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BioAssay: AID 2194

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 ..
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AID: 2194
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (M5 PAM NMS ACh Com)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2009-12-09

Data Table ( Complete ):           View Active Data    View All Data
Target
Sequence: muscarinic acetylcholine receptor M5 [Homo sapiens]
Description ..   
Protein Family: Serpentine type 7TM GPCR chemoreceptor Srx

Gene:CHRM5     Related Protein 3D Structures     More BioActivity Data..
BioActive Compound: 1
Related Experiments
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AIDNameTypeComment
2416Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5Summarydepositor-specified cross reference
2665Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5: SAR with AcetylcholineScreeningdepositor-specified cross reference
626Discovery of Novel Allosteric Modulators of the M1 Muscarinic Receptor: Agonist Primary ScreenScreeningsame project related to Summary assay
2186Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5: Fold-shift AssayConfirmatorysame project related to Summary assay
2192Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5: [3H]N-methylscopolamine CompetitionConfirmatorysame project related to Summary assay
2198Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5: PAM SAR with Muscarinic M5Othersame project related to Summary assay
2204Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5: Calcium Flux AssayConfirmatorysame project related to Summary assay
2206Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5: SAR with Muscarinic M1Othersame project related to Summary assay
651776Discovery of Novel Positive Allosteric Modulators (PAM) of the Muscarinic Receptor M5: Fold-shift AssayConfirmatorysame project related to Summary assay
Description:
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.
Protocol
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'.
References:
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.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Conc1 Rep1 (0.0005μM**)value for Ach at 0.0005 uM for rep 1 with compoundFloat
2Conc2 Rep1 (0.0015μM**)value for Ach at 0.0015 uM for rep 1 with compoundFloat
3Conc3 Rep1 (0.0046μM**)value for Ach at 0.0046 uM for rep 1 with compoundFloat
4Conc4 Rep1 (0.0138μM**)value for Ach at 0.0138 uM for rep 1 with compoundFloat
5Conc5 Rep1 (0.0407μM**)value for Ach at 0.0407 uM for rep 1 with compoundFloat
6Conc6 Rep1 (0.123μM**)value for Ach at 0.123 uM for rep 1 with compoundFloat
7Conc7 Rep1 (0.317μM**)value for Ach at 0.3715 uM for rep 1 with compoundFloat
8Conc8 Rep1 (1.122μM**)value for Ach at 1.122 uM for rep 1 with compoundFloat
9Conc9 Rep1 (3.3113μM**)value for Ach at 3.3113 uM for rep 1 with compoundFloat
10Conc10 Rep1 (10μM**)value for Ach at 10 uM for rep 1 with compoundFloat
11Conc11 Rep1 (30.1995μM**)value for Ach at 30.1995 uM for rep 1 with compoundFloat
12Conc12 Rep1 (100μM**)value for Ach at 100 uM for rep 1 with compoundFloat
13Conc13 Rep1 (301.995μM**)value for Ach at 301.9952 uM for rep 1 with compoundFloat
14Conc1 Rep2 (0.0005μM**)value for Ach at 0.0005 uM for rep 2 with compoundFloat
15Conc2 Rep2 (0.0015μM**)value for Ach at 0.0015 uM for rep 2 with compoundFloat
16Conc3 Rep2 (0.0046μM**)value for Ach at 0.0046 uM for rep 2 with compoundFloat
17Conc4 Rep2 (0.0138μM**)value for Ach at 0.0138 uM for rep 2 with compoundFloat
18Conc5 Rep2 (0.0407μM**)value for Ach at 0.0407 uM for rep 2 with compoundFloat
19Conc6 Rep2 (0.123μM**)value for Ach at 0.123 uM for rep 2 with compoundFloat
20Conc7 Rep2 (0.317μM**)value for Ach at 0.3715 uM for rep 2 with compoundFloat
21Conc8 Rep2 (1.122μM**)value for Ach at 1.122 uM for rep 2 with compoundFloat
22Conc9 Rep2 (3.3113μM**)value for Ach at 3.3113 uM for rep 2 with compoundFloat
23Conc10 Rep2 (10μM**)value for Ach at 10 uM for rep 2 with compoundFloat
24Conc11 Rep2 (30.1995μM**)value for Ach at 30.1995 uM for rep 2 with compoundFloat
25Conc12 Rep2 (100μM**)value for Ach at 100 uM for rep 2 with compoundFloat
26Conc13 Rep2 (301.995μM**)value for Ach at 301.9952 uM for rep 2 with compoundFloat
27Conc1 Rep3 (0.0005μM**)value for Ach at 0.0005 uM for rep 3 with compoundFloat
28Conc2 Rep3 (0.0015μM**)value for Ach at 0.0015 uM for rep 3 with compoundFloat
29Conc3 Rep3 (0.0046μM**)value for Ach at 0.0046 uM for rep 3 with compoundFloat
30Conc4 Rep3 (0.0138μM**)value for Ach at 0.0138 uM for rep 3 with compoundFloat
31Conc5 Rep3 (0.0407μM**)value for Ach at 0.0407 uM for rep 3 with compoundFloat
32Conc6 Rep3 (0.123μM**)value for Ach at 0.123 uM for rep 3 with compoundFloat
33Conc7 Rep3 (0.317μM**)value for Ach at 0.3715 uM for rep 3 with compoundFloat
34Conc8 Rep3 (1.122μM**)value for Ach at 1.122 uM for rep 3 with compoundFloat
35Conc9 Rep3 (3.3113μM**)value for Ach at 3.3113 uM for rep 3 with compoundFloat
36Conc10 Rep3 (10μM**)value for Ach at 10 uM for rep 3 with compoundFloat
37Conc11 Rep3 (30.1995μM**)value for Ach at 30.1995 uM for rep 3 with compoundFloat
38Conc12 Rep3 (100μM**)value for Ach at 100 uM for rep 3 with compoundFloat
39Conc13 Rep3 (301.995μM**)value for Ach at 301.9952 uM for rep 3 with compoundFloat
40BottomCalculated bottom for compoundFloat
41TopCalculated top for compoundFloat
42LogEC50Calculated logEC50 for compoundFloatμM
43EC50_uM*Calculated EC50 for compoundFloatμM
44KICalculated KI for compoundFloatμM
45Std. Error BottomStd Error of bottom for compoundFloat
46Std. ErrorTopStd Error of top for compoundFloat
47Std. Error LogEC50Std Error of LogEC50 for compoundFloat
4895% Confidence Intervals Bottom95% confidence intervals for bottom for compoundString
4995% Confidence Intervals Top95% confidence intervals for top for compoundString
5095% Confidence Intervals LogEC5095% confidence intervals for LogEC50 for compoundString
5195% Confidence Intervals EC5095% confidence intervals for EC50 for compoundString
5295% Confidence Intervals KI95% confidence intervals for KI for compoundString
53Degrees of FreedomDegrees of freedom for EC50 calculation for compoundFloat
54RsquaredR-squared for EC50 calculation for compoundFloat
55AbsSumOfSquaresAbsolute sum of squares for EC50 calculation for compoundFloat
56Conc1 Rep1 Vhl (0.0005μM**)value for Ach at 0.0005 uM for rep 1 with vehicleFloat
57Conc2 Rep1 Vhl (0.0015μM**)value for Ach at 0.0015 uM for rep 1 with vehicleFloat
58Conc3 Rep1 Vhl (0.0046μM**)value for Ach at 0.0046 uM for rep 1 with vehicleFloat
59Conc4 Rep1 Vhl (0.0138μM**)value for Ach at 0.0138 uM for rep 1 with vehicleFloat
60Conc5 Rep1 Vhl (0.0407μM**)value for Ach at 0.0407 uM for rep 1 with vehicleFloat
61Conc6 Rep1 Vhl (0.123μM**)value for Ach at 0.123 uM for rep 1 with vehicleFloat
62Conc7 Rep1 Vhl (0.317μM**)value for Ach at 0.3715 uM for rep 1 with vehicleFloat
63Conc8 Rep1 Vhl (1.122μM**)value for Ach at 1.122 uM for rep 1 with vehicleFloat
64Conc9 Rep1 Vhl (3.3113μM**)value for Ach at 3.3113 uM for rep 1 with vehicleFloat
65Conc10 Rep1 Vhl (10μM**)value for Ach at 10 uM for rep 1 with vehicleFloat
66Conc11 Rep1 Vhl (30.1995μM**)value for Ach at 30.1995 uM for rep 1 with vehicleFloat
67Conc12 Rep1 Vhl (100μM**)value for Ach at 100 uM for rep 1 with vehicleFloat
68Conc13 Rep1 Vhl (301.995μM**)value for Ach at 301.9952 uM for rep 1 with vehicleFloat
69Conc1 Rep2 Vhl (0.0005μM**)value for Ach at 0.0005 uM for rep 1 with vehicleFloat
70Conc2 Rep2 Vhl (0.0015μM**)value for Ach at 0.0015 uM for rep 2 with vehicleFloat
71Conc3 Rep2 Vhl (0.0046μM**)value for Ach at 0.0046 uM for rep 2 with vehicleFloat
72Conc4 Rep2 Vhl (0.0138μM**)value for Ach at 0.0138 uM for rep 2 with vehicleFloat
73Conc5 Rep2 Vhl (0.0407μM**)value for Ach at 0.0407 uM for rep 2 with vehicleFloat
74Conc6 Rep2 Vhl (0.123μM**)value for Ach at 0.123 uM for rep 2 with vehicleFloat
75Conc7 Rep2 Vhl (0.317μM**)value for Ach at 0.3715 uM for rep 2 with vehicleFloat
76Conc8 Rep2 Vhl (1.122μM**)value for Ach at 1.122 uM for rep 2 with vehicleFloat
77Conc9 Rep2 Vhl (3.3113μM**)value for Ach at 3.3113 uM for rep 2 with vehicleFloat
78Conc10 Rep2 Vhl (10μM**)value for Ach at 10 uM for rep 2 with vehicleFloat
79Conc11 Rep2 Vhl (30.1995μM**)value for Ach at 30.1995 uM for rep 2 with vehicleFloat
80Conc12 Rep2 Vhl (100μM**)value for Ach at 100 uM for rep 2 with vehicleFloat
81Conc13 Rep2 Vhl (301.995μM**)value for Ach at 301.9952 uM for rep 2 with vehicleFloat
82Conc1 Rep3 Vhl (0.0005μM**)value for Ach at 0.0005 uM for rep 2 with vehicleFloat
83Conc2 Rep3 Vhl (0.0015μM**)value for Ach at 0.0015 uM for rep 3 with vehicleFloat
84Conc3 Rep3 Vhl (0.0046μM**)value for Ach at 0.0046 uM for rep 3 with vehicleFloat
85Conc4 Rep3 Vhl (0.0138μM**)value for Ach at 0.0138 uM for rep 3 with vehicleFloat
86Conc5 Rep3 Vhl (0.0407μM**)value for Ach at 0.0407 uM for rep 3 with vehicleFloat
87Conc6 Rep3 Vhl (0.123μM**)value for Ach at 0.123 uM for rep 3 with vehicleFloat
88Conc7 Rep3 Vhl (0.317μM**)value for Ach at 0.3715 uM for rep 3 with vehicleFloat
89Conc8 Rep3 Vhl (1.122μM**)value for Ach at 1.122 uM for rep 3 with vehicleFloat
90Conc9 Rep3 Vhl (3.3113μM**)value for Ach at 3.3113 uM for rep 3 with vehicleFloat
91Conc10 Rep3 Vhl (10μM**)value for Ach at 10 uM for rep 3 with vehicleFloat
92Conc11 Rep3 Vhl (30.1995μM**)value for Ach at 30.1995 uM for rep 3 with vehicleFloat
93Conc12 Rep3 Vhl (100μM**)value for Ach at 100 uM for rep 3 with vehicleFloat
94Conc13 Rep3 Vhl (301.995μM**)value for Ach at 301.9952 uM for rep 3 with vehicleFloat
95Bottom VhlCalculated bottom for vehicleFloat
96Top VhlCalculated top for vehicleFloat
97LogEC50 VhlCalculated logEC50 for vehicleFloatμM
98EC50_uM VhlCalculated EC50 for vehicleFloatμM
99KI VhlCalculated KI for vehicleFloatμM
100Std. Error Bottom VhlStd Error of bottom for vehicleFloat
101Std. ErrorTop VhlStd Error of top for vehicleFloat
102Std. Error LogEC50 VhlStd Error of LogEC50 for vehicleFloat
10395% Confidence Intervals Bottom Vhl95% confidence intervals for bottom for vehicleString
10495% Confidence Intervals Top Vhl95% confidence intervals for top for vehicleString
10595% Confidence Intervals LogEC50 Vhl95% confidence intervals for LogEC50 for vehicleString
10695% Confidence Intervals EC50 Vhl95% confidence intervals for EC50 for vehicleString
10795% Confidence Intervals KI Vhl95% confidence intervals for KI for vehicleString
108Degrees of Freedom VhlDegrees of freedom for EC50 calculation for vehicleFloat
109Rsquared VhlR-squared for EC50 calculation for vehicleFloat
110AbsSumOfSquares VhlAbsolute sum of squares for EC50 calculation for vehicleFloat

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH077606

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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