Fig. 5. Tx24 is a probe-selective PAM for antagonists.

(A) Allosteric modulator activity of Tx24 toward antagonist atropine. The cells expressing the NanoBiT–G protein and the test GPCR were treated with titrated atropine followed by addition of Tx24 or vehicle. Luminescent signals were measured before and after ligand stimulation (10 μM ACh). (B) HEK293 cells transiently expressing the NanoBiT-G_i1 protein and M₂AChR were loaded with coelenterazine and pretreated with titrated antagonist (atropine, NMS, or tiotropium), followed by addition of Tx24 (100 nM, 500 nM, or 2 μM). Luminescent signals were measured before and after ACh treatment (10 μM). Changes in luminescent signals were normalized to those with vehicle treatment. The chemical structure of each antagonist compound is placed next to the respective curve, with the aromatic head group highlighted in blue. Symbols and error bars represent means and SEM of three to five independent experiments, each performed in duplicate (see tables S2 and S3 for statistics).