title: Characterization of a Novel M1 Muscarinic Acetylcholine Receptor Positive Allosteric Modulator Radioligand, [3H]PT-1284.
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 ([(3)H]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 [(3)H]N-methylscopolamine ([(3)H]NMS) binding to M1, left-shifting the ACh Ki approximately 19-fold at 10 μM. Saturation analysis of a human M1 mAChR stable cell line showed that [(3)H]PT-1284 bound to M1 mAChR in the presence of 1 mM ACh with Kd, 4.23 nM, and saturable binding capacity (Bmax), 6.38 pmol/mg protein. M1 selective PAMs were shown to inhibit [(3)H]PT-1284 binding in a concentration-responsive manner, whereas M1 allosteric and orthosteric agonists showed weak affinity (>30 μM). A strong positive correlation (R(2) = 0.86) was found to exist between affinity values generated for nineteen M1 PAMs in the [(3)H]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 [(3)H]PT-1284 can serve as a tool for pharmacological investigation of M1 mAChR PAMs.
authors.affiliation: Neuroscience and Pain Research Unit (D.L.S., J.R.E., S.N., S.G.) and Worldwide Medicinal Chemistry (J.E.D., L.Z.), Pfizer Inc., Cambridge, Massachusetts; Primary Pharmacology Group (J.T.L.) and Worldwide Medicinal Chemistry (C.-W.L.), Pfizer Inc., Groton, Connecticut Deborah.L.Smith@Pfizer.com.
authors.forename: Deborah L
authors.initials: DL
authors.lastname: Smith
authors.affiliation: Neuroscience and Pain Research Unit (D.L.S., J.R.E., S.N., S.G.) and Worldwide Medicinal Chemistry (J.E.D., L.Z.), Pfizer Inc., Cambridge, Massachusetts; Primary Pharmacology Group (J.T.L.) and Worldwide Medicinal Chemistry (C.-W.L.), Pfizer Inc., Groton, Connecticut.
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Davoren
authors.affiliation: Neuroscience and Pain Research Unit (D.L.S., J.R.E., S.N., S.G.) and Worldwide Medicinal Chemistry (J.E.D., L.Z.), Pfizer Inc., Cambridge, Massachusetts; Primary Pharmacology Group (J.T.L.) and Worldwide Medicinal Chemistry (C.-W.L.), Pfizer Inc., Groton, Connecticut.
authors.forename: Jeremy R
authors.initials: JR
authors.lastname: Edgerton
authors.affiliation: Neuroscience and Pain Research Unit (D.L.S., J.R.E., S.N., S.G.) and Worldwide Medicinal Chemistry (J.E.D., L.Z.), Pfizer Inc., Cambridge, Massachusetts; Primary Pharmacology Group (J.T.L.) and Worldwide Medicinal Chemistry (C.-W.L.), Pfizer Inc., Groton, Connecticut.
authors.forename: John T
authors.initials: JT
authors.lastname: Lazzaro
authors.affiliation: Neuroscience and Pain Research Unit (D.L.S., J.R.E., S.N., S.G.) and Worldwide Medicinal Chemistry (J.E.D., L.Z.), Pfizer Inc., Cambridge, Massachusetts; Primary Pharmacology Group (J.T.L.) and Worldwide Medicinal Chemistry (C.-W.L.), Pfizer Inc., Groton, Connecticut.
authors.forename: Che-Wah
authors.initials: CW
authors.lastname: Lee
authors.affiliation: Neuroscience and Pain Research Unit (D.L.S., J.R.E., S.N., S.G.) and Worldwide Medicinal Chemistry (J.E.D., L.Z.), Pfizer Inc., Cambridge, Massachusetts; Primary Pharmacology Group (J.T.L.) and Worldwide Medicinal Chemistry (C.-W.L.), Pfizer Inc., Groton, Connecticut.
authors.forename: Sarah
authors.initials: S
authors.lastname: Neal
authors.affiliation: Neuroscience and Pain Research Unit (D.L.S., J.R.E., S.N., S.G.) and Worldwide Medicinal Chemistry (J.E.D., L.Z.), Pfizer Inc., Cambridge, Massachusetts; Primary Pharmacology Group (J.T.L.) and Worldwide Medicinal Chemistry (C.-W.L.), Pfizer Inc., Groton, Connecticut.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Neuroscience and Pain Research Unit (D.L.S., J.R.E., S.N., S.G.) and Worldwide Medicinal Chemistry (J.E.D., L.Z.), Pfizer Inc., Cambridge, Massachusetts; Primary Pharmacology Group (J.T.L.) and Worldwide Medicinal Chemistry (C.-W.L.), Pfizer Inc., Groton, Connecticut.
authors.forename: Sarah
authors.initials: S
authors.lastname: Grimwood
mesh_terms: D000109:Acetylcholine; D000494:Allosteric Regulation; D000818:Animals; D001345:Autoradiography; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D055724:Electrophysiological Phenomena; D005470:Fluorometry; D057809:HEK293 Cells; D006624:Hippocampus / Q000502:physiology; D006801:Humans; D054833:Isoindoles / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008566:Membranes / Q000378:metabolism; D019832:N-Methylscopolamine / Q000378:metabolism; D049268:Positron-Emission Tomography; D011725:Pyridines / Q000378:metabolism*; D011869:Radioligand Assay; D017207:Rats, Sprague-Dawley; D043643:Receptor, Muscarinic M1 / Q000378:metabolism*
chemical_list: D054833:Isoindoles; C000618266:PT-1284; D011725:Pyridines; D043643:Receptor, Muscarinic M1; D000109:Acetylcholine; D019832:N-Methylscopolamine