Structure-Activity Analysis of Biased Agonism at the Human Adenosine A₃ Receptor

Abstract

Biased agonism at G protein-coupled receptors (GPCRs) has significant implications for current drug discovery, but molecular determinants that govern ligand bias remain largely unknown. The adenosine A 3 GPCR (A 3 AR) is a potential therapeutic target for various conditions, including cancer, inflammation, and ischemia, but for which biased agonism remains largely unexplored. We now report the generation of bias "fingerprints" for prototypical ribose containing A 3 AR agonists and rigidified (N)-methanocarba 59-N-methyluronamide nucleoside derivatives with regard to their ability to mediate different signaling pathways. Relative to the reference prototypical agonist IB-MECA, (N)-methanocarba 59-Nmethyluronamide nucleoside derivatives with significant N 6 or C2 modifications, including elongated aryl-ethynyl groups, exhibited biased agonism. Significant positive correlation was observed between the C2 substituent length (in Å) and bias toward cell survival. Molecular modeling suggests that extended C2 substituents on (N)-methanocarba 59-N-methyluronamide nucleosides promote a progressive outward shift of the A 3 AR transmembrane domain 2, which may contribute to the subset of A 3 AR conformations stabilized on biased agonist binding.

Design and validation of the first cell‐impermeant melatonin receptor agonist

“…Ligand bias for two pathways, relative to melatonin, was expressed as 10 ΔΔlog(τ/ KA ) as previously described (van der Westhuizen et al , ) (Supporting Information Table S2). No bias was observed for ICOA‐9 and ICOA‐13 for most receptors, with the exception of hMT 1 for which ICOA‐9 showed significant bias (25‐ and 57‐fold) on the cAMP over β‐arrestin and ERK pathways, respectively, when using extrapolated EC 50 and E max values for ERK activation as previously reported (Baltos et al , ) (see section) (Figure C, Supporting Information Table S2). Taken together, this analysis shows that the cell‐permeable ICOA‐9 shows bias at hMT 1 and the cell‐impermeable ICOA‐13 does not display any detectable signalling bias compared with the cell‐permeable melatonin.…”

“…The significance of the ligand biases was determined by Student's two‐tailed unpaired t ‐test. Because we did not reach a plateau for the two fluorescent compounds for ERK activation, we used the extrapolated data for EC 50 and E max as derived from GraphPad Prism using a non‐linear regression analysis to obtain an estimate of the bias factor at hMT 1 receptors, following the same procedure as previously applied by the Christopoulos lab (Baltos et al , ). The estimated pEC 50 values were −6.35 ± 0.52 (E max = 112 ± 6) and −5.60 ± 0.32 (E max = 128 ± 15) for ICOA‐9 and ICOA‐13 respectively.…”

A3 Adenosine Receptors as Modulators of Inflammation: From Medicinal Chemistry to Therapy

“…Thus, a hybrid homology model in which TM2 assumed its highly displaced position in opsin was required to dock biphenyl derivative MRS5679 26, and the other TMs followed their orientation in the agonist-bound A 2A AR. 226 This proposed outward movement of TM2 was also associated with the degree of activational bias of C2-extended A 3 AR agonists for cell survival, in a comparison of five different functional readouts. 131 Molecular dynamics (MD) simulation of the A 3 AR in complex with agonists have been reported.…”

“…An outward movement of TM2, as observed in several other active GPCR structures such as the β 2 ‐adrenergic receptor and opsin, was therefore hypothesized to occur also for the A 3 AR. Thus, a hybrid homology model in which TM2 assumed its highly displaced position in opsin was required to dock biphenyl derivative MRS5679 26 , and the other TMs followed their orientation in the agonist‐bound A 2A AR . This proposed outward movement of TM2 was also associated with the degree of activational bias of C2‐extended A 3 AR agonists for cell survival, in a comparison of five different functional readouts …”

New paradigms in adenosine receptor pharmacology: allostery, oligomerization and biased agonism

“…The ability of capadenoson and VCP746 to stimulate potent A 2B receptor‐mediated cAMP accumulation in particular may lead to a desirable activity profile within cardiac cells (Vecchio et al, ; Baltos et al, ; Vecchio et al, ). Studies at the A 3 receptor have detected bias both within G protein‐dependent pathways (Baltos et al, ) and also with respect to β‐arrestin translocation (Gao and Jacobson, ). Moreover, biased allosteric modulation has also been demonstrated with respect to the efficacy modulation mediated by the PAM LUF6000 (Gao et al, ).…”