Influence of fluorophore and linker composition on the pharmacology of fluorescent adenosine A 1 receptor ligands

Jillian G Baker, Richard Middleton, Luke Anthony Adams, Lauren Therese May, Stephen J Briddon, Barrie Kellam, Stephen J Hill

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Background and purpose: The introduction of fluorescence-based techniques, and in particular the development of fluorescent ligands, has allowed the study of G protein-coupled receptor pharmacology at the single cell and single molecule level. This study evaluated how the physicochemical nature of the linker and the fluorophore affected the pharmacological properties of fluorescent agonists and antagonists. Experimental approach: Chinese hamster ovary cells stably expressing the human adenosine A 1 receptor and a cyclic 3 ,5 adenosine monophosphate response element-secreted placental alkaline phosphatase (CRE-SPAP) reporter gene, together with whole cell [ 3H]-8-cyclopentyl-1,3-dipropylxanthine (DPCPX) radioligand binding, were used to evaluate the pharmacological properties of a range of fluorescent ligands based on the antagonist xanthine amine congener (XAC) and the agonist 5 (N-ethylcarboxamido) adenosine (NECA). Key results: Derivatives of NECA and XAC with different fluorophores, but equivalent linker length, showed significant differences in their binding properties to the adenosine A 1 receptor. The BODIPY 630/650 derivatives had the highest affinity. Linker length also affected the pharmacological properties, depending on the fluorophore used. Particularly in fluorescent agonists, higher agonist potency could be achieved with large or small linkers for dansyl and BODIPY 630/650 derivatives, respectively. Conclusions and implications: The pharmacology of a fluorescent ligand was critically influenced by both the fluorophore and the associated linker. Furthermore, our data strongly suggest that the physicochemical properties of the fluorophore/linker pairing determine where in the environment of the target receptor the fluorophore is placed, and this, together with the environmental sensitivity of the resulting fluorescence, may finally decide its utility as a fluorescent probe.
Original languageEnglish
Pages (from-to)772 - 786
Number of pages15
JournalBritish Journal of Pharmacology
Volume159
Issue number4
DOIs
Publication statusPublished - 2010
Externally publishedYes

Cite this

Baker, Jillian G ; Middleton, Richard ; Adams, Luke Anthony ; May, Lauren Therese ; Briddon, Stephen J ; Kellam, Barrie ; Hill, Stephen J. / Influence of fluorophore and linker composition on the pharmacology of fluorescent adenosine A 1 receptor ligands. In: British Journal of Pharmacology. 2010 ; Vol. 159, No. 4. pp. 772 - 786.
@article{a93d3c2b6bdc49fc8d945f0d25e719f2,
title = "Influence of fluorophore and linker composition on the pharmacology of fluorescent adenosine A 1 receptor ligands",
abstract = "Background and purpose: The introduction of fluorescence-based techniques, and in particular the development of fluorescent ligands, has allowed the study of G protein-coupled receptor pharmacology at the single cell and single molecule level. This study evaluated how the physicochemical nature of the linker and the fluorophore affected the pharmacological properties of fluorescent agonists and antagonists. Experimental approach: Chinese hamster ovary cells stably expressing the human adenosine A 1 receptor and a cyclic 3 ,5 adenosine monophosphate response element-secreted placental alkaline phosphatase (CRE-SPAP) reporter gene, together with whole cell [ 3H]-8-cyclopentyl-1,3-dipropylxanthine (DPCPX) radioligand binding, were used to evaluate the pharmacological properties of a range of fluorescent ligands based on the antagonist xanthine amine congener (XAC) and the agonist 5 (N-ethylcarboxamido) adenosine (NECA). Key results: Derivatives of NECA and XAC with different fluorophores, but equivalent linker length, showed significant differences in their binding properties to the adenosine A 1 receptor. The BODIPY 630/650 derivatives had the highest affinity. Linker length also affected the pharmacological properties, depending on the fluorophore used. Particularly in fluorescent agonists, higher agonist potency could be achieved with large or small linkers for dansyl and BODIPY 630/650 derivatives, respectively. Conclusions and implications: The pharmacology of a fluorescent ligand was critically influenced by both the fluorophore and the associated linker. Furthermore, our data strongly suggest that the physicochemical properties of the fluorophore/linker pairing determine where in the environment of the target receptor the fluorophore is placed, and this, together with the environmental sensitivity of the resulting fluorescence, may finally decide its utility as a fluorescent probe.",
author = "Baker, {Jillian G} and Richard Middleton and Adams, {Luke Anthony} and May, {Lauren Therese} and Briddon, {Stephen J} and Barrie Kellam and Hill, {Stephen J}",
year = "2010",
doi = "10.1111/j.1476-5381.2009.00488.x",
language = "English",
volume = "159",
pages = "772 -- 786",
journal = "British Journal of Pharmacology",
issn = "1476-5381",
publisher = "Wiley-Blackwell",
number = "4",

}

Influence of fluorophore and linker composition on the pharmacology of fluorescent adenosine A 1 receptor ligands. / Baker, Jillian G; Middleton, Richard; Adams, Luke Anthony; May, Lauren Therese; Briddon, Stephen J; Kellam, Barrie; Hill, Stephen J.

In: British Journal of Pharmacology, Vol. 159, No. 4, 2010, p. 772 - 786.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Influence of fluorophore and linker composition on the pharmacology of fluorescent adenosine A 1 receptor ligands

AU - Baker, Jillian G

AU - Middleton, Richard

AU - Adams, Luke Anthony

AU - May, Lauren Therese

AU - Briddon, Stephen J

AU - Kellam, Barrie

AU - Hill, Stephen J

PY - 2010

Y1 - 2010

N2 - Background and purpose: The introduction of fluorescence-based techniques, and in particular the development of fluorescent ligands, has allowed the study of G protein-coupled receptor pharmacology at the single cell and single molecule level. This study evaluated how the physicochemical nature of the linker and the fluorophore affected the pharmacological properties of fluorescent agonists and antagonists. Experimental approach: Chinese hamster ovary cells stably expressing the human adenosine A 1 receptor and a cyclic 3 ,5 adenosine monophosphate response element-secreted placental alkaline phosphatase (CRE-SPAP) reporter gene, together with whole cell [ 3H]-8-cyclopentyl-1,3-dipropylxanthine (DPCPX) radioligand binding, were used to evaluate the pharmacological properties of a range of fluorescent ligands based on the antagonist xanthine amine congener (XAC) and the agonist 5 (N-ethylcarboxamido) adenosine (NECA). Key results: Derivatives of NECA and XAC with different fluorophores, but equivalent linker length, showed significant differences in their binding properties to the adenosine A 1 receptor. The BODIPY 630/650 derivatives had the highest affinity. Linker length also affected the pharmacological properties, depending on the fluorophore used. Particularly in fluorescent agonists, higher agonist potency could be achieved with large or small linkers for dansyl and BODIPY 630/650 derivatives, respectively. Conclusions and implications: The pharmacology of a fluorescent ligand was critically influenced by both the fluorophore and the associated linker. Furthermore, our data strongly suggest that the physicochemical properties of the fluorophore/linker pairing determine where in the environment of the target receptor the fluorophore is placed, and this, together with the environmental sensitivity of the resulting fluorescence, may finally decide its utility as a fluorescent probe.

AB - Background and purpose: The introduction of fluorescence-based techniques, and in particular the development of fluorescent ligands, has allowed the study of G protein-coupled receptor pharmacology at the single cell and single molecule level. This study evaluated how the physicochemical nature of the linker and the fluorophore affected the pharmacological properties of fluorescent agonists and antagonists. Experimental approach: Chinese hamster ovary cells stably expressing the human adenosine A 1 receptor and a cyclic 3 ,5 adenosine monophosphate response element-secreted placental alkaline phosphatase (CRE-SPAP) reporter gene, together with whole cell [ 3H]-8-cyclopentyl-1,3-dipropylxanthine (DPCPX) radioligand binding, were used to evaluate the pharmacological properties of a range of fluorescent ligands based on the antagonist xanthine amine congener (XAC) and the agonist 5 (N-ethylcarboxamido) adenosine (NECA). Key results: Derivatives of NECA and XAC with different fluorophores, but equivalent linker length, showed significant differences in their binding properties to the adenosine A 1 receptor. The BODIPY 630/650 derivatives had the highest affinity. Linker length also affected the pharmacological properties, depending on the fluorophore used. Particularly in fluorescent agonists, higher agonist potency could be achieved with large or small linkers for dansyl and BODIPY 630/650 derivatives, respectively. Conclusions and implications: The pharmacology of a fluorescent ligand was critically influenced by both the fluorophore and the associated linker. Furthermore, our data strongly suggest that the physicochemical properties of the fluorophore/linker pairing determine where in the environment of the target receptor the fluorophore is placed, and this, together with the environmental sensitivity of the resulting fluorescence, may finally decide its utility as a fluorescent probe.

U2 - 10.1111/j.1476-5381.2009.00488.x

DO - 10.1111/j.1476-5381.2009.00488.x

M3 - Article

VL - 159

SP - 772

EP - 786

JO - British Journal of Pharmacology

JF - British Journal of Pharmacology

SN - 1476-5381

IS - 4

ER -