The influence of linker length and fluorophore on the pharmacology of fluorescent-NECA 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.

We have previously described the synthesis of a novel fluorescent adenosine A1-receptor agonist (ABEA-X-BY630) with a four carbon linker between the parent NECA molecule and the BODIPY-X-630/650 fluorophore (Briddon et al., 2002). The aim of this study was to examine the effect of different linker lengths and different fluorophores on the pharmacological properties of these analogues using a cyclic AMP response element (CRE) reporter gene.

Linkers composed of three (APrEA-X-BY630), five (APEA-X-BY630) and eight (AOEA-X-BY630) carbon atoms or an 8 carbon equivalent polyethylene glycol linker (AOPEA-X-BY630) were synthesised in a similar manner to ABEA-X-BY630 (Briddon et al., 2002). Variants of ABEA (i.e. NECA with a four carbon linker) conjugated to Texas Red (TXR; Molecular Probes), Cy5 (GE Healthcare) and EVOblue (Evotec) were also synthesised. CHO cells stably expressing the human A1-adenosine receptor and a CRE-SPAP reporter gene were used and CRE-gene transcription measurements made as previously described (Baker and Hill 2006).

NECA produced a biphasic response that was made up of an initial decrease in forskolin-stimulated CRE-gene transcription at low concentrations of agonist (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 concentrations (Baker and Hill, 2006). APrEA-X-BY630, ABEA-X-BY630, APEA-X-BY630, AOEA-X-BY630 and AOPEA-X-630 produced similar biphasic responses (log IC50s = -8.21±0.11; -7.92±0.06; -7.97±0.07; -7.32±0.09; -7.20±0.17, n=6-8) although only the more potent agonists were able to stimulate the Gs component (log EC50 = -6.18±0.13; -6.38±0.16; -6.23±0.13 n=5-8 for APrEA-X-BY630, ABEA-X-BY630 and APEA-X-BY630 respectively). AOEA-X-BY630 and AOPEA-X-BY630 did not cause any stimulation of the Gs-conformation of the receptor. All of the Gi-coupled responses were inhibited by DPCPX, XAC and CGS 15943 to yield similar log KD values to that obtained with the parent NECA (see Table). ABEA-Cy5, ABEA-EVOblue and ABEA-X-TXR stimulated PTX-sensitive decreases in forskolin-stimulated gene transcription but the log IC50 values were greater than -6. ABEA-X-TXR was the most potent of the three yielding a log IC50 of -5.89±0.25 (n=3).

Table. Antagonist log KD values (mean + s.e.mean, n)

In summary, increasing the chain length between NECA and the BODIPY-X-630/650 fluorophore decreased the potency of the fluorescent NECA-analogue. However all of the BODIPY-X-630/650 analogues retained excellent agonist activity. Substitution of the BODIPY fluorophore for TXR, Cy5 and EVOblue, however, resulted in substantial loss of agonist activity and potency.

Baker JG and Hill SJ (2006) J. Pharmacol. Exp. Ther.In press
Briddon SJ et al (2002) Br. J. Pharmacol. 138, 126P

JGB holds a Wellcome Trust Clinician Scientist Fellowship.