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1.
J Physiol ; 581(Pt 2): 479-93, 2007 Jun 01.
Article in English | MEDLINE | ID: mdl-17363390

ABSTRACT

The human ether-a-go-go related gene (hERG) potassium channel is expressed in a variety of tissues including the heart, neurons and some cancer cells. hERG channels are modulated by several intracellular signalling pathways and these provide important mechanisms for regulating cellular excitability. In this study, we investigated muscarinic modulation of hERG currents and direct phosphorylation of channel subunits expressed in HEK-293 cells at physiologically relevant temperatures by protein kinase C (PKC). Activation of G(alpha q/11)-coupled M(3)-muscarinic receptors with methacholine, reduced current amplitudes at all potentials with minor effects on the voltage dependence of activation and inactivation. The response to methacholine was insensitive to intracellular BAPTA, but was attenuated by either acute inhibition of PKC with 300 nm bisindolylmaleimide-1 (bis-1) or chronic down-regulation of PKC isoforms by 24 h pretreatment of cells with phorbol 12-myristate 13-acetate (PMA). Stimulation of PKC with 1-oleoyl 2-acetylglycerol (OAG), an analogue of diacylglycerol (DAG), mimicked the actions of muscarinic receptor stimulation. Direct phosphorylation of hERG was measured by [(32)P]orthophosphate labelling of immunoprecipitated protein with an anti-hERG antibody. Basal phosphorylation was high in unstimulated cells and further increased by OAG. The OAG dependent increase was abolished by bis-1 and down-regulation of PKC, but basal levels of phosphorylation were unchanged. Deletion of the amino-terminus of hERG prevented both the modulation of channel activity and the increase of phosphorylation by OAG. Our results are consistent with calcium and/or DAG sensitive isotypes of PKC modulating hERG currents through a mechanism that involves direct phosphorylation of sites on the amino terminus of hERG.


Subject(s)
Ether-A-Go-Go Potassium Channels/metabolism , Ion Channel Gating , Potassium/metabolism , Protein Kinase C/metabolism , Receptor, Muscarinic M3/metabolism , Signal Transduction , Calcium/metabolism , Cell Line , Diglycerides/pharmacology , ERG1 Potassium Channel , Enzyme Activators/pharmacology , Ether-A-Go-Go Potassium Channels/drug effects , Ether-A-Go-Go Potassium Channels/genetics , Humans , Indoles/pharmacology , Ion Channel Gating/drug effects , Ionomycin/pharmacology , Ionophores/pharmacology , Maleimides/pharmacology , Membrane Potentials , Methacholine Chloride/pharmacology , Muscarinic Agonists/pharmacology , Mutation , Phosphorylation , Protein Kinase C/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Receptor, Muscarinic M3/agonists , Signal Transduction/drug effects , Tetradecanoylphorbol Acetate/pharmacology , Transfection
2.
J Pharmacol Exp Ther ; 316(2): 860-8, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16227470

ABSTRACT

The human ether-a-go-go-related gene (hERG) potassium channel is expressed in a variety of cell types, including neurons, tumor cells, and cardiac myocytes. In the heart, it is important for repolarization of the cardiac action potential. Attenuation of hERG current can cause long QT syndrome and cardiac arrhythmias such as torsades de pointes. Caffeine is frequently used as a pharmacological tool to study calcium-dependent transduction pathways in cellular preparations. It raises cytosolic calcium by opening ryanodine receptors and may also inhibit phosphodiesterases to increase cytosolic cAMP. In this study, we show 5 mM caffeine rapidly and reversibly attenuates hERG currents expressed in human embryonic kidney 293 cells to 61.1 +/- 2.2% of control. Caffeine-dependent inhibition of hERG current is not altered by raising cAMP with forskolin, buffering cytosolic calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, or inhibition of protein kinase C. Thus, the effects of caffeine are unlikely to be mediated by cAMP or intracellular calcium-dependent mechanisms. Further experiments showed caffeine directly blocks hERG in an open state-dependent manner. Furthermore, caffeine inhibition is greatly reduced by the pore mutants Y562A and F656A hERG, which disrupt block of most previously tested hERG antagonists. Thus, caffeine attenuates hERG currents by binding to a drug receptor located within the inner cavity of the channel. Dietary intake of caffeine is unlikely to cause long QT syndrome because plasma concentrations do not reach sufficiently high levels to significantly inhibit hERG currents. However, the effects of caffeine have implications for its use in examining calcium-dependent pathways in cellular preparations expressing hERG.


Subject(s)
Caffeine/pharmacology , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Gene Expression/drug effects , Potassium Channel Blockers/pharmacology , Animals , Calcium/metabolism , Cell Line , Cyclic AMP/metabolism , Cytosol/drug effects , Cytosol/metabolism , Ether-A-Go-Go Potassium Channels/genetics , Humans
3.
Cancer Res ; 61(19): 7196-203, 2001 Oct 01.
Article in English | MEDLINE | ID: mdl-11585755

ABSTRACT

The type I receptor tyrosine kinases constitute a family of transmembrane proteins involved in various aspects of cell growth and survival and have been implicated in the initiation and progression of several types of human malignancies. The best characterized of these proteins are the epidermal growth factor receptor (EGFR) and ErbB-2 (HER-2/neu). We have developed potent quinazoline and pyrido-[3,4-d]-pyrimidine small molecules that are dual inhibitors of ErbB-2 and EGFR. The compounds demonstrate potent in vitro inhibition of the ErbB-2 and EGFR kinase domains with IC(50)s <80 nM. Growth of ErbB-2- and EGFR-expressing tumor cell lines is inhibited at concentrations <0.5 microM. Selectivity for tumor cell growth inhibition versus normal human fibroblast growth inhibition ranges from 10- to >75-fold. Tumor growth in mouse s.c. xenograft models of the BT474 and HN5 cell lines is inhibited in a dose-responsive manner using oral doses of 10 and 30 mg/kg twice per day. In addition, the tested compounds caused a reduction of ErbB-2 and EGFR autophosphorylation in tumor fragments from these xenograft models. These data indicate that these compounds have potential use as therapy in the broad population of cancer patients overexpressing ErbB-2 and/or EGFR.


Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , ErbB Receptors/antagonists & inhibitors , Quinazolines/pharmacology , Receptor, ErbB-2/antagonists & inhibitors , Animals , Cell Division/drug effects , Drug Screening Assays, Antitumor , Female , Growth Inhibitors/pharmacology , Humans , Mice , Mice, SCID , Structure-Activity Relationship , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
4.
Bioorg Med Chem Lett ; 11(15): 1993-5, 2001 Aug 06.
Article in English | MEDLINE | ID: mdl-11454465

ABSTRACT

The synthesis and antiviral evaluation of unsymmetrical indolocarbazole derivatives of Arcyriaflavin A, substituted with a range of alkyl groups at the indole nitrogen, is described. Structure-activity relationships in this series against human cytomegalovirus (HCMV) replication in cell culture are reported. Compound 4b was identified as potent inhibitor of HCMV (IC(50)=19 nM), which retained activity against a range of HCMV strains including ganciclovir resistant isolates.


Subject(s)
Antiviral Agents/chemical synthesis , Antiviral Agents/pharmacology , Carbazoles/pharmacology , Cytomegalovirus/drug effects , Indoles/pharmacology , Virus Replication/drug effects , Carbazoles/chemical synthesis , Cells, Cultured , Drug Resistance/genetics , Drug Resistance/physiology , Ganciclovir/pharmacology , Humans , Indoles/chemical synthesis , Inhibitory Concentration 50 , Protein Kinase C/antagonists & inhibitors , Structure-Activity Relationship
5.
Bioorg Med Chem Lett ; 11(11): 1401-5, 2001 Jun 04.
Article in English | MEDLINE | ID: mdl-11378364

ABSTRACT

Described herein is the design and synthesis of indazolylaminopyridopyrimidines and quinazolines as inhibitors of the class 1 tyrosine kinase receptor family. Data is presented for N(4)-(1-benzyl-1H-indazol-5-yl)-N(6),N(6)-dimethylpyrido[3,4-d]pyrimidine-4,6-diamine 3B. This compound inhibited EGFr and c-erbB-2 enzymes selectively over other kinases. It inhibited the proliferation of a range of tumour cell lines in vitro and the growth of BT474 xenografts in SCID mice.


Subject(s)
Antineoplastic Agents/pharmacology , ErbB Receptors/antagonists & inhibitors , Pyrimidines/pharmacology , Quinazolines/pharmacology , Receptor, ErbB-2/antagonists & inhibitors , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Cell Division/drug effects , Disease Models, Animal , Mice , Mice, SCID , Pyrimidines/chemical synthesis , Pyrimidines/chemistry , Pyrimidines/therapeutic use , Quinazolines/chemical synthesis , Quinazolines/chemistry , Quinazolines/therapeutic use , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
6.
Bioorg Med Chem ; 7(6): 1067-74, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10428375

ABSTRACT

In our search for new, safer anti-HCMV agents, we discovered that the natural product Arcyriaflavin A (la) was a potent inhibitor of HCMV replication in cell culture. A series of analogues (symmetrical indolocarbazoles) was synthesised to investigate structure activity relationships in this series against a range of herpes viruses (HCMV, VZV, HSV1, and 2). This identified a number of novel, selective and potent inhibitors of HCMV, 12,13-dihydro-2,10-difluoro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazol e-5,7-(6H)-dione (1d) being the best example (IC50=40 nM, therapeutic index > 1450). Compounds described in this series were generally poor inhibitors of protein kinase C betaII, and no correlation was found between the ability to inhibit HCMV and the enzyme PKC.


Subject(s)
Antiviral Agents/chemical synthesis , Carbazoles/pharmacology , Cytomegalovirus/drug effects , Virus Replication/drug effects , Animals , Antiviral Agents/pharmacology , Carbazoles/chemistry , Cell Division/drug effects , Chlorocebus aethiops , Cytomegalovirus/physiology , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Herpesviridae/drug effects , Protein Kinase C/antagonists & inhibitors , Rats , Structure-Activity Relationship , Vero Cells
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