A fluorescent approach for identifying P2X1 ligands

Marc-David Ruepp, James A Brozik, Iwan J P de Esch, Richard W Farndale, Ruth D Murrell-Lagnado, Andrew J Thompson

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

There are no commercially available, small, receptor-specific P2X1 ligands. There are several synthetic derivatives of the natural agonist ATP and some structurally-complex antagonists including compounds such as PPADS, NTP-ATP, suramin and its derivatives (e.g. NF279, NF449). NF449 is the most potent and selective ligand, but potencies of many others are not particularly high and they can also act at other P2X, P2Y and non-purinergic receptors. While there is clearly scope for further work on P2X1 receptor pharmacology, screening can be difficult owing to rapid receptor desensitisation. To reduce desensitisation substitutions can be made within the N-terminus of the P2X1 receptor, but these could also affect ligand properties. An alternative is the use of fluorescent voltage-sensitive dyes that respond to membrane potential changes resulting from channel opening. Here we utilised this approach in conjunction with fragment-based drug-discovery. Using a single concentration (300 μM) we identified 46 novel leads from a library of 1443 fragments (hit rate = 3.2%). These hits were independently validated by measuring concentration-dependence with the same voltage-sensitive dye, and by visualising the competition of hits with an Alexa-647-ATP fluorophore using confocal microscopy; confocal yielded kon (1.142 × 10(6) M(-1) s(-1)) and koff (0.136 s(-1)) for Alexa-647-ATP (Kd = 119 nM). The identified hit fragments had promising structural diversity. In summary, the measurement of functional responses using voltage-sensitive dyes was flexible and cost-effective because labelled competitors were not needed, effects were independent of a specific binding site, and both agonist and antagonist actions were probed in a single assay. The method is widely applicable and could be applied to all P2X family members, as well as other voltage-gated and ligand-gated ion channels. This article is part of the Special Issue entitled 'Fluorescent Tools in Neuropharmacology'.

Original languageEnglish
Pages (from-to)13-21
Number of pages9
JournalNeuropharmacology
Volume98
DOIs
Publication statusPublished - Nov 2015

Fingerprint

Purinergic P2X1 Receptors
Adenosine Triphosphate
Ligands
Coloring Agents
Purinergic P2Y Receptors
Neuropharmacology
Ligand-Gated Ion Channels
Suramin
Drug Discovery
Confocal Microscopy
Membrane Potentials
Libraries
Binding Sites
Pharmacology
Costs and Cost Analysis
4,4,',4'',4'''-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis(benzene-1,3-disulfonate)

Keywords

  • Adenosine Triphosphate
  • Benzenesulfonates
  • Binding Sites
  • Carbocyanines
  • Dose-Response Relationship, Drug
  • Fluorescence
  • Gene Expression
  • HEK293 Cells
  • Humans
  • Ligands
  • Microscopy, Confocal
  • Purinergic P2X Receptor Agonists
  • Purinergic P2X Receptor Antagonists
  • Receptors, Purinergic P2X1
  • Transfection
  • Voltage-Sensitive Dye Imaging
  • Journal Article
  • Research Support, Non-U.S. Gov't

Cite this

Ruepp, M-D., Brozik, J. A., de Esch, I. J. P., Farndale, R. W., Murrell-Lagnado, R. D., & Thompson, A. J. (2015). A fluorescent approach for identifying P2X1 ligands. Neuropharmacology, 98, 13-21. https://doi.org/10.1016/j.neuropharm.2015.05.016
Ruepp, Marc-David ; Brozik, James A ; de Esch, Iwan J P ; Farndale, Richard W ; Murrell-Lagnado, Ruth D ; Thompson, Andrew J. / A fluorescent approach for identifying P2X1 ligands. In: Neuropharmacology. 2015 ; Vol. 98. pp. 13-21.
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year = "2015",
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language = "English",
volume = "98",
pages = "13--21",
journal = "Neuropharmacology",
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Ruepp, M-D, Brozik, JA, de Esch, IJP, Farndale, RW, Murrell-Lagnado, RD & Thompson, AJ 2015, 'A fluorescent approach for identifying P2X1 ligands' Neuropharmacology, vol. 98, pp. 13-21. https://doi.org/10.1016/j.neuropharm.2015.05.016

A fluorescent approach for identifying P2X1 ligands. / Ruepp, Marc-David; Brozik, James A; de Esch, Iwan J P; Farndale, Richard W; Murrell-Lagnado, Ruth D; Thompson, Andrew J.

In: Neuropharmacology, Vol. 98, 11.2015, p. 13-21.

Research output: Contribution to JournalArticleAcademicpeer-review

TY - JOUR

T1 - A fluorescent approach for identifying P2X1 ligands

AU - Ruepp, Marc-David

AU - Brozik, James A

AU - de Esch, Iwan J P

AU - Farndale, Richard W

AU - Murrell-Lagnado, Ruth D

AU - Thompson, Andrew J

N1 - Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

PY - 2015/11

Y1 - 2015/11

N2 - There are no commercially available, small, receptor-specific P2X1 ligands. There are several synthetic derivatives of the natural agonist ATP and some structurally-complex antagonists including compounds such as PPADS, NTP-ATP, suramin and its derivatives (e.g. NF279, NF449). NF449 is the most potent and selective ligand, but potencies of many others are not particularly high and they can also act at other P2X, P2Y and non-purinergic receptors. While there is clearly scope for further work on P2X1 receptor pharmacology, screening can be difficult owing to rapid receptor desensitisation. To reduce desensitisation substitutions can be made within the N-terminus of the P2X1 receptor, but these could also affect ligand properties. An alternative is the use of fluorescent voltage-sensitive dyes that respond to membrane potential changes resulting from channel opening. Here we utilised this approach in conjunction with fragment-based drug-discovery. Using a single concentration (300 μM) we identified 46 novel leads from a library of 1443 fragments (hit rate = 3.2%). These hits were independently validated by measuring concentration-dependence with the same voltage-sensitive dye, and by visualising the competition of hits with an Alexa-647-ATP fluorophore using confocal microscopy; confocal yielded kon (1.142 × 10(6) M(-1) s(-1)) and koff (0.136 s(-1)) for Alexa-647-ATP (Kd = 119 nM). The identified hit fragments had promising structural diversity. In summary, the measurement of functional responses using voltage-sensitive dyes was flexible and cost-effective because labelled competitors were not needed, effects were independent of a specific binding site, and both agonist and antagonist actions were probed in a single assay. The method is widely applicable and could be applied to all P2X family members, as well as other voltage-gated and ligand-gated ion channels. This article is part of the Special Issue entitled 'Fluorescent Tools in Neuropharmacology'.

AB - There are no commercially available, small, receptor-specific P2X1 ligands. There are several synthetic derivatives of the natural agonist ATP and some structurally-complex antagonists including compounds such as PPADS, NTP-ATP, suramin and its derivatives (e.g. NF279, NF449). NF449 is the most potent and selective ligand, but potencies of many others are not particularly high and they can also act at other P2X, P2Y and non-purinergic receptors. While there is clearly scope for further work on P2X1 receptor pharmacology, screening can be difficult owing to rapid receptor desensitisation. To reduce desensitisation substitutions can be made within the N-terminus of the P2X1 receptor, but these could also affect ligand properties. An alternative is the use of fluorescent voltage-sensitive dyes that respond to membrane potential changes resulting from channel opening. Here we utilised this approach in conjunction with fragment-based drug-discovery. Using a single concentration (300 μM) we identified 46 novel leads from a library of 1443 fragments (hit rate = 3.2%). These hits were independently validated by measuring concentration-dependence with the same voltage-sensitive dye, and by visualising the competition of hits with an Alexa-647-ATP fluorophore using confocal microscopy; confocal yielded kon (1.142 × 10(6) M(-1) s(-1)) and koff (0.136 s(-1)) for Alexa-647-ATP (Kd = 119 nM). The identified hit fragments had promising structural diversity. In summary, the measurement of functional responses using voltage-sensitive dyes was flexible and cost-effective because labelled competitors were not needed, effects were independent of a specific binding site, and both agonist and antagonist actions were probed in a single assay. The method is widely applicable and could be applied to all P2X family members, as well as other voltage-gated and ligand-gated ion channels. This article is part of the Special Issue entitled 'Fluorescent Tools in Neuropharmacology'.

KW - Adenosine Triphosphate

KW - Benzenesulfonates

KW - Binding Sites

KW - Carbocyanines

KW - Dose-Response Relationship, Drug

KW - Fluorescence

KW - Gene Expression

KW - HEK293 Cells

KW - Humans

KW - Ligands

KW - Microscopy, Confocal

KW - Purinergic P2X Receptor Agonists

KW - Purinergic P2X Receptor Antagonists

KW - Receptors, Purinergic P2X1

KW - Transfection

KW - Voltage-Sensitive Dye Imaging

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1016/j.neuropharm.2015.05.016

DO - 10.1016/j.neuropharm.2015.05.016

M3 - Article

VL - 98

SP - 13

EP - 21

JO - Neuropharmacology

JF - Neuropharmacology

SN - 0028-3908

ER -

Ruepp M-D, Brozik JA, de Esch IJP, Farndale RW, Murrell-Lagnado RD, Thompson AJ. A fluorescent approach for identifying P2X1 ligands. Neuropharmacology. 2015 Nov;98:13-21. https://doi.org/10.1016/j.neuropharm.2015.05.016