Discovery of Novel Allosteric Modulators of the M1 Muscarinic Receptor: PAM Calcium Assay SAR
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 more ..
BioActive Compounds: 15
Depositor Specified Assays
Assay Provider: P. Jeffery Conn
Assay Provider Affiliation: Vanderbilt University
Grant Title: Discovery of novel allosteric modulators of the M1 Muscarinic receptor
Grant Number: 1 R03 MH077606-01
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.
CHO-K1 cells stably transfected with rat M1 were loaded with calcium indicator dye (2mM Fluo-4 AM) for 45-60 min at 37 degrees C. 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% DMSO. This stock was then added to the assay plate for a final DMSO concentration of 0.3%. Acetylcholine (ACh) submax concentration (~EC20) was prepared at a 10X stock solution in assay buffer prior to addition to assay plates. Calcium mobilization was measured at 25 degrees C 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 (~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). The percentage of ACh maximum response values reported represent the Prism-calculated top of the concentration-response curve for three replicates analyzed together. The M1 EC50 mean values reported represent the Prism-calculated EC50 values from concentration-response curves for three replicates analyzed together.
Compounds with mean EC50s greater than 10uM were considered inactive (0). Active compounds with mean EC50s from 5-10uM were assigned a score of '50' and less than 5uM were assigned a score of '100'.
* Activity Concentration.
Data Table (Concise)