Discovery of Novel Positive Allosteric Modulators (PAM) of the Muscarinic Receptor M5: Fold-shift Assay
Muscarinic acetylcholine receptors are family A GPCRs comprised of five distinct mammalian subtypes (mAChR1-5 or M1-M5), which are expressed differentially throughout the body and play an important role in a variety of physiological processes. Among the mAChRs, M1 and M4 have been historically considered attractive targets for small molecule treatments of numerous CNS disorders such as more ..
BioActive Compound: 1
Assay Provider: P. Jeffrey Conn
Assay Provider Affiliation: Vanderbilt University
Muscarinic acetylcholine receptors are family A GPCRs comprised of five distinct mammalian subtypes (mAChR1-5 or M1-M5), which are expressed differentially throughout the body and play an important role in a variety of physiological processes. Among the mAChRs, M1 and M4 have been historically considered attractive targets for small molecule treatments of numerous CNS disorders such as Alzheimer's disease and schizophrenia due to their respective localization and involvement in regulation of certain aspects of learning, memory, sleep, motor control, reward, and pain, among others. However, discovery of subtype-selective small molecules has proven highly difficult due to the conservation of the orthosteric binding-site across the mAChRs. This has contributed to the failure of muscarinic agonists in clinical trials and has also hampered pharmacological investigation into the role(s) of each mAChR in basic neurobiology.
Among the mAChRs, M5 has remained perhaps the most challenging to investigate pharmacologically due in part to its extremely low expression level and a complete lack of M5-selective ligands. Interestingly, studies using M5-KO mice suggest that M5 is the sole mediator of acetylcholine-induced cerebrovasodilation, which has led to the hypothesis that an M5 activator would have therapeutic efficacy in treatment of cerebrovascular dementias and ischemic stroke. Furthermore, M5-KO mice show dramatically reduced reward responses to drugs of abuse, consistent with its putative localization on midbrain dopaminergic neurons of the nigrostriatal and mesolimbic pathways. This suggests that M5 antagonism or negative modulation may have utility in treatment of illicit drug addiction and withdrawal. Despite these and other related findings from M5-KO mice, there remains a strong need for small molecule tools to probe M5 function and test M5-related hypotheses in order to advance the state of the mAChR research field and provide critical proof-of-concept studies for drug discovery aims.
Assay Info: CHO-K1 cells stably expressing human M5 were loaded with calcium indicator dye (2mM Fluo-4 AM) for 45-60 min at 37 degrees C. Dye was removed and replaced with the appropriate volume of assay buffer, pH 7.4 (1X HBSS (Hanks' Balanced Salt Solution), supplemented with 20 mM HEPES and 2.5 mM probenecid). All compounds were diluted in assay buffer for a 60 uM 2X stock concentration (30 uM final concentration) in 0.6% DMSO. This stock was then added to the assay plate for a final DMSO concentration of 0.3%. Acetylcholine CRC serial dilutions were 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 one of eight concentrations of the Acetylcholine CRC. The signal amplitude was first normalized to baseline and then as a percentage of the maximal response to acetylcholine. EC50 values for the Acetylcholine CRC alone (i.e. plus Vehicle) and in the presence of a fixed high concentration (30 uM final concentration) of each test compound were determined using GraphPad Prism (4.0c), which fit curves using standard non-linear regression (variable slope).
This compound shifted the EC50 of acetylcholine in the calcium flux assay by greater than 13 fold. The compound was assigned 'Outcome' as 'Active' and 'Score' as '100'.
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* Activity Concentration. ** Test Concentration.
Data Table (Concise)