Fig. 1.

Initial identification of LSN3172176. A cassette of nonradiolabeled putative tracer molecules was assessed in filtration binding assays using membranes expressing the human M1 mAChR. The most promising of these compounds was (A) LSN3172176. (B) Single-point binding assay using two different concentrations of LSN3172176 (1 and 10 nM). Nonspecific binding was determined in the presence of 10 μM atropine (C) saturation assay from which specific binding was calculated by subtracting the mean nonspecific binding in the presence of 10 μM atropine from the mean total binding at each ligand concentration performed in triplicate and then expressed as nanograms ligand per gram protein. For both (B and C) after filtration, bound compound was extracted and measured using LC/MS/MS. Data points represent mean ± S.E.M. from three independent experiments.