Selective or Specific M1 Muscarinic Receptor Antagonists Exhibit Biased Agonism at the M1 Receptor via β‐arrestin Signaling

Abstract

Introduction Selective (pirenzepine; PZ) and specific (muscarinic toxin 7; MT7) antagonists of the muscarinic acetylcholine type 1 receptor (M1R) reverse nerve degeneration in rodent models of peripheral neuropathy. These drugs drive axonal outgrowth of adult sensory neurons and signaling is mediated via extracellular-regulated protein kinase (ERK) and β-arrestin. In order to understand the pharmacological mechanism of action of PZ and MT7, we hypothesized that these drugs exhibited biased agonism at the M1R mediated via b-arrestin signaling. Methods Nano bioluminescence resonance energy transfer (NanoBRET) assay: Human embryonic kidney (HEK293) cells co-expressing human M1R-Nluc and Halo-tagged β-arrestin 2 were treated with different doses of M1R agonists (muscarine or carbachol) or antagonists (PZ and MT7)at different time points. NanoBRET assay was employed to measure ligand-induced β-arrestin 2 recruitment to the M1R. Assays were performed in triplicate with 3 independent experiments completed. Data are presented as corrected milli-BRET (mBRET) units. Substrate was added 2 min before BRET detection, and drugs were applied at various time points. Data are reported as the mean ± SD (statistics by 1-way ANOVA with Tukey's post-hoc test). Inositol-phosphate one (IP1) measurement: Intracellular IP1 levels were measured using IP-One assay (Cisbio Bioassays, USA). HEK293 cells co-expressing hM1R-Nluc and Halo-tagged β-arrestin 2 were plated onto 384-well white microplates (20000 cells per well) and were treated with stimulation buffer with or without 1) different doses of M1R agonist (muscarine), 2) M1R antagonists (PZ and MT7) or 3) both M1R antagonist (different doses of PZ) and agonist (muscarine, 1mM) for different time points at 37 °C. Cells were lysed by addition of the supplied buffer containing d2-labeled IP1, followed by addition of terbium cryptate-labeled anti-IP1 antibody, according to the manufacturer's instructions. Plates were incubated for 1 h at room temperature and fluorescence signals were measured at 620 and 665 nm using the Biotek Synergy Neo2 plate reader. HTRF ratio 665 nm/620 nm of each well were extrapolated on the standard curve to obtain IP1 concentrations. Results Both carbachol (Fig. 1A) and muscarine (Fig. 1B) induced Halo-tagged β-arrestin 2 recruitment to hM1R-Nluc in a dose-dependent manner at 5 min of treatment. pirenzepine (Fig. 1C) and MT7 (Fig. 1D) induced Halo-tagged β-arrestin 2 recruitment to hM1R-Nluc in a dose-dependent manner at longer time points (e.g. after 30 min). Interestingly, over short periods, e.g. 2-3 min, PZ and MT7 acted as classical antagonists with blockade of muscarine-induced b-arrestin 2 association with M1R. Unlike MT7 and PZ, muscarine increased IP1 levels in transfected cells (Fig. 2A). Also, PZ dose-dependently inhibited the formation of IP1 following muscarine (1mM) treatment (Fig. 2B). Conclusion Thisstudy for the first time provides molecular evidence to support a role for selective/specific muscarinic receptor antagonists acting as biased agonists at the M1R. This novel signaling pathway may contribute to driving axonal regeneration in adult sensory neurons and could be mobilized for nerve repair in neuropathic disease.