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Biochemical Pharmacology (v.84, #3)
Where rilpivirine meets with tenofovir, the start of a new anti-HIV drug combination era by Erik De Clercq (pp. 241-248).
The triple-drug once-daily combination pill containing tenofovir, emtricitabine and rilpivirine for HIV treatment was launched in 2011, both in the USA (Complera®) and the E.U. (Eviplera®). The active ingredients of Complera or Eviplera are the nucleotide reverse transcriptase inhibitor (NtRTI) tenofovir, the nucleoside reverse transcriptase inhibitor (NRTI) emtricitabine, and the non-nucleoside reverse transcriptase inhibitor (NNRTI) rilpivirine. Rilpivirine originated from a collaborative research I had started with Dr. Paul Janssen in 1987, whereas tenofovir emanated from a collaborative research with Dr. Antonin Holý and Gilead Sciences. Exactly twenty-five years later rilpivirine and tenofovir joined each other, together with emtricitabine, in the same “combo” pill, representing a full treatment regimen for AIDS (HIV infection) based on a single once-daily pill.
Keywords: Rilpivirine; Tenfovir; Emtricitabine; Complera; ®; Eviplera; ®
G protein-coupled receptor signalling in astrocytes in health and disease: A focus on metabotropic glutamate receptors by Sophie J. Bradley; R.A. John Challiss (pp. 249-259).
Work published over the past 10–15 years has caused the neuroscience community to engage in a process of constant re-evaluation of the roles of glial cells in the mammalian central nervous system. Recent emerging evidence suggests that, in addition to carrying out various homeostatic functions within the CNS, astrocytes can also engage in a two-way dialogue with neurons. Astrocytes possess many of the receptors, and some of the ion channels, present in neurons endowing them with an ability to sense and respond to an array of neuronal signals. In addition, an expanding number of small molecules and proteins have been shown to be released by astrocytes in both health and disease. In this commentary we will highlight advances in our understanding of G protein-coupled receptor signalling in astrocytes, with a particular emphasis on metabotropic glutamate (mGlu) receptors. Discussion will focus on the major mGlu receptors expressed in astrocytes, mGlu3 and mGlu5, how these receptors can influence different aspects of astrocyte physiology, and how signalling by these G protein-coupled receptors might change under pathophysiological circumstances.
Keywords: Glutamate; Metabotropic glutamate receptor (mGlu3, mGlu5); Glia; Astrocyte; Signal transduction; Calcium signalling
Interaction of the EGFR inhibitors gefitinib, vandetanib, pelitinib and neratinib with the ABCG2 multidrug transporter: Implications for the emergence and reversal of cancer drug resistance by Csilla Hegedüs; Krisztina Truta-Feles; Géza Antalffy; György Várady; Katalin Német; Csilla Özvegy-Laczka; György Kéri; László Őrfi; Gergely Szakács; Jeffrey Settleman; András Váradi; Balázs Sarkadi (pp. 260-267).
(A) ABCG2 confers gefitinib or pelitinib resistance. Intracellular efficiency of neratinib or vandetanib is not restricted by ABCG2. (B) EGFR inhibitors block ABCG2 function thereby reversing MDR.Human ABCG2 is a plasma membrane glycoprotein that provides physiological protection against xenobiotics. ABCG2 also significantly influences biodistribution of drugs through pharmacological tissue barriers and confers multidrug resistance to cancer cells. Moreover, ABCG2 is the molecular determinant of the side population that is characteristically enriched in normal and cancer stem cells. Numerous tumors depend on unregulated EGFR signaling, thus inhibition of this receptor by small molecular weight inhibitors such as gefitinib, and the novel second generation agents vandetanib, pelitinib and neratinib, is a promising therapeutic option. In the present study, we provide detailed biochemical characterization regarding the interaction of these EGFR inhibitors with ABCG2. We show that ABCG2 confers resistance to gefitinib and pelitinib, whereas the intracellular action of vandetanib and neratinib is unaltered by the presence of the transporter. At higher concentrations, however, all these EGFR inhibitors inhibit ABCG2 function, thereby promoting accumulation of ABCG2 substrate drugs. We also report enhanced expression of ABCG2 in gefitinib-resistant non-small cell lung cancer cells, suggesting potential clinical relevance of ABCG2 in acquired drug resistance. Since ABCG2 has important impact on both the pharmacological properties and anti-cancer efficiencies of drugs, our results regarding the novel EGFR inhibitors should provide useful information about their therapeutic applicability against ABCG2-expressing cancer cells depending on EGFR signaling. In addition, the finding that these EGFR inhibitors efficiently block ABCG2 function may help to design novel drug-combination therapeutic strategies.
Keywords: ABCG2; Multidrug resistance; Receptor tyrosine kinase; EGFR inhibitor; Cancer stem cell
Inhibition of Bcl-2 improves effect of LCL161, a SMAC mimetic, in hepatocellular carcinoma cells by Kuen-Feng Chen; Jing-Ping Lin; Chung-Wai Shiau; Wei-Tien Tai; Chun-Yu Liu; Hui-Chuan Yu; Pei-Jer Chen; Ann-Lii Cheng (pp. 268-277).
In this study, we investigated the effect of LCL161, a SMAC mimetic, in hepatocellular carcinoma (HCC). LCL161 showed differential effects on apoptosis in four HCC cell lines, and the endogenous level of Bcl-2 determined the sensitivity of HCC cells to LCL161. Cytotoxicity and apoptosis were observed in sensitive PLC5 and Hep3B cells that express lower levels of Bcl-2, but not in resistant Huh-7 and SK-Hep1 cells with higher Bcl-2 expression. Down regulation of Bcl-2 by small interference RNA overcame the resistance to LCL161 in Huh-7, and the apoptotic effect was rescued in Bcl-2-expressing Hep3B. To test the hypothesis that Bcl-2 determines the sensitivity of HCC cells to LCL161, we assayed the biological effect of SC-2001, a novel Bcl-2 inhibitor derived from obatoclax, in LCL161-resistant cell lines. Huh-7 cells co-treated with LCL161 and SC-2001 showed a significant dose-dependent apoptotic effect demonstrated by sub-G1 assay and cleavage of PARP. Furthermore, the combination index (CI) of LCL161 and SC-2001 showed a convincing synergism in resistant Huh-7. In addition, the combinational therapy showed significant growth inhibition in Huh-7-bearing xenograft tumors. Notably, down regulation of Bcl-2 was observed in a tumor sample treated with LCL161 and SC-2001. In conclusion, targeting Bcl-2 with SC-2001 overcomes drug resistance to LCL161 in HCC cells thus suggesting a new anti-IAP combinational therapy for HCC.
Keywords: Abbreviations; HCC; hepatocellular carcinoma; IAP; inhibitor of apoptosis; SMAC; second mitochondrial activator of caspases; TRAIL; tumor necrosis factor-related apoptosis-inducing ligand; PARP; polypolymerase; DMEM; Dulbecco's modified Eagle's medium; FBS; fetal bovine serumLCL161; SC-2001; IAP; Bcl-2; HCC
Structure–activity relationships for the binding of polymyxins with human α-1-acid glycoprotein by Mohammad A.K. Azad; Johnny X. Huang; Matthew A. Cooper; Kade D. Roberts; Philip E. Thompson; Roger L. Nation; Jian Li; Tony Velkov (pp. 278-291).
Here, for the first time, we have characterized binding properties of the polymyxin class of antibiotics for human α-1-acid glycoprotein (AGP) using a combination of biophysical techniques. The binding affinity of colistin, polymyxin B, polymyxin B3, colistin methansulfonate, and colistin nona-peptide was determined by isothermal titration calorimetry (ITC), surface plasma resonance (SPR) and fluorometric assay methods. All assay techniques indicated colistin, polymyxin B and polymyxin B3 display a moderate binding affinity for AGP. ITC and SPR showed there was no detectable binding affinity for colistin methansulfonate and colistin nona-peptide, suggesting both the positive charges of the diaminobutyric acid (Dab) side chains and the N-terminal fatty acyl chain of the polymyxin molecule are required to drive binding to AGP. In addition, the ITC and fluorometric data suggested that endogenous lipidic substances bound to AGP provide part of the polymyxin binding surface. A molecular model of the polymyxin B3–AGP F1*S complex was presented that illustrates the pivotal role of the N-terminal fatty acyl chain and the D-Phe6-L-Leu7 hydrophobic motif of polymyxin B3 for binding to the cleft-like ligand binding cavity of AGP F1*S variant. The model conforms with the entropy driven binding interaction characterized by ITC which suggests hydrophobic interactions coupled to desolvation events and conformational changes are the primary driving force for polymyxins binding to AGP. Collectively, the data are consistent with a role of this acute-phase reactant protein in the transport of polymyxins in plasma.
Keywords: Abbreviations; ANS; 1-anilino-8-naphthalene sulfonic acid; AO; Auramine O; AGP; human α-1-acid glycoprotein; CAPs; cationic antimicrobial peptides; CMS; colistin methanesulfonate; Fmoc; fluorenylmethyloxycarbonyl; Dab; diaminobutyric-acid; ITC; isothermal titration calorimetry; LPS; lipopolysaccharide; OM; outer-membrane; colistin NP; colistin; nona; -peptide; PmB; polymyxin B; NR; Nile Red; NDM-1; New Delhi metallo-beta-lactamase 1; SAR; structure–activity relationships; SPR; surface plasmon resonanceHuman α-1-acid glycoprotein; Binding affinity; Polymyxin; Colistin
Uncoupling of K+ and Cl− transport across the cell membrane in the process of regulatory volume decrease by Linjie Yang; Linyan Zhu; Yue Xu; Haifeng Zhang; Wencai Ye; Jianwen Mao; Lixin Chen; Liwei Wang (pp. 292-302).
It is accepted that K+ and Cl− flows are coupled tightly in regulatory volume decrease (RVD). However, using self referencing microelectrodes, we proved that K+ and Cl− transport mainly by channels in RVD was uncoupled in nasopharyngeal carcinoma CNE-2Z cells, with the transient K+ efflux activated earlier and sustained Cl− efflux activated later. Hypotonic challenges decreased intracellular pH (pHi), and activated a proton pump-dependent H+ efflux, resulting in a decline of extracellular pH (pHo). Modest decreases of pHo inhibited the volume-activated K+ outflow and RVD, but not the Cl− outflow, while inhibition of H+ efflux or increase of pHo buffer ability promoted K+ efflux and RVD. The results suggest that the temporal dynamics of K+ channel activities is different from that of Cl− channels in RVD, due to differential sensitivity of K+ and Cl− channels to pHo. H+ efflux may play important roles in cell volume regulation, and may be a therapeutic target for human nasopharyngeal carcinoma.
Keywords: Ion-selective electrode; Patch-clamp technique; Ion transport; Cell size; Tumor cell
Functional and pharmacological characterization of a VEGF mimetic peptide on reparative angiogenesis by Federica Finetti; Anna Basile; Domenica Capasso; Sonia Di Gaetano; Rossella Di Stasi; Maria Pascale; Caterina Maria Turco; Marina Ziche; Lucia Morbidelli; Luca Domenico D’Andrea (pp. 303-311).
Vascular endothelial growth factor (VEGF) is the main regulator of physiological and pathological angiogenesis. Low molecular weight molecules able to stimulate angiogenesis have interesting medical application for example in regenerative medicine, but at present none has reached the clinic. We reported that a VEGF mimetic helical peptide, QK, designed on the VEGF helix sequence 17–25, is able to bind and activate the VEGF receptors, producing angiogenesis. In this study we evaluate the pharmacological properties of peptide QK with the aim to propose it as a VEGF-mimetic drug to be employed in reparative angiogenesis. We show that the peptide QK is able to recapitulate all the biological activities of VEGF in vivo and on endothelial cells. In experiments evaluating sprouting from aortic ring and vessel formation in an in vivo angiogenesis model, the peptide QK showed biological effects comparable with VEGF. At endothelial level, the peptide up-regulates VEGF receptor expression, activates intracellular pathways depending on VEGFR2, and consistently it induces endothelial cell proliferation, survival and migration. When added to angiogenic factors (VEGF and/or FGF-2), QK produces an improved biological action, which resulted in reduced apoptosis and accelerated in vitro wound healing. The VEGF-like activity of the short peptide QK, characterized by lower cost of production and easier handling compared to the native glycoprotein, suggests that it is an attractive candidate to be further developed for application in therapeutic angiogenesis.
Keywords: Angiogenesis; Endothelial cell; Peptide; VEGF; Nitric oxide; Reparative medicine
Degradation, insulin secretion, glucose-lowering and GIP additive actions of a palmitate-derivatised analogue of xenin-25 by Christine M.A. Martin; Victor A. Gault; Stephen McClean; Peter R. Flatt; Nigel Irwin (pp. 312-319).
Xenin-25, a K-cell derived peptide co-secreted with glucose-dependent insulinotropic polypeptide (GIP), has recently been shown to have glucose homeostatic actions and potentiate the insulinotropic effect of GIP. However, the biological actions of xenin-25 are brief due to rapid metabolism, yet little is known regarding enzymatic degradation of this peptide. Therefore, the present study has fully characterised the plasma enzymatic degradation products of xenin-25. We have also generated a novel acylated xenin-25 analogue, xenin-25(Lys13PAL), and evaluated its stability, biological action profile and therapeutic applicability compared to the native peptide. In contrast to xenin-25, xenin-25(Lys13PAL) was completely resistant to plasma enzyme degradation. Insulinotropic responses of xenin-25(Lys13PAL) in clonal beta-cells were similar to native xenin-25, moreover xenin-25(Lys13PAL) significantly ( p<0.05 to p<0.001) potentiated the insulin releasing action of (D-Ala2)GIP. When administered together with glucose to normal mice, the glycaemic excursion was significantly ( p<0.05) less and overall insulin secretory effect significantly ( p<0.05) greater for xenin-25(Lys13PAL) when compared to xenin-25 mice. Glucose-lowering and insulin releasing effects of both peptides was less prominent in high fat fed mice and ob/ ob mice. However, xenin-25 significantly ( p<0.05) augmented the glucose-lowering action of (D-Ala2)GIP in both groups of mice. Similarly, xenin-25(Lys13PAL) potentiated ( p<0.05) the gluco-regulatory effect of (D-Ala2)GIP. Overall, these data indicate that palmitate-derivatised analogues of xenin-25 represent a novel class of GIP potentiator drugs for possible type 2 diabetes therapy.
Keywords: Abbreviations; ESI; electrospray ionisation; FASTA; FAST-All; GIP; glucose-dependent insulinotropic polypeptide; GLP-1; glucagon-like peptide-1; HPLC; high performance liquid chromatography; Lys; lysine; MALDI-TOF; matrix-assisted laser desorption ionisation-time of flight; MS; mass spectrometry; obese diabetic mice; (; ob; /; ob; ) mice; PAL; palmitate; AUC; area under curveGlucose-dependent insulinotropic polypeptide; ob; /; ob; mice; Glucose homeostasis; Analogues; Type 2 diabetes; Xenin
MiR-139 inhibits invasion and metastasis of colorectal cancer by targeting the type I insulin-like growth factor receptor by Ke Shen; Qiannan Liang; Ke Xu; Daling Cui; Lin Jiang; Peihao Yin; Yanhua Lu; Qi Li; Jianwen Liu (pp. 320-330).
MicroRNAs (miRNAs), which are noncoding RNAs that regulate gene expression, are involved in tumor metastasis. In this study, we describe the down-regulation and function of miR-139 in colorectal cancer (CRC) metastasis. MiR-139 was found underexpressed in 34 CRC tissues compared to their corresponding nontumor tissues. Decreased miR-139 in CRC tissue was associated with disease progression and metastasis. Re-expression of miR-139 did not inhibit CRC cell growth but suppresses CRC cell metastasis and invasion in vitro and in vivo. MiR-139 might suppress CRC cells invasion and metastasis by targeting type I insulin-like growth factor receptor (IGF-IR). We also found miR-139 directed migration inactivation of human CRC cells involves down-regulation of matrix metalloproteinase 2 (MMP-2). The IGF-IR/MEK/ERK signaling was inhibited by miR-139 overexpression and then resulted in MMP-2 promoter suppression. Taken together, our results provide evidence that miR-139 might function as a metastasis suppressor in CRC. Targeting miR-139 may provide a strategy for blocking CRC metastasis.
Keywords: Abbreviations; miRNA; microRNA; CRC; colorectal cancer; IGF-IR; type I insulin-like growth factor receptor; UTR; untranslated regionmiR-139; CRC; Metastasis; Invasion; IGF-IR
Echinocystic acid ameliorates lung inflammation in mice and alveolar macrophages by inhibiting the binding of LPS to TLR4 in NF-κB and MAPK pathways by Eun-Ha Joh; Wan Gu; Dong-Hyun Kim (pp. 331-340).
Orally administered lancemaside A, which is isolated from Codonopsis lanceolata (family Campanulaceae), showed anti-colitic effect in mice. However, its metabolite echinocystic acid was absorbed into the blood. Therefore, its anti-inflammatory effects were investigated in lipopolysaccharide (LPS)-stimulated alveolar macrophages in vitro and acute lung injury in vivo. Alveolar macrophages from mice were stimulated with LPS and were treated with echinocystic acid. Acute lung injury was induced by intratracheal administration of LPS in mice. Mice were treated with echinocystic acid or dexamethasone. Echinocystic acid potently suppressed the production of the pro-inflammatory cytokines, TNF-α and IL-1β, as well as of the activations of NF-κB and MAPKS, in LPS-stimulated alveolar macrophages. Echinocystic acid also down-regulated the production of inflammatory markers, which included inducible nitric oxide synthase and cyclooxygenase-2, as well as the inflammatory mediators, nitric oxide and prostaglandin E2, in LPS-stimulated alveolar macrophages. Echinocystic acid also inhibited the activation of IL-1 receptor-associated kinases, and the activation of mitogen-activated protein kinases in LPS-stimulated alveolar macrophages. Furthermore, echinocystic acid potently inhibited the interaction between LPS and TLR4 in alveolar macrophages transfected with or without MyD88 siRNA, although it did not inhibit the binding in the macrophages transfected with TLR4 siRNA. Echinocystic acid suppressed LPS-induced acute lung inflammation in mice, as well as the expression of pro-inflammatory cytokines, such as IL-1β and TNF-α, and their transcription factor, NF-κB. On the basis of these findings, echinocystic acid, a metabolite of lancemaside A, may express anti-inflammatory effects by inhibiting the binding of LPS to TLR4 on macrophages.
Keywords: Echinocystic acid; Inflammation; Acute lung injury; Lipopolysaccharide; Toll-like receptor 4
Histaminergic pharmacology of homo-oligomeric β3 γ-aminobutyric acid type A receptors characterized by surface plasmon resonance biosensor technology by Christian Seeger; Tony Christopeit; Karoline Fuchs; Katharina Grote; Werner Sieghart; U. Helena Danielson (pp. 341-351).
A surface plasmon resonance biosensor assay was established for studying the interactions of 51 histaminergic and 15 GABAergic ligands with homo-oligomeric β3 GABAA receptors. Detergent solubilized receptors were successfully immobilized via affinity-capture on biosensor surfaces. The interaction kinetics of both histaminergic and GABAergic ligands were very rapid but affinities could be determined by steady-state analysis. Binding of several GABAergic ligands was observed, in agreement with previous data. Histamine and 16 histaminergic ligands were detected to directly bind to β3 GABAA receptors with micromolar affinity ( KD<300μM), thus extending previous evidence that β3 GABAA receptors can interact with histaminergic ligands. Histamine exhibited an affinity for these receptors comparable to that for human histamine type 1 (H1) or type 2 (H2) receptors. Furthermore, 13 of these histaminergic ligands appeared to compete with histamine. The discovery that H2, H3 and H4 receptor ligands interact with β3 receptors indicates a unique histaminergic pharmacology of these receptors. Due to their low affinity for the homo-pentameric β3 receptors these histaminergic drugs are not expected to modulate these receptors at clinically relevant concentrations. The results support the use of the new biosensor assay for the identification of drugs interacting with full length receptors and for fragment-based drug discovery of high affinity ligands for β3 receptors. Drugs with high affinity and selectivity for these receptors can be used to clarify the question whether β3 receptors do exist in the brain, and provide new avenues for the development of therapeutically active compounds targeting this novel histamine binding site.
Keywords: Abbreviations; GABA; γ-aminobutyric acid; GABA; A; receptors; GABA type A receptors; SPR; surface plasmon resonanceLigand-gated ion channel; γ-Aminobutyric acid type A receptor; Histamine; Surface plasmon resonance; Competition analysis
The nicotinic acetylcholine receptors of zebrafish and an evaluation of pharmacological tools used for their study by Roger L. Papke; Fumihito Ono; Clare Stokes; Jason M. Urban; R. Thomas Boyd (pp. 352-365).
Zebrafish ( Danio rerio) have been used to study multiple effects of nicotine, for example on cognition, locomotion, and stress responses, relying on the assumption that pharmacological tools will operate similarly upon molecular substrates in the fish and mammalian systems. We have cloned the zebrafish nicotinic acetylcholine receptor (nAChR) subunits and expressed key nAChR subtypes in Xenopus oocytes including neuronal (α4β2, α2β2, α3β4, and α7) and muscle (α1β1bɛδ) nAChR. Consistent with studies of mammalian nAChR, nicotine was relatively inactive on muscle-type receptors, having both low potency and efficacy. It had high efficacy but low potency for α7 receptors, and the best potency and good efficacy for α4β2 receptors. Cytisine, a key lead compound for the development of smoking cessation agents, is a full agonist for both mammalian α7 and α3β4 receptors, but a full agonist only for the fish α7, with surprisingly low efficacy for α3β4. The efficacy of cytisine for α4β2 was somewhat greater than typically reported for mammalian α4β2. The ganglionic blocker mecamylamine was most potent for blocking α3β4 receptors, least potent for α7, and roughly equipotent for the muscle receptors and the β2-containing nAChR. However, the block of β2-containing receptors was slowly reversible, consistent with effective targeting of these CNS-type receptors in vivo. Three prototypical α7-selective agonists, choline, tropane, and 4OH-GTS-21, were tested, and these agents were observed to activate both fish α7 and α4β2 nAChR. Our data therefore indicate that while some pharmacological tools used in zebrafish may function as expected, others will not.
Keywords: Drug development; Nicotine dependence; Animal models
Phenylalanine 368 of multidrug resistance-associated protein 4 (MRP4/ABCC4) plays a crucial role in substrate-specific transport activity by Hanneke G.M. Wittgen; Jeroen J.M.W. van den Heuvel; Elmar Krieger; Gijs Schaftenaar; Frans G.M. Russel; Jan B. Koenderink (pp. 366-373).
Multidrug resistance-associated protein 4 (MRP4) is a membrane transporter that mediates the cellular efflux of a wide range of anionic drugs and endogenous molecules. MRP4 transport can influence the pharmacokinetics of drugs and their metabolites, therefore more knowledge about the molecular determinants important for its transport function would be of relevance. Here, we substituted amino acids Phe368, Trp995, and Arg998 with conservative or non-conservative residues, and determined the effect on transport of the model substrates estradiol 17-β-d-glucuronide (E217βG), cyclic guanosine monophosphate (cGMP), methotrexate (MTX), and folic acid into membrane vesicles isolated from baculovirus transduced HEK293 cells overexpressing the mutant MRP4 proteins. This revealed that all Arg998 mutations appeared to be deleterious, whereas the effect of a Phe368 or Trp995 replacement was dependent on the amino acid introduced and the substrate studied. Substitution of Phe368 with Trp (F368W) induced a gain-of-function of E217βG transport and a loss-of-function of MTX transport, which could not be attributed to an altered substrate binding. Moreover, we did not observe any modification in ATP or ADP handling for F368W. These results, in combination with docking of substrates in a homology model of MRP4 in the inward- and outward-facing conformation, suggest that Phe368 and Trp995 do not play an important role in the initial binding of substrates. They, however, might interact with the substrates during rearrangement of helixes for substrate translocation, funneling the substrates to the exit site in the outward-facing conformation.
Keywords: Abbreviations; MRP; multidrug resistance-associated protein; eYFP; enhanced yellow fluorescent protein; HEK293; human embryonic kidney 293 cells; DMEM; Dulbecco Eagle's modified medium; F368/Phe; 368; phenylalanine at position 368 of MRP4; W995/Trp; 995; tryptophan at position 995 of MRP4; R998/Arg; 998; arginine at position 998 of MRP4; Ala/A; alanine; Asp; asparagine; Leu/L; leucine; Lys/K; lysine; Ser/S; serine; Tyr/Y; tyrosine; TM; transmembrane helix; TMD; transmembrane domain; NBD; nucleotide binding domain; E; 2; 17βG; estradiol 17-β-; d; -glucuronide; MTX; methotrexate; cGMP; guanosine 3′,5′-cyclic monophosphate; P-gp; P-glycoprotein; LTC; 4; leukotriene C; 4MRP4; ATP-binding cassette; Membrane transport; Mutagenesis; Baculovirus
Peroxidase-like activity of uncoupled cytochrome P450 by Francesco De Matteis; David P. Ballou; Minor J. Coon; Ronald W. Estabrook; Donovan C. Haines (pp. 374-382).
The NADPH-dependent consumption of O2 by cytochrome P450 BM3 was stimulated by either laurate or perfluorolaurate, but the NADPH/O2 molar consumption ratios were approximately 1 and 2, respectively, indicating that perfluorolaurate does not become oxygenated by BM3 and oxygen undergoes full reduction to water. The nature of this catalytic cycle uncoupled to hydroxylation was explored using bilirubin as a molecular probe. During uncoupling with perfluorolaurate bilirubin was degraded and stimulated O2 uptake by an approximately equimolar amount. No stimulation of oxygen uptake was caused by bilirubin in presence of NADPH alone or in presence of laurate together with NADPH; under these conditions little degradation of bilirubin was observed. Mesobilirubin was also degraded during uncoupling with perfluorolaurate, whereas biliverdin (which lacks the central methene bridge present in rubins) was unaffected. It is suggested that the CYP ferryl oxygen species abstracts a hydrogen atom from the central methene bridge of bilirubin to generate a radical, which is further dehydrogenated to biliverdin or else binds O2 and undergoes fragmentation. We conclude that the uncoupled catalytic cycle of cytochrome P450 has properties resembling those of a peroxidase and that bilirubin is rapidly oxidized as a peroxidase substrate. The potential toxicological significance of cytochrome P450 uncoupling is considered.
Keywords: Abbreviations; BM3; CYP102A1 from; Bacillus megaterium; CYP; cytochrome P450; DMSO; dimethylsulfoxide; PCBs; polychlorinated biphenyls; TCDD; 2,3,7,8,-tetrachlorodibenzo-; p; -dioxinBM3; Cytochrome P450; Uncoupling; Perfluorolaurate; Bilirubin oxidation; NADPH/O; 2; consumption ratio
Residue Ile89 in human plasma membrane monoamine transporter influences its organic cation transport activity and sensitivity to inhibition by dilazep by Horace T.B. Ho; Li Xia; Joanne Wang (pp. 383-390).
Plasma membrane monoamine transporter (PMAT) is a polyspecific organic cation transporter belonging to the equilibrative nucleoside transporter (ENT) family. Despite its distinct substrate specificity from the classic nucleoside transporters ENT1 and 2, PMAT appears to share similar protein architecture with ENT1/2 and retains low affinity binding to classic ENT inhibitors such as nitrobenzylmercaptopurine riboside (NBMPR) and the coronary vasodilators dilazep and dipyridamole. Here we investigated the role of residue Ile89, a position known to be important for ENT interaction with dilazep, dipyridamole, and nucleoside substrates, in PMAT transport function and its interaction with classic ENT inhibitors using Madin–Darby canine kidney (MDCK) cells stably expressing human PMAT. Substitution of Ile89 in PMAT with Met, the counterpart residue in ENT1, resulted in normal plasma membrane localization and protein expression. Transport kinetic analysis revealed that I89M mutant had a 2.7-fold reduction in maximal transport velocity ( Vmax) with no significant change in apparent binding affinity ( Km) towards the prototype PMAT substrate 1-methyl-4-phenylpyridinium (MPP+), suggesting that I89 is an important determinant for the catalytic activity of PMAT. Dose-dependent inhibition studies further showed that the I89M mutation significantly increased PMAT's sensitivity to dilazep by 2.5-fold without affecting its sensitivity to dipyridamole and NBMPR. Located at the extracellular end of transmembrane domain 1 of PMAT, I89 may occupy an important position close to the substrate permeation pathway and may be involved in direct interaction with the vasodilator dilazep.
Keywords: Abbreviations; PMAT; plasma membrane monoamine transporter; SLC29; solute carrier 29; SLC22; solute carrier 22; MPP; +; 1-methyl-4-phenylpyridinium; ENT; equilibrative nucleoside transporter; V; max; maximal transport velocity; K; m; apparent binding affinity; OCT; organic cation transporter; NBMPR; nitrobenzylmercaptopurine riboside; YFP; yellow fluorescence protein; MDCK; Madin–Darby canine kidney; FACS; fluorescence activated cell sorting; TM; transmembrane domainPMAT; SLC29 family; ENT4; Dilazep; Dipyridamole
Human PXR-mediated induction of intestinal CYP3A4 attenuates 1α,25-dihydroxyvitamin D3 function in human colon adenocarcinoma LS180 cells by Xi Emily Zheng; Zhican Wang; Michael Z. Liao; Yvonne S. Lin; Margaret C. Shuhart; Erin G. Schuetz; Kenneth E. Thummel (pp. 391-401).
Oxidative catabolism of 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] is mediated by either CYP24A1 or CYP3A4. In this paper, we tested whether induction of CYP3A4 in the LS180 intestinal cell model enhances clearance of 1α,25(OH)2D3 and blunts its hormonal effect on expression of the apical membrane calcium transport protein, TRPV6. Treatment with the hPXR agonist rifampin significantly increased CYP3A4 mRNA content and catalytic activity, but had no effect on CYP24A1 or TRPV6 mRNA content. Pre-treating cells with rifampin for 48h, prior to a 24h 1α,25(OH)2D3 treatment phase, was associated with a subsequent 48% increase in the elimination of 1α,25(OH)2D3 and a 35% reduction of peak TRPV6 mRNA. Introduction of the CYP3A4 inhibitor, 6′,7′-dihydroxybergamottin, an active inhibitor in grapefruit juice, reversed the effects of rifampin on 1α,25(OH)2D3 clearance and TRPV6 expression. Over-expression of hPXR in LS180 cells greatly enhanced the CYP3A4 responsiveness to rifampin pretreatment, and elicited a greater relative suppression of TRPV6 expression and an increase in 1α,25(OH)2D3 disappearance rate, compared to vector expressed cells, following hormone administration. Together, these results suggest that induction of CYP3A4 in the intestinal epithelium by hPXR agonists can result in a greater metabolic clearance of 1α,25(OH)2D3 and reduced effects of the hormone on the intestinal calcium absorption, which may contribute to an increased risk of drug-induced osteomalacia/osteoporosis in patients receiving chronic therapy with potent hPXR agonists. Moreover, ingestion of grapefruit juice in the at-risk patients could potentially prevent this adverse drug effect.
Keywords: Cytochrome P450 3A4; TRPV6; 1α,25-Dihydroxyvitamin D; 3; Human pregnane X receptor; Rifampin; Osteomalacia
Amurensin G, a novel SIRT1 inhibitor, sensitizes TRAIL-resistant human leukemic K562 cells to TRAIL-induced apoptosis by Hak-Bong Kim; Mi-Ju Kim; Su-Hoon Lee; Jae-Won Lee; Jae-Ho Bae; Dong-Wan Kim; Trong Tuan Dao; Won Keun Oh; Chi-Dug Kang; Sun-Hee Kim (pp. 402-410).
Amurensin G, a potent new SIRT1 inhibitor, augmented the effect of TRAIL-mediated apoptosis in TRAIL-resistant human K562 cells by up-regulation of DR5 and c-Myc and down-regulation of c-FLIP and Mcl-1Many types of cancer cells remain resistant towards TRAIL-induced cytotoxicity by the blockade of apoptotic signaling cascades. Thus, sensitizers are needed to enhance the effect of TRAIL-based cancer therapies. Although synergistic tumor cell death has been reported when various HDAC inhibitors were administered with TRAIL in a variety of human cancers, the effect of inhibitors of Class III HDAC such as SIRT1 have not been reported. We reported here for the first time that inhibition of SIRT1 augmented the cytotoxic and apoptotic effects of TRAIL on human leukemic K562 cells. Knockdown of SIRT1 or treatment with amurensin G, a potent new SIRT1 inhibitor, up-regulated the levels of DR5 and c-Myc and down-regulated the level of c-FLIPL/S. Furthermore, knockdown of SIRT1 or treatment with amurensin G augmented the molecular responses to TRAIL, including activation of caspase-8, -9 and -3, PARP cleavage, up-regulation of Bax, and down-regulation of Bcl-2. Amurensin G-enhanced TRAIL-induced apoptosis was abrogated by caspase inhibitor Z-VAD-FMK. These findings suggest that the suppression of SIRT1 with siRNA or amurensin G sensitize the TRAIL-resistant K562 cell to TRAIL-induced apoptosis, possibly by the up-regulation of c-Myc and DR5 surface expression and the down-regulations of c-FLIP and Mcl-1. In addition, amurensin G, a potent new SIRT1 inhibitor, would be used as a sensitizer of TRAIL in TRAIL-resistant leukemic cells.
Keywords: TRAIL; Amurensin G; SIRT1; c-Myc; K562 cells