Abstract

Selective activation of the M₁ muscarinic acetylcholine receptor (mAChR) via a positive allosteric modulator (PAM) is a new approach for the treatment of the cognitive impairments associated with schizophrenia and Alzheimer’s disease. Herein, we describe the characterization of an M₁ PAM radioligand, 8-((1S,2S)-2-hydroxycyclohexyl)-5-((6-(methyl-t3)pyridin-3-yl)methyl)-8,9-dihydro-7H-pyrrolo[3,4-hour]quinolin-7-one ([³H]PT-1284), as a tool for characterizing the M₁ allosteric binding site, as well as profiling novel M₁ PAMs. 8-((1S,2S)-2-Hydroxycyclohexyl)-5-((6-methylpyridin-3-yl)methyl)-8,9-dihydro-7H-pyrrolo[3,4-hour]quinolin-7-one (PT-1284 (1)) was shown to potentiate acetylcholine (ACh) in an M₁ fluorometric imaging plate reader (FLIPR) functional assay (EC₅₀, 36 nM) and carbachol in a hippocampal slice electrophysiology assay (EC₅₀, 165 nM). PT-1284 (1) also reduced the concentration of ACh required to inhibit [³H]N-methylscopolamine ([³H]NMS) binding to M₁, left-shifting the ACh K_i approximately 19-fold at 10 μM. Saturation analysis of a human M₁ mAChR stable cell line showed that [³H]PT-1284 bound to M₁ mAChR in the presence of 1 mM ACh with K_d, 4.23 nM, and saturable binding capacity (B_max), 6.38 pmol/mg protein. M₁ selective PAMs were shown to inhibit [³H]PT-1284 binding in a concentration-responsive manner, whereas M₁ allosteric and orthosteric agonists showed weak affinity (>30 μM). A strong positive correlation (R² = 0.86) was found to exist between affinity values generated for nineteen M₁ PAMs in the [³H]PT-1284 binding assay and the EC₅₀ values of these ligands in a FLIPR functional potentiation assay. These data indicate that there is a strong positive correlation between M₁ PAM binding affinity and functional activity, and that [³H]PT-1284 can serve as a tool for pharmacological investigation of M₁ mAChR PAMs.