The Design and Characterisation of High Affinity Fluorescent Dipeptide-linked Propranolol Derivatives for the Human β2–Adrenoceptors.

β-adrenoceptors belong to the super family of G protein-coupled receptors and remain an important target for drug discovery for the treatment of cardiovascular diseases (1). The development of selective fluorescent ligands targeted at human β1 and β2–adrenoceptors may facilitate drug discovery programs in terms of understanding receptor pharmacology and receptor localisation in primary cells, the latter from both healthy and diseased tissue (2, 3). Highly selective fluorescent probes may offer an excellent tool for imaging of β-adrenoceptors in native systems where more than one receptor subtype is present. Baker et al have previously described the synthesis and characterisation of a series of β-adrenoceptor fluorescent ligands with the aim to quantify specific interactions between the ligand and receptor in real-time in living cells (2). Whilst the fluorescently labelled propranolol, alprenolol and pindolol derivatives reported displayed high potency and selectivity for the β2-AR over the β1AR, there remains the need to better understand which part of the molecule plays a role in the selectivity and to improve their imaging properties (2). Vernall et al have successfully developed peptide-modified adenosine receptor fluorescent ligands, which demonstrated enhanced binding affinity and imbued a measurable selectivity for the A3 adenosine receptor along with improved properties for imaging (3). This provided an excellent justification for the attempted design and synthesis of subtype-selective β-adrenoceptor fluorescent ligands.

A series of propranolol-based dipeptide congeners were synthesised and coupled to commercially available BODIPY-X-630/650 or BODIPY–FL to yield a series of four red and four green emitting fluorescent ligands for the human β1 and β2–adrenoceptors. Pharmacological characterization of the fluorescent ligands was carried in CHO cells expressing the human β1 and β2-ARs and a cyclic AMP reponse element reporter gene (CRE-SPAP). Cells were grown to confluence in clear plastic tissue culture treated 96-well plates, and CRE-SPAP secretion into the media was measured between 5 and 6 h after the addition of cimaterol. The absorbance at 405 nm was measured using a Dynex MRX plate reader (Chelmsford, MA). For competition binding, cells were seeded as previous and treated with various concentrations of the fluorescent ligands with a fixed concentration of 10 nm of 3HCGP12177. Plates were counted on a Topcount (Perkin Elmer).

All compounds retained affinity at the β2-AR andaside from propranolol–Gly-L-Phe-BY630 retained affinity for the β1-AR in the functional SPAP-based reporter gene assay and radio ligand binding assayrelative to propranolol [pA2 = 8.62 ± 0.10 &7.0± 0.05 (n=5)]. Importantly, two of these compounds (propranolol-Gly-L-Ala-FLandpropranolol-Gly-L-Ser-FL) showed a >70 fold selectivity for the β2–adrenoceptor with the SPAP assay (pA2 = 9.16 ± 0.10 (n=5) & 7.31±0.14 (n=6) and 8.89 ± 0.05 (n=9) & 7.05± 0.09 (n=7); pKi = 8.37 ±10 & 6.77±0.16 (n=6) and 8.64 ± 0.13 & 6.91±0.19 (n=6) respectively) when compared with the β1–adrenoceptor. In cells expressing β2-AR, specific membrane labelling was observed using confocal microscopy, which could be inhibited by 10 µM ICI 118551.

In conclusion, we have synthesised a range of red and green emitting fluorescent ligands that have high affinity for the β2-AR. Manoeuvres to enhance receptor sub-type selectivity even further are on-going within our laboratory.

(1) Tehan, B. G. et al. (2014). Pharmacol. Ther. 143: 51-60

(2) Baker, J.G. et al. (2011). J. Med. Chem. 54: 6874-6887;

(3) Vernall, A. J. et al. (2012). J. Med. Chem. 55: 1771-1782