Truncated (N)-Methanocarba Nucleosides as Partial Agonists at Mouse and Human A₃ Adenosine Receptors: Affinity Enhancement by N⁶-(2-Phenylethyl) Substitution

Abstract

Dopamine-derived N 6 -substituents, compared to N 6 -(2-phenylethyl), in truncated (N)-methanocarba (bicyclo[3.1.0]hexyl) adenosines favored high A 3 adenosine receptor (AR) affinity/selectivity, e.g., C2-phenylethynyl analogue 15 (MRS7591, K i = 10.9/17.8 nM, at human/mouse A 3 AR). 15 was a partial agonist in vitro (hA 3 AR, cAMP inhibition, 31% E max ; mA 3 AR, [ 35 S]GTP-γ-S binding, 16% E max ) and in vivo and also antagonized hA 3 AR in vitro. Distal H-bonding substitutions of the N 6 -(2-phenylethyl) moiety particularly enhanced mA 3 AR affinity by polar interactions with the extracellular loops, predicted using docking and molecular dynamics simulation with newly constructed mA 3 AR and hA 3 AR homology models. These hybrid models were based on an inactive antagonistbound hA 1 AR structure for the upper part of TM2 and an agonistbound hA 2A AR structure for the remaining TM portions. These species-independent A 3 AR-selective nucleosides are low efficacy partial agonists and novel, nuanced modulators of the A 3 AR, a drug target of growing interest.

Purinergic Signaling: Impact of GPCR Structures on Rational Drug Design

“…To compensate for this loss, we noted that by strategically designing the N 6 group of truncated analogues we could access polar groups that are particularly prominent in mA 3 AR ELs. Following MD simulation of agonists bound to hA 3 AR and mA 3 AR homology models, nucleoside synthesis confirmed the enhanced mA 3 AR affinity in a series of partial agonists bearing H‐bonding (OH/OMe) groups at a distal point on a hydrophobic N 6– 2‐phenylethyl substituent [78] . MD simulations of a 4′ truncated (N)‐methanocarba nucleoside MRS7591 23 (Figure 3) have predicted its distal polar interaction at both h and mA 3 AR models (Figure 6).…”

“…Receptor structural plasticity was proposed, with a displacement of TM2 and an outward movement from the TM bundle, that was not possible in the A 2A AR due to the constraints from 3 disulfide bonds in ELs. This was accomplished by employing hybrid models, using an opsin, β 2 ‐adrenergic [77] or A 1 adenosine [78] receptor structure for TM2, and an A 2A AR structure for the rest of the receptor. This would permit docking of nucleoside ligands to the binding site while retaining the conserved agonist contacts observed in A 1 AR and A 2A AR structures.…”

“… Pose of MRS7591 23 at the A) human and B) mouse A 3 AR hybrid models, built using hA 2A AR and hA 1 AR X‐ray (for TM2) structures [78] . A) Last frame of a 30‐ns MD simulation starting from the docking pose of MRS7591 (lime) at the hA 3 AR model (light gray).…”

In Silico Drug Design for Purinergic GPCRs: Overview on Molecular Dynamics Applied to Adenosine and P2Y Receptors

“…The first models that were based on this hypothesis were built using an intermediate state agonist-bound structure of A 2A AR, and opsin or β 2 -adrenergic receptor for TM2 [68]. Recently, a revised nucleoside-bound A 3 AR model was constructed, based on an intermediate state agonist-bound structure of A 2A AR and an inactive antagonist-bound structure of A 1 AR for TM2, which is outwardly displaced from the TM bundle [69].…”

“…Human (h) and mouse (m) A 3 AR hybrid models that were built in this way were recently applied to rationalize the effective contribution of polar substituents on the N 6 -2-phenylethyl moiety of 4 -truncated (N)-methanocarba 2-phenylethynyl adenosine partial-agonists [69]. In particular, derivatization at the N 6 position with a dopamine-like moiety increased both h and mA 3 AR binding affinity, with a particular increase in the case of mA 3 AR with respect to the unsubstituted N 6 -2-phenylethyl.…”