Sequence: muscarinic acetylcholine receptor M4 [Rattus norvegicus]

Gene:CHRM4     Conserved Domain     Related Protein 3D Structures

AID	Name	Type	Comment
2543	Discovery of novel allosteric modulators of the M1 muscarinic receptor: PAM Summary	summary
2651	Discovery of Novel Allosteric Modulators of the M1 Muscarinic Receptor: PAM Calcium Assay SAR	confirmatory
2626	Discovery of novel allosteric modulators of the M1 Muscarinic receptor: PAM Activity with Acetylcholine	screening

Selective M1 activation is an attractive therapeutic approach for the treatment of cognitive impairment, Alzheimer's disease, schizophrenia and a number of other CNS disorders. Until recently, no highly selective M1 activators existed, and those that claimed to be highly M1 selective were either not centrally penetrant or possessed significant ancillary pharmacology which prohibited their use as probes to study M1 receptor function. We have identified that different M1 PAM chemotypes display different modes of activity on downstream receptor signaling. Thus, all allosteric M1 activation is not equivalent, and additional tool compounds representing diverse chemotypes are required to truly dissect and study M1 function in the CNS.

Assay Info: CHO-K1 cells stably transfected with rat M4/Gqi5 were loaded with calcium indicator dye (2mM Fluo-4 AM) for 45-60 min at 37C. Dye was removed and replaced with assay buffer, pH 7.4 (1X HBSS (Hanks' Balanced Salt Solution), supplemented with 20 mM HEPES and 2.5 mM probenecid). All compounds were serially diluted in assay buffer for a final 2X stock in 0.6 percent DMSO. This stock was then added to the assay plate for a final DMSO concentration of 0.3 percent. Acetylcholine (ACh) submax concentration (ca. EC20) was prepared at a 10X stock solution in assay buffer prior to addition to assay plates. Calcium mobilization was measured at 25C using a FLEXstation II (Molecular Devices, Sunnyvale, CA) according to the following protocol. Cells were preincubated with test compound (or vehicle) for 1.5 min prior to the addition of the agonist, acetylcholine. Cells were then stimulated for 50 sec with a submaximal ACh concentration (ca. EC20). The signal amplitude was first normalized to baseline and then as a percentage of the maximal response to acetylcholine. EC50 values for each compound were determined using GraphPad Prism (4.0c), which fit curves using standard non-linear regression (variable slope). Compound dose-response curves with R2 > 0.5 were assigned as 'Active.' The 'Score' was assigned as '100' for compounds with EC50 < 1uM and as '50' for compounds with EC50 > 1uM. All other compounds were assigned as 'Inactive' with a 'Score' of '0'.

Grant Number: MH077606