Smith Deborah L, Davoren Jennifer E, Edgerton Jeremy R, Lazzaro John T, Lee Che-Wah, Neal Sarah, Zhang Lei, Grimwood Sarah
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
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.
Mol Pharmacol. 2016 Sep;90(3):177-87. doi: 10.1124/mol.116.104737. Epub 2016 Jul 5.
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.
通过正变构调节剂（PAM）选择性激活M1毒蕈碱型乙酰胆碱受体（mAChR）是治疗与精神分裂症和阿尔茨海默病相关的认知障碍的一种新方法。在此，我们描述了一种M1 PAM放射性配体8-((1S,2S)-2-羟基环己基)-5-((6-(甲基-t3)吡啶-3-基)甲基)-8,9-二氢-7H-吡咯并[3,4-小时]喹啉-7-酮（[(3)H]PT-1284）的特性，它可作为表征M1变构结合位点以及分析新型M1 PAMs的工具。8-((1S,2S)-2-羟基环己基)-5-((6-甲基吡啶-3-基)甲基)-8,9-二氢-7H-吡咯并[3,4-小时]喹啉-7-酮（PT-1284 (1: )）在M1荧光成像板读数器（FLIPR）功能测定中显示可增强乙酰胆碱（ACh）（EC50，36 nM），在海马切片电生理测定中可增强卡巴胆碱（EC50，165 nM）。PT-1284 (1: )还降低了抑制[(3)H]N-甲基东莨菪碱（[(3)H]NMS）与M1结合所需的ACh浓度，在10 μM时使ACh Ki左移约19倍。对人M1 mAChR稳定细胞系的饱和分析表明，[(3)H]PT-1284在1 mM ACh存在下与M1 mAChR结合，Kd为4.23 nM，饱和结合容量（Bmax）为6.38 pmol/mg蛋白质。M1选择性PAMs显示以浓度响应方式抑制[(3)H]PT-1284结合，而M1变构和正构激动剂显示出弱亲和力（>30 μM）。在[(3)H]PT-1284结合测定中为19种M1 PAMs生成的亲和力值与FLIPR功能增强测定中这些配体的EC50值之间发现存在强正相关（R(2)=0.86）。这些数据表明M1 PAM结合亲和力与功能活性之间存在强正相关，并且[(3)H]PT-1284可作为M1 mAChR PAMs药理学研究的工具。
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