Discovery of Novel Positive Allosteric Modulators (PAM) of the Muscarinic Receptor M5: CRC 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 ..
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.
CHO-K1 cells stably transfected with rat M1, human M2/Gqi5, human M3, rat M4/Gqi5, or 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 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 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 2.5 min and then stimulated with the EC20 of acetylcholine for 50 seconds. 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).
These compounds had EC50s less than 10 micromolar for the muscarinic M5 receptor in the calcium flux assay, and therefore, 'Outcome' was assigned as 'Active' (PAM).
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* Activity Concentration. ** Test Concentration.
Data Table (Concise)