Pharmacological Analysis and Structure Determination of 7-Methylcyanopindolol–Boundβ₁-Adrenergic Receptor

Abstract

Comparisons between structures of the b 1 -adrenergic receptor (AR) bound to either agonists, partial agonists, or weak partial agonists led to the proposal that rotamer changes of Ser 5.46 , coupled to a contraction of the binding pocket, are sufficient to increase the probability of receptor activation. (RS)-4-[3-(tertbutylamino)-2-hydroxypropoxy]-1H-indole-2-carbonitrile (cyanopindolol) is a weak partial agonist of b 1 AR and, based on the hypothesis above, we predicted that the addition of a methyl group to form 4-[(2S)-3-(tert-butylamino)-2-hydroxypropoxy]-7-methyl-1H-indole-2-carbonitrile (7-methylcyanopindolol) would dramatically reduce its efficacy. An eight-step synthesis of 7-methylcyanopindolol was developed and its pharmacology was analyzed. 7-Methylcyanopindolol bound with similar affinity to cyanopindolol to both b 1 AR and b 2 AR. As predicted, the efficacy of 7-methylcyanopindolol was reduced significantly compared with cyanopindolol, acting as a very weak partial agonist of turkey b 1 AR and an inverse agonist of human b 2 AR. The structure of 7-methylcyanopindolol-bound b 1 AR was determined to 2.4-Å resolution and found to be virtually identical to the structure of cyanopindolol-bound b 1 AR. The major differences in the orthosteric binding pocket are that it has expanded by 0.3 Å in 7-methylcyanopindolol-bound b 1 AR and the hydroxyl group of Ser 5.46 is positioned 0.8 Å further from the ligand, with respect to the position of the Ser 5.46 side chain in cyanopindololbound b 1 AR. Thus, the molecular basis for the reduction in efficacy of 7-methylcyanopindolol compared with cyanopindolol may be regarded as the opposite of the mechanism proposed for the increase in efficacy of agonists compared with antagonists.

Insight into partial agonism by observing multiple equilibria for ligand-bound and Gs-mimetic nanobody-bound β1-adrenergic receptor

“…To examine the effect of ligand binding, 1 H, 13 C HMQC spectra were recorded at 308 K for β 1 AR-MetΔ5 in the apo form and in the presence of saturating amounts of 7-methylcyanopindolol (very weak partial agonist) 35 , carvedilol (weak partial agonist), cyanopindolol (weak partial agonist), salbutamol (partial agonist), isoprenaline (full agonist) and adrenaline (full agonist), respectively (Supplementary Table 2 ) 36 . Apo- and ligand-bound spectra (Supplementary Fig.…”

Structural determinants of 5-HT2B receptor activation and biased agonism

“…Although residue 5.46 has been implicated as an important determinant of activation in β adrenergic receptors 21,32 , the role of the highly conserved Thr 3.37 is less understood. In fact, crystallographic evidence that shows direct ligand engagement with residue 3.37 in TM3 is scarce.…”

A Systematic Review of Inverse Agonism at Adrenoceptor Subtypes

“…IA at β 1 -AR has been explored using transfected cell lines; this was mostly done in HEK cells, although CHO [ 78 ] and CHW cells were also used [ 54 ]. They were mostly transfected with the human receptor, but in some cases, also with the turkey receptor [ 78 , 79 ].…”

“…IA at β 1 -AR has been explored using transfected cell lines; this was mostly done in HEK cells, although CHO [ 78 ] and CHW cells were also used [ 54 ]. They were mostly transfected with the human receptor, but in some cases, also with the turkey receptor [ 78 , 79 ]. Transfections were mostly conducted with the WT β 1 -AR, but often also with CAM [ 80 ] and/or naturally occurring variants of the receptor [ 30 , 54 , 55 ].…”

“…IA has also been reported for CGP 20,712 [ 54 ], whereas the detection of IA by propranolol with the human receptor [ 14 ] was not confirmed with the turkey ortholog [ 79 ]. Two studies from the same group also did not observe IA by ICI 118,551 at the turkey β 1 -AR [ 78 , 79 ]. Bucindolol at the human receptor [ 14 ] and carazolol at the turkey receptor [ 78 ] were reported to be partial agonists, with the latter being noteworthy because it has consistently been shown to exhibit IA at the β 2 -AR (see below).…”