Synthesis and characterization of high-affinity 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-labeled fluorescent ligands for human β-adrenoceptors

The growing practice of exploiting noninvasive fluorescence-based techniques to study G protein-coupled receptor pharmacology at the single cell and single molecule level demands the availability of high-quality fluorescent ligands. To this end, this study evaluated a new series of red-emitting ligands for the human β-adrenoceptor family. Upon the basis of the orthosteric ligands propranolol, alprenolol, and pindolol, the synthesized linker-modified congeners were coupled to the commercially available fluorophore BODIPY 630/650-X. This yielded high-affinity β-adrenoceptor fluorescent ligands for both the propranolol and alprenolol derivatives; however, the pindolol-based products displayed lower affinity. A fluorescent diethylene glycol linked propranolol derivative (18a) had the highest affinity (log KD of -9.53 and -8.46 as an antagonist of functional β2- and β1-mediated responses, respectively). Imaging studies with this compound further confirmed that it can be employed to selectively label the human β2-adrenoceptor in single living cells, with receptor-associated binding prevented by preincubation with the nonfluorescent β2-selective antagonist 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]-butan-2-ol (ICI 118551) (J. Cardiovasc. Pharmacol. 1983, 5, 430-437.

Effects of (−)-RO363 at human atrial β-adrenoceptor subtypes, the human cloned β(3)-adrenoceptor and rodent intestinal β(3)-adrenoceptors

1. Chronic treatment of patients with β-blockers causes atrial inotropic hyperresponsiveness through β(2)-adrenoceptors, 5-HT(4) receptors and H(2)-receptors but apparently not through β(1)-adrenoceptors despite data claiming an increased β(1)-adrenoceptor density from homogenate binding studies. We have addressed the question of β(1)-adrenoceptor sensitivity by determining the inotropic potency and intrinsic activity of the β(1)-adrenoceptor selective partial agonist (−)-RO363 and by carrying out both homogenate binding and quantitative β-adrenoceptor autoradiography in atria obtained from patients treated or not treated with β-blockers. In the course of the experiments it became apparent that (−)-RO363 also may cause agonistic effects through the third atrial β-adrenoceptor. To assess whether (−)-RO363 also caused agonistic effects through β(3)-adrenoceptors we studied its relaxant effects in rat colon and guinea-pig ileum, as well as receptor binding and adenylyl cyclase stimulation of chinese hamster ovary (CHO) cells expressing human β(3)-adrenoceptors. 2. β-Adrenoceptors were labelled with (−)-[(125)I]-cyanopindolol. The density of both β(1)- and β(2)-adrenoceptors was unchanged in the 2 groups, as assessed with both quantitative receptor autoradiography and homogenate binding. The affinities of (−)-RO363 for β(1)-adrenoceptors (pK(i)=8.0–7.7) and β(2)-adrenoceptors (pK(i)=6.1–5.8) were not significantly different in the two groups. 3. (−)-RO363 increased atrial force with a pEC(50) of 8.2 (β-blocker treated) and 8.0 (non-β-blocker treated) and intrinsic activity with respect to (−)-isoprenaline of 0.80 (β-blocker treated) and 0.54 (non-β-blocker treated) (P<0.001) and with respect to Ca(2+) (7 mM) of 0.65 (β-blocker treated) and 0.45 (non-β-blocker treated) (P<0.01). The effects of (−)-RO363 were resistant to antagonism by the β(2)-adrenoceptor antagonist, ICI 118,551 (50 nM). The effects of 0.3–10 nM (−)-RO363 were antagonized by 3–10 nM of the β(1)-adrenoceptor selective antagonist CGP 20712A. The effects of 20–1000 nM (−)-RO363 were partially resistant to antagonism by 30–300 nM CGP 20712A. 4. (−)-RO363 relaxed the rat colon, partially precontracted by 30 mM KCl, with an intrinsic activity of 0.97 compared to (−)-isoprenaline. The concentration-effect curve to (−)-RO363 revealed 2 components, one antagonized by (−)-propranolol (200 nM) with pEC(50)=8.5 and fraction 0.66, the other resistant to (−)-propranolol (200 nM) with pEC(50)=5.6 and fraction 0.34 of maximal relaxation. 5. (−)-RO363 relaxed the longitudinal muscle of guinea-pig ileum, precontracted by 0.5 μM histamine, with intrinsic activity of 1.0 compared to (−)-isoprenaline and through 2 components, one antagonized by (−)-propranolol (200 nM) with pEC(50)=8.7 and fraction 0.67, the other resistant to (−)-propranolol with pEC(50)=4.9 and fraction 0.33 of maximal relaxation. 6. (−)-RO363 stimulated the adenylyl cyclase of CHO cells expressing human β(3)-adrenoceptors with pEC(50)=5.5 and intrinsic activity 0.74 with respect to (−)-isoprenaline (pEC(50)=5.9). (−)-RO363 competed for binding with [(125)I]-cyanopindolol at human β(3)-adrenoceptors transfected into CHO cells with pK(i)=4.5. (−)-Isoprenaline (pK(i)=5.2) and (−)-CGP 12177A (pK(i)=6.1) also competed for binding at human β(3)-adrenoceptors. 7. We conclude that under conditions used in this study, (−)-RO363 is a potent partial agonist for human β(1)- and β(3)-adrenoceptors and appears also to activate the third human atrial β-adrenoceptor. (−)-RO363 relaxes mammalian gut through both β(1)- and β(3)-adrenoceptors. (−)-RO363, used as a β(1)-adrenoceptor selective tool, confirms previous findings with (−)-noradrenaline that β(1)-adrenoceptor-mediated atrial effects are only slightly enhanced by chronic treatment of patients with β-blockers. Chronic treatment with β(1)-adrenoceptor-selective blockers does not significantly increase the density of human atrial β(1)- and β(2)-adrenoceptors