26 results on '"Fonfria, E."'
Search Results
2. The role of TRPM channels in cell death
- Author
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McNulty, S. and Fonfria, E.
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- 2005
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3. Cloning and pharmacological characterization of the dog P2X7 receptor
- Author
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Roman, S, Cusdin, F S, Fonfria, E, Goodwin, J A, Reeves, J, Lappin, S C, Chambers, L, Walter, D S, Clay, W C, and Michel, A D
- Published
- 2009
- Full Text
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4. Cloning and pharmacological characterization of the guinea pig P2X7 receptor orthologue
- Author
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Fonfria, E, Clay, W C, Levy, D S, Goodwin, J A, Roman, S, Smith, G D, Condreay, J P, and Michel, A D
- Published
- 2008
- Full Text
- View/download PDF
5. Agonist potency at P2X7 receptors is modulated by structurally diverse lipids
- Author
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Michel, A D and Fonfria, E
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- 2007
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6. Species and response dependent differences in the effects of MAPK inhibitors on P2X7 receptor function
- Author
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Michel, A D, Thompson, K M, Simon, J, Boyfield, I, Fonfria, E, and Humphrey, P P A
- Published
- 2006
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7. Amyloid β-peptide(1–42) and hydrogen peroxide-induced toxicity are mediated by TRPM2 in rat primary striatal cultures
- Author
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Fonfria, E., Marshall, I. C. B., Boyfield, I., Skaper, S. D., Hughes, J. P., Owen, D. E., Zhang, W., Miller, B. A., Benham, C. D., and McNulty, S.
- Published
- 2005
8. Mercury interaction with the GABA A receptor modulates the benzodiazepine binding site in primary cultures of mouse cerebellar granule cells
- Author
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Fonfrı́a, E, Rodrı́guez-Farré, E, and Suñol, C
- Published
- 2001
- Full Text
- View/download PDF
9. Agonist potency at P2X7receptors is modulated by structurally diverse lipids
- Author
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Michel, A D, primary and Fonfria, E, additional
- Published
- 2007
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10. Amyloid beta-peptide(1-42) and hydrogen peroxide-induced toxicity are mediated by TRPM2 in rat primary striatal cultures
- Author
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Fonfria, E., primary, Marshall, I. C. B., additional, Boyfield, I., additional, Skaper, S. D., additional, Hughes, J. P., additional, Owen, D. E., additional, Zhang, W., additional, Miller, B. A., additional, Benham, C. D., additional, and McNulty, S., additional
- Published
- 2005
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11. Agonist potency at P2X7 receptors is modulated by structurally diverse lipids.
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Michel, A. D. and Fonfria, E.
- Subjects
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LIPIDS , *MATERIAL plasticity , *INTERLEUKIN-1 , *RADIOLIGAND assay , *CELL receptors , *PATHOLOGICAL physiology - Abstract
Background and purpose:The P2X7 receptor exhibits a high degree of plasticity with agonist potency increasing after prolonged receptor activation. In this study we investigated the ability of lipids to modulate agonist potency at P2X7 receptors.Experimental approach:A variety of lipids, including lysophosphatidylcholine, sphingosylphosphorylcholine and hexadecylphosphorylcholine were studied for their effect on P2X7 receptor-stimulated ethidium bromide accumulation in cells expressing human recombinant P2X7 receptors and on P2X7 receptor-stimulated interleukin-1β (IL1β) release from THP-1 cells. The effects of the lipids were also assessed in radioligand binding studies on human P2X7 receptors.Key results:At concentrations (3-30 μM) below the threshold to cause cell lysis, the lipids increased agonist potency and/or maximal effects at P2X7 receptors in both ethidium accumulation and IL1β release studies. There was little structure activity relationship (SAR) for this effect and sub-lytic concentrations of Triton X-100 partially mimicked the effects of the lipids. The lipids caused cell lysis and increased intracellular calcium at higher concentrations (30-100 μM) which complicated interpretation of their effects in functional studies. However, the lipids (3-100 μM) also increased agonist potency 30-100 fold in radioligand binding studies.Conclusions and implications:This study demonstrates that a diverse range of lipids increase agonist potency at the P2X7 receptor in functional and binding studies. The broad SAR, including the effect of Triton X-100, suggests this may reflect changes in membrane properties rather than a direct effect on the P2X7 receptor. Since many of the lipids studied accumulate in disease states they may enhance P2X7 receptor function under pathophysiological conditions.British Journal of Pharmacology (2007) 152, 523–537; doi:10.1038/sj.bjp.0707417; published online 13 August 2007 [ABSTRACT FROM AUTHOR]
- Published
- 2007
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12. Mercury interaction with the GABAA receptor modulates the benzodiazepine binding site in primary cultures of mouse cerebellar granule cells
- Author
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Fonfria, E., Rodre, E., and Sunol, C.
- Published
- 2001
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13. Replacement of the Mouse LD 50 Assay for Determination of the Potency of AbobotulinumtoxinA with a Cell-Based Method in Both Powder and Liquid Formulations.
- Author
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Fonfria E, Marks E, Foulkes LM, Schofield R, Higazi D, Coward S, and Kippen A
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- Animals, Mice, Powders, Lethal Dose 50, Neurotoxins, Botulinum Toxins, Type A toxicity
- Abstract
Botulinum neurotoxins (BoNTs) are important therapeutic agents. The in vivo median lethal dose (LD
50 ) assay has been commonly used to measure the potency of BoNT commercial preparations. As an alternative, we developed cell-based assays for abobotulinumtoxinA in both powder (Dysport® , Azzalure® ) and liquid (Alluzience® ) formulations using the in vitro BoCell® system. The assays demonstrated linearity over 50-130% of the expected relative potency, with a correlation coefficient of 0.98. Mean recoveries of 90-108% of the stated potency were observed over this range. The coefficients of variation for powder and liquid formulations, respectively, were 3.6% and 4.0% for repeatability and 8.3% and 5.0% for intermediate precision. A statistically powered comparability assessment of the BoCell® and LD50 assays was performed. Equivalence was demonstrated between the assays for the liquid formulation at release and end of shelf life using a paired equivalence test with predefined equivalence margins. For the powder formulation, the assays were also shown to be equivalent for release samples and when determining loss of potency following thermal degradation. The BoCell® assay was approved for establishing the potency of abobotulinumtoxinA for both powder and liquid formulations in Europe and for the powder formulation only in the USA.- Published
- 2023
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14. hiPSC-Derived Neurons Provide a Robust and Physiologically Relevant In Vitro Platform to Test Botulinum Neurotoxins.
- Author
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Lamotte JD, Roqueviere S, Gautier H, Raban E, Bouré C, Fonfria E, Krupp J, and Nicoleau C
- Abstract
Botulinum neurotoxins (BoNTs) are zinc metalloproteases that block neurotransmitter release at the neuromuscular junction (NMJ). Their high affinity for motor neurons combined with a high potency have made them extremely effective drugs for the treatment of a variety of neurological diseases as well as for aesthetic applications. Current in vitro assays used for testing and developing BoNT therapeutics include primary rodent cells and immortalized cell lines. Both models have limitations concerning accuracy and physiological relevance. In order to improve the translational value of preclinical data there is a clear need to use more accurate models such as human induced Pluripotent Stem Cells (hiPSC)-derived neuronal models. In this study we have assessed the potential of four different human iPSC-derived neuronal models including Motor Neurons for BoNT testing. We have characterized these models in detail and found that all models express all proteins needed for BoNT intoxication and showed that all four hiPSC-derived neuronal models are sensitive to both serotype A and E BoNT with Motor Neurons being the most sensitive. We showed that hiPSC-derived Motor Neurons expressed authentic markers after only 7 days of culture, are functional and able to form active synapses. When cultivated with myotubes, we demonstrated that they can innervate myotubes and induce contraction, generating an in vitro model of NMJ showing dose-responsive sensitivity BoNT intoxication. Together, these data demonstrate the promise of hiPSC-derived neurons, especially Motor Neurons, for pharmaceutical BoNT testing and development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. All authors are Ipsen employees., (Copyright © 2021 Duchesne De Lamotte, Roqueviere, Gautier, Raban, Bouré, Fonfria, Krupp and Nicoleau.)
- Published
- 2021
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15. A comparison of biological activity of commercially available purified native botulinum neurotoxin serotypes A1 to F1 in vitro, ex vivo, and in vivo.
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Donald S, Elliott M, Gray B, Hornby F, Lewandowska A, Marlin S, Favre-Guilmard C, Périer C, Cornet S, Kalinichev M, Krupp J, and Fonfria E
- Subjects
- Animals, Biological Assay methods, Body Weight drug effects, Botulinum Toxins genetics, Botulinum Toxins isolation & purification, Diaphragm innervation, Dose-Response Relationship, Drug, Female, Male, Mice, Models, Animal, Neurons, Neurotoxins genetics, Neurotoxins isolation & purification, Phrenic Nerve drug effects, Primary Cell Culture, Rats, Rats, Sprague-Dawley, Species Specificity, Botulinum Toxins pharmacology, Clostridium botulinum genetics, Muscle Relaxation drug effects, Neurotoxins pharmacology, Serogroup
- Abstract
Botulinum neurotoxin (BoNT) is a major therapeutic agent. Of seven native BoNT serotypes (A to G), only A and B are currently used in the clinic. Here we compared the potency of commercially available purified native serotypes A1 to F1 across in vitro, ex vivo, and in vivo assays. BoNT potency in vitro was assessed in rat primary cells (target protein cleavage and neurotransmitter release assays) in supraspinal, spinal, and sensory systems. BoNT potency ex vivo was measured in the mouse phrenic nerve hemidiaphragm (PNHD) assay, measuring muscle contractility. In vivo , BoNT-induced muscle relaxation in mice and rats was assessed in the Digit Abduction Score (DAS) test, while effects on body weight (BW) gain were used to assess tolerability. In all assays, all BoNT serotypes were potent toxins, except serotype D1 in vivo which failed to produce significant muscle flaccidity in mice and rats. In rats, all serotypes were well-tolerated, whereas in mice, reductions in BW were detected at high doses. Serotype A1 was the most potent serotype across in vitro, ex vivo, and in vivo assays. The rank order of potency of the serotypes revealed differences among assays. For example, species-specificity was seen for serotype B1, and to a lesser extent for serotype C1. Serotypes F1 and C1, not currently in the clinic, showed preference for sensory over motor models and therefore could be considered for development in conditions involving the somatosensory system.
- Published
- 2018
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16. Engineering Botulinum Toxins to Improve and Expand Targeting and SNARE Cleavage Activity.
- Author
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Fonfria E, Elliott M, Beard M, Chaddock JA, and Krupp J
- Subjects
- Animals, Humans, Protein Engineering, Recombinant Proteins metabolism, Recombinant Proteins pharmacology, SNARE Proteins metabolism, Botulinum Toxins genetics, Botulinum Toxins metabolism, Botulinum Toxins pharmacology, Neurotoxins genetics, Neurotoxins metabolism, Neurotoxins pharmacology
- Abstract
Botulinum neurotoxins (BoNTs) are highly successful protein therapeutics. Over 40 naturally occurring BoNTs have been described thus far and, of those, only 2 are commercially available for clinical use. Different members of the BoNT family present different biological properties but share a similar multi-domain structure at the molecular level. In nature, BoNTs are encoded by DNA in producing clostridial bacteria and, as such, are amenable to recombinant production through insertion of the coding DNA into other bacterial species. This, in turn, creates possibilities for protein engineering. Here, we review the production of BoNTs by the natural host and also recombinant production approaches utilised in the field. Applications of recombinant BoNT-production include the generation of BoNT-derived domain fragments, the creation of novel BoNTs with improved performance and enhanced therapeutic potential, as well as the advancement of BoNT vaccines. In this article, we discuss site directed mutagenesis, used to affect the biological properties of BoNTs, including approaches to alter their binding to neurons and to alter the specificity and kinetics of substrate cleavage. We also discuss the target secretion inhibitor (TSI) platform, in which the neuronal binding domain of BoNTs is substituted with an alternative cellular ligand to re-target the toxins to non-neuronal systems. Understanding and harnessing the potential of the biological diversity of natural BoNTs, together with the ability to engineer novel mutations and further changes to the protein structure, will provide the basis for increasing the scope of future BoNT-based therapeutics.
- Published
- 2018
- Full Text
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17. The Expanding Therapeutic Utility of Botulinum Neurotoxins.
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Fonfria E, Maignel J, Lezmi S, Martin V, Splevins A, Shubber S, Kalinichev M, Foster K, Picaut P, and Krupp J
- Subjects
- Animals, Drug Administration Routes, Drug Compounding, Humans, Serogroup, Botulinum Toxins therapeutic use, Neurotoxins therapeutic use, Peripheral Nervous System Agents therapeutic use
- Abstract
Botulinum neurotoxin (BoNT) is a major therapeutic agent that is licensed in neurological indications, such as dystonia and spasticity. The BoNT family, which is produced in nature by clostridial bacteria, comprises several pharmacologically distinct proteins with distinct properties. In this review, we present an overview of the current therapeutic landscape and explore the diversity of BoNT proteins as future therapeutics. In recent years, novel indications have emerged in the fields of pain, migraine, overactive bladder, osteoarthritis, and wound healing. The study of biological effects distal to the injection site could provide future opportunities for disease-tailored BoNT therapies. However, there are some challenges in the pharmaceutical development of BoNTs, such as liquid and slow-release BoNT formulations; and, transdermal, transurothelial, and transepithelial delivery. Innovative approaches in the areas of formulation and delivery, together with highly sensitive analytical tools, will be key for the success of next generation BoNT clinical products.
- Published
- 2018
- Full Text
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18. Botulinum neurotoxin A and an engineered derivate targeted secretion inhibitor (TSI) A enter cells via different vesicular compartments.
- Author
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Fonfria E, Donald S, and Cadd VA
- Subjects
- Cytoplasm metabolism, Humans, Neuroblastoma metabolism, Neuroblastoma pathology, Neurotoxins chemistry, Neurotoxins pharmacology, Protein Transport, Synaptosomal-Associated Protein 25 metabolism, Tumor Cells, Cultured, Botulinum Toxins, Type A chemistry, Botulinum Toxins, Type A pharmacology, ErbB Receptors antagonists & inhibitors, Neuroblastoma drug therapy, Synaptic Vesicles metabolism
- Abstract
Botulinum neurotoxins (BoNTs) are highly potent multi-domain proteins, responsible for botulism in animals and humans. The modular structural organization of BoNTs has led to the development of novel engineered bio-therapeutic proteins called targeted secretion inhibitors (TSIs). We report here that botulinum neurotoxin A (BoNT/A) and a TSI/A in which the neuronal binding domain of BoNT/A has been substituted by an epidermal growth factor (EGF) ligand, named EGFR-targeted TSI/A, exploit different routes to gain entry in the same in vitro neuroblastoma cell system, SiMa cells. We found that the EGF ligand conferred the affinity to the EGFR-targeted TSI/A at the EGF receptor when compared to an untargeted TSI/A and also the ability to internalize into the cells and cleave its cytosolic target protein SNAP-25. Using high content analysis we found that both BoNT/A and the EGFR-targeted TSI/A enter the cell in a concentration-dependent manner and in compartments which are able to translocate the proteins into the cytosol within 4 h. The EGFR-targeted TSI/A internalized into a compartment which gave a punctate staining pattern by immunofluorescence and partially overlapped with structures positive for the early endosomal marker EAA1; whereas BoNT/A did not internalize into a punctate compartment but did so in an acidifying compartment consistent with local synaptic vesicle recycling. These findings show that the BoNT/A translocation domain, common to both BoNT/A and the EGFR-targeted TSI/A, is a versatile tool for cytosolic delivery from distinct intracellular vesicular compartments.
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- 2016
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19. Identification of 2-oxo-N-(phenylmethyl)-4-imidazolidinecarboxamide antagonists of the P2X(7) receptor.
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Abberley L, Bebius A, Beswick PJ, Billinton A, Collis KL, Dean DK, Fonfria E, Gleave RJ, Medhurst SJ, Michel AD, Moses AP, Patel S, Roman SA, Scoccitti T, Smith B, Steadman JG, and Walter DS
- Subjects
- Administration, Oral, Animals, Biological Availability, Half-Life, Haplorhini, Imidazolines administration & dosage, Imidazolines chemistry, Imidazolines pharmacokinetics, Purinergic Antagonists administration & dosage, Purinergic Antagonists chemistry, Purinergic Antagonists pharmacokinetics, Rats, Imidazolines pharmacology, Purinergic Antagonists pharmacology, Receptors, Purinergic P2X7 drug effects
- Abstract
A backup molecule to compound 2 was sought by targeting the most likely metabolically vulnerable site in this molecule. Compound 18 was subsequently identified as a potent P2X(7) antagonist with very low in vivo clearance and high oral bioavailability in all species examined. Some evidence to support the role of P2X(7) in the etiology of pain is also presented., (Copyright © 2010 Elsevier Ltd. All rights reserved.)
- Published
- 2010
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20. Discovery and structure-activity relationships of a series of pyroglutamic acid amide antagonists of the P2X7 receptor.
- Author
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Abdi MH, Beswick PJ, Billinton A, Chambers LJ, Charlton A, Collins SD, Collis KL, Dean DK, Fonfria E, Gleave RJ, Lejeune CL, Livermore DG, Medhurst SJ, Michel AD, Moses AP, Page L, Patel S, Roman SA, Senger S, Slingsby B, Steadman JG, Stevens AJ, and Walter DS
- Subjects
- Amides chemistry, Drug Discovery, Models, Molecular, Purinergic P2 Receptor Antagonists chemistry, Structure-Activity Relationship, Amides pharmacology, Purinergic P2 Receptor Antagonists pharmacology, Pyrrolidonecarboxylic Acid chemistry, Receptors, Purinergic P2X7 drug effects
- Abstract
A computational lead-hopping exercise identified compound 4 as a structurally distinct P2X(7) receptor antagonist. Structure-activity relationships (SAR) of a series of pyroglutamic acid amide analogues of 4 were investigated and compound 31 was identified as a potent P2X(7) antagonist with excellent in vivo activity in animal models of pain, and a profile suitable for progression to clinical studies., (Copyright 2010 Elsevier Ltd. All rights reserved.)
- Published
- 2010
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21. Synthesis and biological activity of a series of tetrasubstituted-imidazoles as P2X(7) antagonists.
- Author
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Gleave RJ, Walter DS, Beswick PJ, Fonfria E, Michel AD, Roman SA, and Tang SP
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- Animals, Anti-Inflammatory Agents, Non-Steroidal chemistry, Anti-Inflammatory Agents, Non-Steroidal pharmacology, Humans, Imidazoles chemical synthesis, Imidazoles pharmacology, Pyrazoles chemistry, Rats, Receptors, Purinergic P2 metabolism, Receptors, Purinergic P2X7, Structure-Activity Relationship, Anti-Inflammatory Agents, Non-Steroidal chemical synthesis, Imidazoles chemistry, Purinergic P2 Receptor Antagonists
- Abstract
A series of analogues of the pyrazole lead 1 were synthesized in which the heterocyclic core was replaced with an imidazole. A number of potent antagonists were identified and structure-activity relationships (SAR) were investigated both with respect to activity at the P2X(7) receptor and in vitro metabolic stability. Compound 10 was identified as a potent P2X(7) antagonist with reduced in vitro metabolism and high solubility., (2010 Elsevier Ltd. All rights reserved.)
- Published
- 2010
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22. Structure-activity relationships and in vivo activity of (1H-pyrazol-4-yl)acetamide antagonists of the P2X(7) receptor.
- Author
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Beswick PJ, Billinton A, Chambers LJ, Dean DK, Fonfria E, Gleave RJ, Medhurst SJ, Michel AD, Moses AP, Patel S, Roman SA, Roomans S, Senger S, Stevens AJ, and Walter DS
- Subjects
- Acetamides chemical synthesis, Acetamides therapeutic use, Administration, Oral, Animals, Disease Models, Animal, Humans, Pain drug therapy, Pyrazoles chemical synthesis, Rats, Receptors, Purinergic P2X7 metabolism, Structure-Activity Relationship, Acetamides chemistry, Purinergic P2X Receptor Antagonists, Pyrazoles chemistry
- Abstract
Structure-activity relationships (SAR) of analogues of lead compound 1 were investigated and compound 16 was selected for further study in animal models of pain. Compound 16 was shown to be a potent antihyperalgesic agent in both the rat acute complete Freund's adjuvant (CFA) model of inflammatory pain [Iadarola, M. J.; Douglass, J.; Civelli, O.; Naranjo, J. R. rain Res.1988, 455, 205] and the knee joint model of chronic inflammatory pain [Wilson, A. W.; Medhurst, S. J.; Dixon, C. I.; Bontoft, N. C.; Winyard, L. A.; Brackenborough, K. T.; De Alba, J.; Clarke, C. J.; Gunthorpe, M. J.; Hicks, G. A.; Bountra, C.; McQueen, D. S.; Chessell, I. P. Eur. J. Pain2006, 10, 537]., (Copyright 2010 Elsevier Ltd. All rights reserved.)
- Published
- 2010
- Full Text
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23. Synthesis and structure-activity relationships of a series of (1H-pyrazol-4-yl)acetamide antagonists of the P2X7 receptor.
- Author
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Chambers LJ, Stevens AJ, Moses AP, Michel AD, Walter DS, Davies DJ, Livermore DG, Fonfria E, Demont EH, Vimal M, Theobald PJ, Beswick PJ, Gleave RJ, Roman SA, and Senger S
- Subjects
- Acetamides chemical synthesis, Acetamides pharmacokinetics, Administration, Oral, Animals, Anti-Infective Agents chemistry, Anti-Infective Agents pharmacokinetics, High-Throughput Screening Assays, Humans, Injections, Intravenous, Pyrazoles chemistry, Pyrazoles pharmacokinetics, Rats, Receptors, Purinergic P2 metabolism, Receptors, Purinergic P2X7, Structure-Activity Relationship, Acetamides chemistry, Anti-Infective Agents chemical synthesis, Purinergic P2 Receptor Antagonists, Pyrazoles chemical synthesis
- Abstract
High-throughput screening identified compound 1 as a potent P2X(7) receptor antagonist suitable for lead optimisation. Structure-activity relationships (SAR) of a series of (1H-pyrazol-4-yl)acetamides were investigated and compound 32 was identified as a potent P2X(7) antagonist with enhanced potency and favourable physicochemical and pharmacokinetic properties., (Copyright 2010 Elsevier Ltd. All rights reserved.)
- Published
- 2010
- Full Text
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24. TRPM2 is elevated in the tMCAO stroke model, transcriptionally regulated, and functionally expressed in C13 microglia.
- Author
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Fonfria E, Mattei C, Hill K, Brown JT, Randall A, Benham CD, Skaper SD, Campbell CA, Crook B, Murdock PR, Wilson JM, Maurio FP, Owen DE, Tilling PL, and McNulty S
- Subjects
- Animals, Base Sequence, Calcium Signaling drug effects, Cell Line, Central Nervous System metabolism, Disease Models, Animal, Gene Expression Regulation drug effects, Humans, Hydrogen Peroxide pharmacology, Interleukin-1 pharmacology, Male, Microglia drug effects, Middle Cerebral Artery, RNA, Antisense administration & dosage, RNA, Antisense genetics, RNA, Messenger genetics, RNA, Messenger metabolism, Rats, Rats, Sprague-Dawley, Tissue Distribution, Microglia metabolism, Stroke genetics, Stroke metabolism, TRPM Cation Channels genetics, TRPM Cation Channels metabolism
- Abstract
We report the detailed expression profile of TRPM2 mRNA within the human central nervous system (CNS) and demonstrate increased TRPM2 mRNA expression at 1 and 4 weeks following ischemic injury in the rat transient middle cerebral artery occlusion (tMCAO) stroke model. Microglial cells play a key role in pathology produced following ischemic injury in the CNS and possess TRPM2, which may contribute to stroke-related pathological responses. We show that TRPM2 mRNA is present in the human C13 microglial cell line and is reduced by antisense treatment. Activation of C13 cells by interleukin-1beta leads to a fivefold increase of TRPM2 mRNA demonstrating transcriptional regulation. To confirm mRNA distribution correlated with functional expression, we combined electrophysiology, Ca2+ imaging, and antisense approaches. C13 microglia exhibited, when stimulated with hydrogen peroxide (H2O2), increased [Ca2+]i, which was reduced by antisense treatment. Moreover, patch-clamp recordings from C13 demonstrated that increased intracellular adenosine diphosphoribose (ADPR) or extracellular H2O2 induced an inward current, consistent with activation of TRPM2. In addition we confirm the functional expression of a TRPM2-like conductance in primary microglial cultures derived from rats. Activation of TRPM2 in microglia during ischemic brain injury may mediate key aspects of microglial pathophysiological responses.
- Published
- 2006
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25. Tissue distribution profiles of the human TRPM cation channel family.
- Author
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Fonfria E, Murdock PR, Cusdin FS, Benham CD, Kelsell RE, and McNulty S
- Subjects
- Base Sequence, DNA Primers genetics, Female, Gene Expression Profiling, Humans, Male, RNA, Messenger genetics, RNA, Messenger metabolism, Reverse Transcriptase Polymerase Chain Reaction methods, Reverse Transcriptase Polymerase Chain Reaction statistics & numerical data, Sensitivity and Specificity, TRPM Cation Channels classification, Tissue Distribution, TRPM Cation Channels genetics
- Abstract
Eight members of the TRP-melastatin (TRPM) subfamily have been identified, whose physiological functions and distribution are poorly characterized. Although tissue expression and distribution patterns have been reported for individual TRPM channels, comparisons between individual studies are not possible because of variations in analysis techniques and tissue selection. We report here a comparative analysis of the expression patterns of all of the human TRPM channels in selected peripheral tissues and the central nervous system (CNS) using two distinct but complimentary approaches: TaqMan and SYBR Green real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). These techniques generated comparative distribution profiles and demonstrated tissue-specific co-expression of TRPM mRNA species, indicating significant potential for the formation of heteromeric channels. TRPM channels 2, 4, 5, 6, and 7 in contrast to 1, 3, and 8 are widely distributed in the CNS and periphery. The tissues demonstrating highest expression for individual family members were brain (TRPM1), brain and bone marrow (TRPM2), brain and pituitary (TRPM3), intestine and prostate (TRPM4), intestine, pancreas, and prostate (TRPM5), intestine and brain (TRPM6), heart, pituitary, bone, and adipose tissue (TRPM7), and prostate and liver (TRPM8). The data reported here will guide the elucidation of TRPM channel physiological functions.
- Published
- 2006
- Full Text
- View/download PDF
26. TRPM2 channel opening in response to oxidative stress is dependent on activation of poly(ADP-ribose) polymerase.
- Author
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Fonfria E, Marshall IC, Benham CD, Boyfield I, Brown JD, Hill K, Hughes JP, Skaper SD, and McNulty S
- Subjects
- Adenosine Diphosphate Ribose pharmacology, Animals, Blotting, Western, Calcium metabolism, Cell Death drug effects, Cell Line, Cell Line, Tumor, Cell Membrane drug effects, Electrophysiology, Enzyme Activation, Fluorescent Dyes, Fluorometry, Humans, Hydrogen Peroxide pharmacology, Insulinoma metabolism, Organic Chemicals, Pancreatic Neoplasms metabolism, Rats, TRPM Cation Channels, Tetrazolium Salts, Thiazoles, Ion Channel Gating physiology, Ion Channels drug effects, Membrane Proteins drug effects, Oxidative Stress physiology, Poly(ADP-ribose) Polymerases metabolism
- Abstract
1. TRPM2 (melastatin-like transient receptor potential 2 channel) is a nonselective cation channel that is activated under conditions of oxidative stress leading to an increase in intracellular free Ca(2+) concentration ([Ca(2+)](i)) and cell death. We investigated the role of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) on hydrogen peroxide (H(2)O(2))-mediated TRPM2 activation using a tetracycline-inducible TRPM2-expressing cell line. 2. In whole-cell patch-clamp recordings, intracellular adenine 5'-diphosphoribose (ADP-ribose) triggered an inward current in tetracycline-induced TRPM2-human embryonic kidney (HEK293) cells, but not in uninduced cells. Similarly, H(2)O(2) stimulated an increase in [Ca(2+)](i) (pEC(50) 4.54+/-0.02) in Fluo-4-loaded TRPM2-expressing HEK293 cells, but not in uninduced cells. Induction of TRPM2 expression caused an increase in susceptibility to plasma membrane damage and mitochondrial dysfunction in response to H(2)O(2). These data demonstrate functional expression of TRPM2 following tetracycline induction in TRPM2-HEK293 cells. 3. PARP inhibitors SB750139-B (patent number DE10039610-A1 (Lubisch et al., 2001)), PJ34 (N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide) and DPQ (3, 4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone) inhibited H(2)O(2)-mediated increases in [Ca(2+)](i) (pIC(50) vs 100 microm H(2)O(2): 7.64+/-0.38; 6.68+/-0.28; 4.78+/-0.05, respectively), increases in mitochondrial dysfunction (pIC(50) vs 300 microm H(2)O(2): 7.32+/-0.23; 6.69+/-0.22; 5.44+/-0.09, respectively) and decreases in plasma membrane integrity (pIC(50) vs 300 microm H(2)O(2): 7.45+/-0.27; 6.35+/-0.18; 5.29+/-0.12, respectively). The order of potency of the PARP inhibitors in these assays (SB750139>PJ34>DPQ) was the same as for inhibition of isolated PARP enzyme. 4. SB750139-B, PJ34 and DPQ had no effect on inward currents elicited by intracellular ADP-ribose in tetracycline-induced TRPM2-HEK293 cells, suggesting that PARP inhibitors are not interacting directly with the channel. 5. SB750139-B, PJ34 and DPQ inhibited increases in [Ca(2+)](i) in a rat insulinoma cell line (CRI-G1 cells) endogenously expressing TRPM2 (pIC(50) vs 100 microm H(2)O(2): 7.64+/-0.38; 6.68+/-0.28; 4.78+/-0.05, respectively). 6. These data suggest that oxidative stress causes TRPM2 channel opening in both recombinant and endogenously expressing cell systems via activation of PARP enzymes.
- Published
- 2004
- Full Text
- View/download PDF
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