The influence of different fluorphores on the pharmacology of fluorescent-XAC analogues at the human A1-adenosine receptor

Jillian G Baker1, Stephen J Hill1, Barrie Kellam2 and Richard Middleton2. 1Institute of Cell Signalling, Queen’s Medical Centre, 2School of Pharmacy, Centre for Biomolecular Sciences, University of Nottingham, Nottingham, NG7 2UH.

Previous studies involving fluorescent analogues of existing antagonists have shown that the pharmacology of the original molecule changes substantially when a fluorophore is conjugated to it (Baker et al., 2003, Briddon et al., 2004). The aim of this study was to examine the effect of different fluorophores on the pharmacological properties of the adenosine A1-receptor antagonist xanthine aminer congener (XAC).

The primary amine of XAC was acylated using BODIPY 630/650-X (BY630) or Texas Red-X (TXR) succinimidyl esters (all Molecular Probes) as described previously (Briddon et al., 2004). A 7-carboxyoctanoyl linker was attached to the amine of XAC, activated as its corresponding succinimidyl ester and subsequently coupled with BODIPY-FL ethylene diamine (BY-FL-EDA) to afford XAC-BY-FL. XAC-Dansyl (XAC-DAN) was prepared by reacting XAC with dansyl chloride. All products were purified by RP-HPLC and their structures confirmed by mass spectrometry. CRE-SPAP reporter gene transcription measurements were made in CHO cells expressing the human A1-adenosine receptor as previously described (Baker & Hill 2006).

NECA produced a biphasic response made up of an initial decrease in forskolin-stimulated CRE-gene transcription at low agonist concentrations (log IC50 -8.19; Gi-mediated) followed by a pertussis toxin (PTX) resistant increase in CRE- gene transcription (log EC50 -6.35; Gs-mediated) at higher agonist concentrations (Baker & Hill, 2006). XAC antagonized both the Gi and Gs- components of the NECA concentration response curve to yield log KD values of -7.77 and -8.05. XAC-BY 630 antagonised the NECA responses to yield log KD values of -6.67±0.10 and -6.91±0.10 (n=3) for the Gi and Gs responses respectively. XAC-DAN yielded log KD values of -7.20±0.15 (Gi) and -7.35±0.04 (Gs; n=3). XAC-TXR (1μM) had minimal antagonist activity and XAC-BY-FL did not cause any antagonism of the NECA response up to concentrations of 1μM (i.e. within the solubility of the ligand). When the Gs-response was isolated by pre-incubation of cells with PTX (100ng/ml for 24 hours), the log K D values obtained were -8.01 (Baker & Hill, 2006), -7.21±0.07; -7.52±0.24; -6.19±0.14 (n=3) for the parent XAC, XAC-BY 630, XAC-DAN and XAC-TXR respectively. Once again XAC-BY-FL had no effect.

When the effect of these ligands alone was examined, XAC was confirmed to be an inverse agonist, stimulating an increase in forskolin-stimulated CRE-gene transcription of 1.53±0.07 fold over basal (log EC50 = -7.09±0.12, n=4). This response was absent following pre-incubation with PTX confirming a Gi-inverse agonist effect. XAC-BY 630 and XAC-DAN also retained this inverse agonist property yielding log EC50 values of -7.11±0.19 and-6.55±0.18 (n=3) respectively. XAC-TXR gave a small stimulatory effect although the log EC50 value was greater than -6. No response was seen with XAC-BY-FL.

This study confirms that XAC is an antagonist of both the Gi and Gs-coupled states of the human adenosine A1-receptor, whilst having some inverse agonist properties. The fluorescent analogues XAC-BY 630 and XAC-DAN were also A1-receptor inverse agonists. XAC-TXR and XAC-BY-FL had minimal or no activity respectively. The choice of fluorophore therefore has a major impact on the pharmacology of the final fluorescent XAC conjugate.

Baker JG and Hill SJ (2006) J. Pharmacol. Exp. Ther. (In press)
Baker JG et al (2003) Br. J. Pharmacol. 139, 232-242.
Briddon SJ et al (2004). Proc. Natl. Aced. Sci. 101, 4673-4678.

J.G.B. is a Wellcome Trust Clinician Scientist Fellow.