binding model of PAB revealed common pharmacophoric features between PAB and colchicinoids, not readily apparent from their chemical structures.

The therapeutic effects of rGel/BLyS fusion toxin in in vitro and in vivo models of mantle cell lymphoma by Mi-Ae Lyu; Lan V. Pham; Bokyung Sung; Archito T. Tamayo; Kwang Seok Ahn; Walter N. Hittelman; Lawrence H. Cheung; John W. Marks; Min-Jeong Cho; Richard J. Ford; Bharat B. Aggarwal; Michael G. Rosenblum (pp. 451-458).

Mantle cell lymphoma (MCL) is an incurable, aggressive histo-type of B-cell non-Hodgkin lymphoma associated with both high relapsed rates and relatively short survival. Because MCL over-expresses receptors for B lymphocyte stimulator (BLyS) and displays constitutively active NF-κB, agents targeting these pathways may be of therapeutic relevance in this disease. To investigate the potential clinical use of the rGel/BLyS fusion toxin in combination with bortezomib, we evaluated this fusion toxin for its ability to inhibit MCL growth in severe combined immunodeficiency (SCID) xenograft model. Compared with PBS-treated mice, mice treated with this fusion toxin prolonged both median (84 days vs. 125 days) and overall survival (0% vs. 40%) ( p=0.0027). Compared with bortezomib alone-treated mice, mice treated with rGel/BLyS plus bortezomib showed significantly increased median (91 days vs. 158 days) and overall survival (0% vs. 20%) ( p=0.0127). Histopathologic analysis of peritoneal intestinal mesentery from MCL-SCID mice showed no demonstrable microscopic lymphomatous involvement at 225 days after treatment with rGel/BLyS. Combination treatment resulted in a synergistic growth inhibition, down-regulation of NF-κB DNA-binding activity, inhibition of cyclin D1, Bcl-x_L, p-Akt, Akt, p-mTOR, and p-Bad, up-regulation of Bax, and induction of cellular apoptosis. Our findings demonstrate that rGel/BLyS is an effective therapeutic agent for both primary and salvage treatment of aggressive MCL expressing constitutively active NF-κB and BLyS receptors and may be an excellent candidate for clinical development.

Keywords: Mantle cell lymphoma; BLyS; BLyS receptor; Bortezomib; Combination

Characterization of virus strains resistant to the herpes virus helicase–primase inhibitor ASP2151 (Amenamevir) by Koji Chono; Kiyomitsu Katsumata; Toru Kontani; Kimiyasu Shiraki; Hiroshi Suzuki (pp. 459-467).

ASP2151 is an antiherpes agent targeting the helicase–primase complex of herpes simplex virus (HSV)-1, HSV-2, and varicella-zoster virus (VZV). We characterized the ASP2151-resistant HSV-1 and HSV-2 variants or mutants based on findings from sequencing analysis, growth, pathogenicity, and susceptibility testing, identifying several single base-pair substitutions resulting in amino acid changes in the helicase and primase subunit of ASP2151-resistant mutants. Amino acid alterations in the helicase subunit were clustered near helicase motif IV in the UL5 helicase gene of both HSV-1 and HSV-2, while the primase subunit substitution associated with reduced susceptibility, R367H, was found in ASP2151-resistant HSV-1 mutants. However, while susceptibility in the ASP2151-resistant HSV mutants to existing antiherpes agents was equivalent to that in wild-type HSV strains, ASP2151-resistant HSV mutants showed attenuated in vitro growth capability and in vivo pathogenicity compared with the parent strains. Taken together, our present findings demonstrated that important amino acid substitutions associated with reduced susceptibilities of HSV-1 and HSV-2 to ASP2151 exist in both the helicase and primase subunits of the helicase–primase complex, and that mutations in this complex against ASP2151 might confer defects in viral replication and pathogenicity.

Keywords: Abbreviations; HHV; human herpesvirus; HSV-1; herpes simplex virus type 1; HSV-2; herpes simplex virus type 2; VZV; varicella-zoster virus; TK; thymidine kinase; HPI; helicase–primase inhibitor (HPI); ACV; acyclovirHerpes simplex viruses; ASP2151; Amenamevir; Helicase–primase inhibitor

Arctigenin preferentially induces tumor cell death under glucose deprivation by inhibiting cellular energy metabolism by Yuan Gu; Chunting Qi; Xiaoxiao Sun; Xiuquan Ma; Haohao Zhang; Lihong Hu; Junying Yuan; Qiang Yu (pp. 468-476).

Selectively eradicating cancer cells with minimum adverse effects on normal cells is a major challenge in the development of anticancer therapy. We hypothesize that nutrient-limiting conditions frequently encountered by cancer cells in poorly vascularized solid tumors might provide an opportunity for developing selective therapy. In this study, we investigated the function and molecular mechanisms of a natural compound, arctigenin, in regulating tumor cell growth. We demonstrated that arctigenin selectively promoted glucose-starved A549 tumor cells to undergo necrosis by inhibiting mitochondrial respiration. In doing so, arctigenin elevated cellular level of reactive oxygen species (ROS) and blocked cellular energy metabolism in the glucose-starved tumor cells. We also demonstrated that cellular ROS generation was caused by intracellular ATP depletion and played an essential role in the arctigenin-induced tumor cell death under the glucose-limiting condition. Furthermore, we combined arctigenin with the glucose analogue 2-deoxyglucose (2DG) and examined their effects on tumor cell growth. Interestingly, this combination displayed preferential cell-death inducing activity against tumor cells compared to normal cells. Hence, we propose that the combination of arctigenin and 2DG may represent a promising new cancer therapy with minimal normal tissue toxicity.

Keywords: Arctigenin; Glucose deprivation; Tumor cell death; ATP levels; ROS generation

Allosteric interactions within the AT₁ angiotensin receptor homodimer: Role of the conserved DRY motif by Bence Szalai; László Barkai; Gábor Turu; László Szidonya; Péter Várnai; László Hunyady (pp. 477-485).

G protein coupled receptor (GPCR) dimerization has a remarkable impact on the diversity of receptor signaling. Allosteric communication between the protomers of the dimer can alter ligand binding, receptor conformation and interactions with different effector proteins. In this study we investigated the allosteric interactions between wild type and mutant protomers of type 1 angiotensin receptor (AT₁R) dimers transiently expressed in CHO cells. In our experimental setup, one protomer of the dimer was selectively stimulated and the β-arrestin2 binding and conformation alteration of the other protomer was followed. The interaction between β-arrestin2 and the non-stimulated protomer was monitored through a bioluminescence resonance energy transfer (BRET) based method. To measure the conformational alterations in the non-stimulated protomer directly, we also used a BRET based intramolecular receptor biosensor, which was created by inserting yellow fluorescent protein (YFP) into the 3rd intracellular loop of AT₁R and fusing Renilla luciferase (RLuc) to its C terminal region. We have detected β-arrestin2 binding, and altered conformation of the non-stimulated protomer. The cooperative ligand binding of the receptor homodimer was also observed by radioligand dissociation experiments. Mutation of the conserved DRY sequence in the activated protomer, which is also required for G protein activation, abolished all the observed allosteric effects. These data suggest that allosteric interactions in the homodimers of AT₁R significantly affect the function of the non-stimulated protomer, and the conserved DRY motif has a crucial role in these interactions.

Keywords: GPCR; Dimerization; Angiotensin receptor; Allosteric interaction; BRET

Inhibition of ROS-induced apoptosis in endothelial cells by nitrone spin traps via induction of phase II enzymes and suppression of mitochondria-dependent pro-apoptotic signaling by Amlan Das; Bhavani Gopalakrishnan; Oliver H. Voss; Andrea I. Doseff; Frederick A. Villamena (pp. 486-497).

Oxidative stress is the main etiological factor behind the pathogenesis of various diseases including inflammation, cancer, cardiovascular and neurodegenerative disorders. Due to the spin trapping abilities and various pharmacological properties of nitrones, their application as therapeutic agent has been gaining attention. Though the antioxidant properties of the nitrones are well known, the mechanism by which they modulate the cellular defense machinery against oxidative stress is not well investigated and requires further elucidation. Here, we have investigated the mechanisms of cytoprotection of the nitrone spin traps against oxidative stress in bovine aortic endothelial cells (BAEC). Cytoprotective properties of both the cyclic nitrone 5,5-dimethyl-pyrroline N-oxide (DMPO) and linear nitrone α-phenyl N- tert-butyl nitrone (PBN) against H₂O₂-induced cytotoxicity were investigated. Preincubation of BAEC with PBN or DMPO resulted in the inhibition of H₂O₂-mediated cytotoxicity and apoptosis. Nitrone-treatment resulted in the induction and restoration of phase II antioxidant enzymes via nuclear translocation of NF-E2-related factor 2 (Nrf-2) in oxidatively-challenged cells. Furthermore, the nitrones were found to inhibit the mitochondrial depolarization and subsequent activation of caspase-3 induced by H₂O₂. Significant down-regulation of the pro-apoptotic proteins p53 and Bax, and up-regulation of the anti-apoptotic proteins Bcl-2 and p-Bad were observed when the cells were preincubated with the nitrones prior to H₂O₂-treatment. It was also observed that Nrf-2 silencing completely abolished the protective effects of nitrones. Hence, these findings suggest that nitrones confer protection to the endothelial cells against oxidative stress by modulating phase II antioxidant enzymes and subsequently inhibiting mitochondria-dependent apoptotic cascade.

Keywords: Nitrones; Spin traps; Oxidative stress; Endothelial dysfunction; Nrf-2

Effects of the natural flavone trimethylapigenin on cardiac potassium currents by Yi Liu; Xiao-Hui Xu; Zheng Liu; Xin-Ling Du; Kui-Hao Chen; Xin Xin; Zhen-Dong Jin; Ji-Zhong Shen; Yan Hu; Gui-Rong Li; Man-Wen Jin (pp. 498-506).

The natural flavones and polymethylflavone have been reported to have cardiovascular protective effects. In the present study, we determined whether quecertin, apigenin and their methylated compounds (3,7,3′,4′-tetramethylquecertin, 3,5,7,3′,4′-pentamethylquecertin, 7,4′-dimethylapigenin, and 5,7,4′-trimethylapigenin) would block the atrial specific potassium channel hKv1.5 using a whole-cell patch voltage-clamp technique. We found that only trimethylapigenin showed a strong inhibitory effect on hKv1.5 channel current. This compound suppressed hKv1.5 current in HEK 293 cell line (IC₅₀=6.4μM), and the ultra-rapid delayed rectify K⁺ current I_Kur in human atrial myocytes (IC₅₀=8.0μM) by binding to the open channels and showed a use- and frequency-dependent manner. In addition, trimethylapigenin decreased transient outward potassium current ( I_to) in human atrial myocytes, inhibited acetylcholine-activated K⁺ current (IC₅₀=6.8μM) in rat atrial myocytes. Interestingly, trimethylapigenin had a weak inhibition of hERG channel current. Our results indicate that trimethyapigenin significantly inhibits the atrial potassium currents hKv1.5/ I_Kur and I_KACh, which suggests that trimethylapigenin may be a potential candidate for anti-atrial fibrillation.

Keywords: Abbreviations; I; _Kur; ultra-rapidly delayed rectifier potassium current; HEK 293 cells; human embryonic kidney 293 cells; I; _KACh; acetylcholine-activated potassium current; I; _to; transient outward potassium currentTrimethylapigenin; hKv1.5; Open channel blocker; Acetylcholine-activated K; ⁺; current

β,β-Dimethylacrylshikonin exerts antitumor activity via Notch-1 signaling pathway in vitro and in vivo by Shao Zhen-Jun; Zhang Yuan-Yuan; Fan Ying-Ying; Jin Shao-Ju; Yan Jiao; Zheng Xiao-Wei; Chen Jian; Xiong Yao; Zhou Li-Ming (pp. 507-512).

β,β-Dimethylacrylshikonin (DA) is a major component of Radix Lithospermum erythrorhizon and has various biological activities. We have investigated the inhibitory effect of DA on the growth of hepatocellular carcinoma in vitro and in vivo. Notch signaling plays a critical role in maintaining the balance between cell proliferation, differentiation and apoptosis. Hence, perturbed Notch signaling may contribute to tumorigenesis. In the present study, we evaluated whether DA could be an effective inhibitor on cell growth in human gastric cancer cell line, and also the molecular mechanisms. Using multiple cellular and molecular approaches such as MTT assay, colony formation assay, DAPI staining, flow cytometry, real-time PCR and Western blot analysis, we found that DA inhibited cell growth in a dose- and time-dependent manner. Biochemical analysis revealed the involvement of cell cycle regulated proteins in DA-mediated of G₀–G₁ arrest of SGC-7901 cells. Furthermore, DA treatment led to reduced Notch-1 activation, expression of Jagged-1 and its downstream target Hes-1 in vitro and in vivo. Our data demonstrated that DA is a potent inhibitor of progression of gastric cancer cells, which could be due to attenuation of Notch-1. We also suggest that DA could be further developed as a potential therapeutic agent for the treatment of gastric cancer.

Keywords: β,β-Dimethylacrylshikonin; Notch-1; Gastric cancer

Ambivalent roles of early growth response 1 in inflammatory signaling following ribosomal insult in human enterocytes by Kee Hun Do; Hye Jin Choi; Juil Kim; Seong-Hwan Park; Hyun-Hong Kim; Chang Gyu Oh; Yuseok Moon (pp. 513-521).

A putative scheme for the dual roles of EGR-1 in response to mucosal ribosomal insults in the presence of endotoxin.NF-κB expression and activity are strictly regulated in gut epithelia to prevent overstimulation of pro-inflammatory responses following exposure to commensal bacteria. The effects of epithelial EGR-1 on responses to bacterial NF-κB-activating lipopolysaccharide (LPS) in intestinal epithelial cells under ribosomal stress were assessed. This was done to determine the potential of EGR-1 as a modulator of epithelial NF-κB signaling. Nuclear translocation of phosphorylated p65 protein was observed in the cells exposed to LPS although chemokine expression was marginally affected. In contrast, simultaneous exposure to LPS and ribosomal insults prevented epithelial NF-κB activation while chemokine expression was enhanced. The effect of EGR-1, another pro-inflammatory signaling mediator, was monitored to determine the involvement of this factor on chemokine production in response to this co-treatment. Similar to the previously reported ribosomal stress response, EGR-1 expression was elevated by ribosomal insults alone and positively affected gene expression of pro-inflammatory chemokines in the intestinal epithelial cells. However, EGR-1 suppression led to super-induction of chemokines by simultaneous treatment with LPS and ribosomal insult, indicating that EGR-1 is a negative modulator of chemokine gene expression. Particularly, mucosal ribosomal insult-triggered EGR-1 mediated PPARγ induction, which counteracted NF-κB activation by LPS. It can be thus concluded that EGR-1 regulates pro-inflammatory NF-κB activation by LPS via PPARγ although EGR-1 is a positive mediator of chemokine expression following ribosomal insult in intestinal epithelial cells.

Keywords: Endotoxin; Ribosomal insults; EGR-1; Intestinal epithelial cells

Fenofibrate lowers lipid accumulation in myotubes by modulating the PPARα/AMPK/FoxO1/ATGL pathway by Wei-Lu Chen; Yu-Lin Chen; Yu-Ming Chiang; Shyang-Guang Wang; Horng-Mo Lee (pp. 522-531).

Fenofibrate, a fibric acid derivative, is known to possess lipid-lowering effects. Although fenofibrate may activate peroxisome proliferator-activated receptor (PPAR)α and regulate the transcription of several genes, the underlying mechanisms are poorly understood. In this study, we demonstrated that incubation of C2C12 myotubes with fenofibrate increased adipose triglyceride lipase (ATGL) expression and suppressed fatty acid synthase (FAS) level, thereby decreasing intracellular triglyceride accumulation when cells were incubated at high-glucose condition. Fenofibrate increased the phosphorylation of AMP-activated protein kinase (AMPK), which subsequently increased fatty acid β-oxidation. AMPK phosphorylation was reduced by pretreatment with GW9662 (a PPARα inhibitor), suggesting that AMPK may be a downstream effector of PPARα. Pretreatment with compound C (an AMPK inhibitor) or GW9662 blocked fenofibrate-induced ATGL expression and the lipid-lowering effect. Our results suggest that AMPK is as an upstream regulator of ATGL. With further exploration, we demonstrated that fenofibrate stimulated FoxO1 translocation from the cytosol to nuclei by immunefluorescence assay, chromatin immuneprecipitation assay, and reporter assay. Furthermore, oral administration of fenofibrate ameliorated the body weight, visceral fat and serum biochemical indexes in db/db mice. Taken together, our results suggest that the lipid-lowering effect of fenofibrate was achieved by activating PPARα and AMPK signaling pathway that resulted in increasing ATGL expression, lipolysis, and fatty acid β-oxidation.

Keywords: Abbreviations; ATGL; adipose triglyceride lipase; FAS; fatty acid synthase; AMPK; AMP-activated protein kinase; PPARα; peroxisome proliferator-activated receptorα; HDL; high-density lipoprotein; VLDL; very-low-density lipoprotein; ACC; acetyl-CoA carboxylase; CPT1; carnitine palmitoyltransferase 1; HSL; Hormone-sensitive lipaseAMP-activated protein kinase; Adipose triglyceride lipase; Fatty acid synthase; Free fatty acid β-oxidation; Lipid metabolism

Ginsenoside Rb₁ induces type I collagen expression through peroxisome proliferator-activated receptor-delta by Hoi-Hin Kwok; Patrick Ying-Kit Yue; Nai-Ki Mak; Ricky Ngok-Shun Wong (pp. 532-539).

Wrinkle formation is one of the primary characteristics of skin aging, the major cause of wrinkle is the loss of structural protein type I collagen in dermal layer of skin. Topical application of natural substances to reduce wrinkle is gaining attention in recent years. Although a number of polyphenoic compounds are suggested to prevent ultraviolet-induced wrinkle, very few of them are able to increase type I collagen synthesis directly. Ginseng has been known in folk medicine of its beneficial effect to skin. The present study investigate the effect of ginsenoside on type I collagen induction in human dermal fibroblasts. Ginsenoside Rb₁ was shown to induce type I collagen expression in dermal fibroblasts in a dose- and time-dependent manner. Recent studies suggest the important post-transcriptional regulatory role of microRNAs; here we demonstrated that miR-25 can directly inhibit type I collagen protein expression, and treatment of fibroblasts with Rb₁ can reduce the inhibition by decreasing miR-25 level. Furthermore, we identified that the nuclear receptor, peroxisome proliferator-activated receptor-delta (PPARδ) is the key mediator of Rb₁-induced type I collagen expression. Knockdown of PPARδ by small-interference RNA abolished the Rb₁-induced type I collagen production and reversed the Rb₁-suppressed miR-25 expression. These results demonstrated that ginsenoside Rb₁ can increase target gene expression through transcriptional pathway, at the same time, inhibit the corresponding miRNA expression to minimize the translation repression. Furthermore, this study provide solid support of ginsenoside Rb₁-induced type I collagen expression, which warrant further study in the dermatological application of ginsenosides in skin disorders.

Keywords: Ginsenoside; Rb; ₁; Type I collagen; PPARδ; miR-25

Isolation, characterization and total regioselective synthesis of the novel μO-conotoxin MfVIA from Conus magnificus that targets voltage-gated sodium channels by Irina Vetter; Zoltan Dekan; Oliver Knapp; David J. Adams; Paul F. Alewood; Richard J. Lewis (pp. 540-548).

The μO-conotoxins are notable for their unique selectivity for Na_v1.8 over other sodium channel isoforms, making them attractive drug leads for the treatment of neuropathic pain. We describe the discovery of a novel μO-conotoxin, MfVIA, from the venom of Conus magnificus using high-throughput screening approaches. MfVIA was found to be a hydrophobic 32-residue peptide (amino acid sequence RDCQEKWEYCIVPILGFVYCCPGLICGPFVCV) with highest sequence homology to μO-conotoxin MrVIB. To overcome the synthetic challenges posed by μO-conotoxins due to their hydrophobic nature and difficult folding, we developed a novel regioselective approach for the synthesis of μO-conotoxins. Performing selective oxidative deprotections of the cysteine side-chain protecting groups of the fully protected peptide allowed manipulations in organic solvents with no chromatography required between steps. Using this approach, we obtained correctly folded MfVIA with increased synthetic yields. Biological activity of MfVIA was assessed using membrane potential-sensitive dyes and electrophysiological recording techniques. MfVIA preferentially inhibits Na_v1.8 (IC₅₀ 95.9±74.3nM) and Na_v1.4 (IC₅₀ 81±16nM), with significantly lower affinity for other Na_v subtypes (IC₅₀ 431–6203nM; Na_v1.5>1.6∼1.7∼1.3∼1.1∼1.2). This improved approach to μO-conotoxin synthesis will facilitate the optimization of μO-conotoxins as novel analgesic molecules to improve pain management.

Keywords: Abbreviations; Na; _v; voltage-gated sodium channel; Acm; acetamidomethyl; Boc; tert; butyloxycarbonyl; CHO; Chinese hamster ovarian; DCM; dichloromethane; DIEA; N,N-diisopropylethylamine; DMF; N,N-dimethylformamide; DRG; dorsal root ganglion; HEK; human embryonic kidney; HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HF; anhydrous hydrogen fluoride; HFIP; 1,1,1,3,3,3-hexafluoroisopropanol; Meb; 4-methylbenzyl; Pbf; 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; RP-HPLC; reversed-phase high-performance liquid chromatography; tBu; tert; butyl; TFA; trifluoroacetic acid; TFE; trifluoroethanol; Trt; triphenylmethyl; TTX-R; tetrodotoxin-resistant; TTX-S; tetrodotoxin-sensitive; ESI-MS; electrospray ionization mass spectrometry; FBS; foetal bovine serum; RPMI; Roswell Park Memorial Institute; DMEM; Dulbecco's Modified Eagle's Medium; MALDI-TOF; Matrix-assisted laser desorption/ionization-time of flight mass spectrometry; CHCA; α-cyano-4-hydroxycinnamic acid; HBTU; 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; FLIPR; fluorescent imaging plate reader; EDTA; ethylenediaminetetraacetic acid; HBSS; Hank's balanced salt solution; DMSO; dimethyl sulfoxide; SEM; standard error of the meanμO-conotoxin; Regioselective disulfide bond synthesis; Voltage-gated sodium channels; FLIPR; Patch-clamp; Electrophysiology

Low nanomolar GABA effects at extrasynaptic α4β1/β3δ GABA_A receptor subtypes indicate a different binding mode for GABA at these receptors by Nasiara Karim; Petrine Wellendorph; Nathan Absalom; Line Haunstrup Bang; Marianne Lerbech Jensen; Maja Michelle Hansen; Ho Joon Lee; Graham A.R. Johnston; Jane R. Hanrahan; Mary Chebib (pp. 549-557).

GABA displays high and low potencies for α4β3δ GABA_A receptors. GABA potency is affected by loop C residues β3Y205, β3R207 and δR218.Ionotropic GABA_A receptors are a highly heterogenous population of receptors assembled from a combination of multiple subunits. The aims of this study were to characterize the potency of GABA at human recombinant δ-containing extrasynaptic GABA_A receptors expressed in Xenopus oocytes using the two-electrode voltage clamp technique, and to investigate, using site-directed mutagenesis, the molecular determinants for GABA potency at α4β3δ GABA_A receptors. α4/δ-Containing GABA_A receptors displayed high sensitivity to GABA, with mid-nanomolar concentrations activating α4β1δ (EC₅₀=24nM) and α4β3δ (EC₅₀=12nM) receptors. In the majority of oocytes expressing α4β3δ subtypes, GABA produced a biphasic concentration-response curve, and activated the receptor with low and high concentrations (EC₅₀(1)=16nM; EC₅₀(2)=1.2μM). At α4β2δ, GABA had low micromolar activity (EC₅₀=1μM). An analysis of 10 N-terminal singly mutated α4β3δ receptors shows that GABA interacts with amino acids different to those reported for α1β2γ2 GABA_A receptors. Residues Y205 and R207 of the β3-subunit significantly affected GABA potency, while the residue F71 of the α4- and the residue Y97 of the β3-subunit did not significantly affect GABA potency. Mutating the residue R218 of the δ-subunit, equivalent to the GABA binding residue R207 of the β2-subunit, reduced the potency of GABA by 670-fold, suggesting a novel GABA binding site at the δ-subunit interface. Taken together, GABA may have different binding modes for extrasynaptic δ-containing GABA_A receptors compared to their synaptic counterparts.

Keywords: Abbreviations; GABA; (γ-aminobutyric acid); (Zn; ²⁺; ); ZincGABA; Extrasynaptic GABA; _A; receptors; Site-directed mutagenesis; GABA binding site; δ-Subunit

Fetal exposure to high isoflurane concentration induces postnatal memory and learning deficits in rats by Fei-Juan Kong; Lei-Lei Ma; Wen-Wen Hu; Wen-Na Wang; Hui-Shun Lu; Shu-Ping Chen (pp. 558-563).

We developed a maternal fetal rat model to study the effects of isoflurane-induced neurotoxicity on the fetuses of pregnant rats exposed in utero. Pregnant rats at gestational day 14 were exposed to 1.3 or 3% isoflurane for 1h. At postnatal day 28, spatial learning and memory of the offspring were examined using the Morris Water Maze. The apoptosis was evaluated by caspase-3 immunohistochemistry in the hippocampal CA1 region. Simultaneously, the ultrastructure changes of synapse in the hippocampal CA1 and dentate gyrus region were observed by transmission electron microscopy (TEM). The 3% isoflurane treatment group showed significantly longer escape latency, less time spent in the third quadrant and fewer original platform crossings in the Morris Water Maze test, significantly increased number and optical densities of caspase-3 neurons. This treatment also produced remarkable changes in synaptic ultrastructure compared with the control and the 1.3% isoflurane groups. There were no differences in the Morris Water Maze test, densities of caspase-3 positive cells, or synaptic ultrastructure between the control and 1.3% isoflurane groups. High isoflurane concentration (3%) exposure during pregnancy caused spatial memory and learning impairments and more neurodegeneration in the offspring rats compared with control or lower isoflurane concentrations.

Keywords: Isoflurane; Fetal rats; Memory and learning deficits; Neuron apoptosis; Synaptic plasticity

Carrier-mediated transport of quercetin conjugates: Involvement of organic anion transporters and organic anion transporting polypeptides by Chi Chun Wong; Yasutoshi Akiyama; Takaaki Abe; Jonathan D. Lippiat; Caroline Orfila; Gary Williamson (pp. 564-570).

Flavonoids modulate cell signaling and inhibit oxidative enzymes. After oral consumption, they circulate in human plasma as amphiphilic glucuronide or sulfate conjugates, but it is unknown how these physiological metabolites permeate into cells. We examined the mechanisms of uptake of these conjugates into hepatocellular carcinoma (HepG2) cells, and found that uptake of quercetin-3′- O-sulfate was saturable and temperature-dependent, indicating the involvement of carrier-mediated transport. Quercetin-3- O-glucuronide was taken up predominantly via passive diffusion in these cells. Quantitative real-time PCR analysis showed high expression of OATP4C1, followed by OAT2, OAT4 and low expression of OATP1B1 in HepG2 cells, and addition of inhibitors of OATs and OATPs resulted in a significant reduction in quercetin-3′- O-sulfate uptake. The accumulation of quercetin-3′- O-sulfate was further evaluated in HEK293 cells expressing OAT2, OAT4 and OATP4C1. Uptake of quercetin-3′- O-sulfate was 2.3- and 1.4-fold higher in cells expressing OAT4 and OATP4C1 at pH 6.0, respectively, than in control HEK293 cells. siRNA knockdown of OATP4C1 expression in HepG2 cells reduced uptake of quercetin-3′- O-sulfate by ∼40%. This study highlights a role for OATs and OATPs in the cellular uptake of biologically active flavonoid conjugates.

Keywords: Abbreviations; COX2; cyclooxygenase-2; HBSS; hank's balanced salt solution; OATs; organic anion transporters; OATPs; organic anion transporting polypeptidesQuercetin; HepG2; Carrier-mediated transport; Organic anion transporter; Organic anion transporting polypeptide

Statins increase cytochrome P450 4F3-mediated eicosanoids production in human liver cells: A PXR dependent mechanism by Emmanuelle Plée-Gautier; Joseph Antoun; Sophie Goulitquer; Catherine Le Jossic-Corcos; Brigitte Simon; Yolande Amet; Jean-Pierre Salaün; Laurent Corcos (pp. 571-579).

In the present study, the ability of lovastatin, a competitive inhibitor of HMG-CoA reductase, to regulate the gene expression and function of Cytochrome P450 4F3B (CYP4F3B) was examined in the well differentiated HepaRG human hepatoma cell line. Statins induced CYP4F3B mRNA, protein and the production of 20-hydroxyeicosatetraenoic acid (20-HETE), a product of arachidonic acid metabolism and a peroxisome proliferator activated receptor (PPAR) ligand. This response was not dependent on cholesterol shortage or on sterol regulatory element binding protein activation. By both a pharmacological and a siRNA approaches, we demonstrated that recruitment of the Pregnane X Receptor (PXR) was required to mediate CYP4F3 induction by lovastatin. Furthermore, the CYP4F3 gene promoter was transcriptionally activated by PXR, and responded to lovastatin. Finally, the expression of fatty acid-responsive genes was increased in response to the statin or 20-HETE in a CYP4F3-dependent way. We propose that metabolites produced by CYP4F3 could modulate lipid metabolism in response to lovastatin. These results suggest the existence of a novel pathway, operating in liver cells, through which statins could lower lipid levels.

Keywords: Abbreviations; 20-HETE; 20-hydroxyeicosatetraenoic acid; 25-OH chol; 25-hydroxycholesterol; ACS; acyl coenzyme A synthase; ApoAI; apoliprotein AI; CAR; constitutive androstane receptor; CYP; cytochrome P450; FAS; fatty acid synthase; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; HMG-CoA Red; HMG-CoA reductase; LDL-R; LDL-receptor; Luc; luciferase; NCI; negative ion chemical ionization; pheno.; phenobarbital; PPAR; peroxisome proliferator activated receptor; PXR; pregnane X receptor; RXR; 9-Cis retinoic acid receptor; SREBP-2; sterol regulatory element binding protein-2; Z9(10)-EpSTA; 9,10-epoxystearic acidCYP4F3; Statin; PXR; HETE; Fatty acids

Corrigendum to “Identification and characterization of small compound inhibitors of human FATP2” [Biochem. Pharmacol. 79 (2010) 990–999] by Angel Sandoval; Aalap Chokshi; Elliot D. Jesch; Paul N. Black; Concetta C. DiRusso (pp. 580-580).

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Biochemical Pharmacology (v.84, #4)