Probing the Existence of a Metastable Binding Site at the β2‑Adrenergic Receptor with Homobivalent Bitopic Ligands

Herein, we report the development of bitopic ligands aimed at targeting the orthosteric binding site (OBS) and a metastable binding site (MBS) within the same receptor unit. Previous molecular dynamics studies on ligand binding to the β2-adrenergic receptor (β2AR) suggested that ligands pause at transient, less-conserved MBSs. We envisioned that MBSs can be regarded as allosteric binding sites and targeted by homobivalent bitopic ligands linking two identical pharmacophores. Such ligands were designed based on docking of the antagonist (S)-alprenolol into the OBS and an MBS and synthesized. Pharmacological characterization revealed ligands with similar potency and affinity, slightly increased β2/β1AR-selectivity, and/or substantially slower β2AR off-rates compared to (S)-alprenolol. Truncated bitopic ligands suggested the major contribution of the metastable pharmacophore to be a hydrophobic interaction with the β2AR, while the linkers alone decreased the potency of the orthosteric fragment. Altogether, the study underlines the potential of targeting MBSs for improving the pharmacological profiles of ligands.

本研究报道了靶向同一受体单元内正构结合位点（orthosteric binding site, OBS）与亚稳结合位点（metastable binding site, MBS）的双位配体（bitopic ligands）的开发工作。既往针对配体与β2肾上腺素能受体（β2-adrenergic receptor, β2AR）结合的分子动力学研究表明，配体可暂驻于瞬时存在、保守性较低的MBS中。我们设想，MBS可作为变构结合位点，由连接两个相同药效团的同二价双位配体进行靶向结合。本研究基于拮抗剂(S)-阿普洛尔（(S)-alprenolol）在OBS与MBS中的分子对接结果设计并合成了此类配体。药理学表征结果显示，与(S)-阿普洛尔相比，此类配体具有相当的效能与亲和力，β2/β1肾上腺素能受体选择性略有提升，且/或β2AR解离速率显著减慢。截短型双位配体实验表明，亚稳药效团的主要贡献为与β2AR发生疏水相互作用，而单独的连接臂则会降低正构片段的效能。综上，本研究证实了靶向MBS可用于改善配体的药理学特性，凸显了该策略的应用潜力。