Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5: [3H]N-methylscopolamine Competition with Acetylcholine
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: Membranes were prepared from M5-CHO cells according to a previously described protocol (Bridges et al., 2009, Marlo et al., 2009, Brady et al., 2008). Binding reactions contained 0.09 nM [3H]N-methylscopolamine ([3H]-NMS) (obtained commercially from Amersham), 15-20 ug of membrane protein, and test compound (30 uM final) or vehicle, and Acetylcholine (13-point CRC from serial dilution) in a total volume of 500 ul assay buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4). 1 uM (final) atropine was used to determine non-specific binding. The KD of [3H]-NMS was determined empirically to be 0.264 nM. Binding reactions were incubated for 2 hours at room temperature on a Lab-Line Titer plate shaker at setting 7 (~750 rpm). Reactions were stopped and membranes collected onto 96-well Barex microplates with GF/B filter (1um pore size) using a Brandel harvester and washed 3X with ice-cold harvesting buffer (50mM Tris-HCl, 0.9% NaCl, pH 7.4). Filter plates were dried overnight and counted in a PerkinElmer TopCount scintillation counter (PerkinElmer Life and Analytical Sciences). True [3H]-NMS concentration was back-calculated after counting aliquots of 5X [3H]-NMS used in the reaction. ACh Ki in the presence and absence of test compound (M5 PAM or vehicle) was determined by the Cheng-Prusoff equation. For all assays, radioligand depletion was kept to approximately 10% or less.
The acetylcholine Ki in the presence of compound SID 85285486 was 10-fold lower than in the presence of vehicle alone. Therefore, the 'Outcome' was assigned as 'Active' and the 'Score' was assigned as '100'.
1.Discovery of the first highly M5-preferring muscarinic acetylcholine receptor ligand, an M5 positive allosteric modulator derived from a series of 5-trifluoromethoxy N-benzyl isatins. Bridges TM, Marlo JE, Niswender CM, Jones CK, Jadhav SB, Gentry PR, Plumley HC, Weaver CD, Conn PJ, Lindsley CW. J Med Chem. 2009 Jun
2.Discovery and characterization of novel allosteric potentiators of M1 muscarinic receptors reveals multiple modes of activity. Marlo JE, Niswender CM, Days EL, Bridges TM, Xiang Y, Rodriguez AL, Shirey JK, Brady AE, Nalywajko T, Luo Q, Austin CA, Williams MB, Kim K, Williams R, Orton D, Brown HA, Lindsley CW, Weaver CD, Conn PJ. Mol Pharmacol. 2009 Mar;75(3):577-88. Epub 2008 Dec 1.
3.Centrally active allosteric potentiators of the M4 muscarinic acetylcholine receptor reverse amphetamine-induced hyperlocomotor activity in rats. Brady AE, Jones CK, Bridges TM, Kennedy JP, Thompson AD, Heiman JU, Breininger ML, Gentry PR, Yin H, Jadhav SB, Shirey JK, Conn PJ, Lindsley CW. J Pharmacol Exp Ther. 2008 Dec;327(3):941-53. Epub 2008 Sep 4.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)