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BioAssay: AID 2192

Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5: [3H]N-methylscopolamine Competition

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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Inactive(1)
 
 
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Inactive(1)
 
 
AID: 2192
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (M5 PAM NMS Competition)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2009-12-09

Data Table ( Complete ):           All
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..
Tested Compound:
Depositor Specified Assays
AIDNameTypeComment
2665Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5: SAR with Acetylcholinescreening
2416Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5summary
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
Methods:
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 or atropine 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. Atropine Ki determined to be 0.21 by Cheng-Prusoff equation. For all assays, radioligand depletion was kept to approximately 10% or less.

The compound did not compete with[3H]-NMS and was inactive in this assay. The 'Outcome' was assigned as 'Inactive' and the 'Score' was assigned as '0'.

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 11;52(11):3445-8.
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
1Conc1 Rep1 (0.000513μM**)Replicate 1 at 0.000513 uMFloat
2Conc2 Rep1 (0.00151μM**)Replicate 1 at 0.00151 uMFloat
3Conc3 Rep1 (0.00457μM**)Replicate 1 at 0.00457 uMFloat
4Conc4 Rep1 (0.0138μM**)Replicate 1 at 0.0138 uMFloat
5Conc5 Rep1 (0.0407μM**)Replicate 1 at 0.0407 uMFloat
6Conc6 Rep1 (0.123μM**)Replicate 1 at 0.123 uMFloat
7Conc7 Rep1 (0.372μM**)Replicate 1 at 0.372 uMFloat
8Conc8 Rep1 (1.12μM**)Replicate 1 at 1.12 uMFloat
9Conc9 Rep1 (3.31μM**)Replicate 1 at 3.31 uMFloat
10Conc10 Rep1 (10μM**)Replicate 1 at 10 uMFloat
11Conc11 Rep1 (30.2μM**)Replicate 1 at 30.2 uMFloat
12Conc1 Rep2 (0.000513μM**)Replicate 2 at 0.000513 uMFloat
13Conc2 Rep2 (0.00151μM**)Replicate 2 at 0.00151 uMFloat
14Conc3 Rep2 (0.00457μM**)Replicate 2 at 0.00457 uMFloat
15Conc4 Rep2 (0.0138μM**)Replicate 2 at 0.0138 uMFloat
16Conc5 Rep2 (0.0407μM**)Replicate 2 at 0.0407 uMFloat
17Conc6 Rep2 (0.123μM**)Replicate 2 at 0.123 uMFloat
18Conc7 Rep2 (0.372μM**)Replicate 2 at 0.372 uMFloat
19Conc8 Rep2 (1.12μM**)Replicate 2 at 1.12 uMFloat
20Conc9 Rep2 (3.31μM**)Replicate 2 at 3.31 uMFloat
21Conc10 Rep2 (10μM**)Replicate 2 at 10 uMFloat
22Conc11 Rep2 (30.2μM**)Replicate 2 at 30.2 uMFloat
23Conc1 Rep3 (0.000513μM**)Replicate 3 at 0.000513 uMFloat
24Conc2 Rep3 (0.00151μM**)Replicate 3 at 0.00151 uMFloat
25Conc3 Rep3 (0.00457μM**)Replicate 3 at 0.00457 uMFloat
26Conc4 Rep3 (0.0138μM**)Replicate 3 at 0.0138 uMFloat
27Conc5 Rep3 (0.0407μM**)Replicate 3 at 0.0407 uMFloat
28Conc6 Rep3 (0.123μM**)Replicate 3 at 0.123 uMFloat
29Conc7 Rep3 (0.372μM**)Replicate 3 at 0.372 uMFloat
30Conc8 Rep3 (1.12μM**)Replicate 3 at 1.12 uMFloat
31Conc9 Rep3 (3.31μM**)Replicate 3 at 3.31 uMFloat
32Conc10 Rep3 (10μM**)Replicate 3 at 10 uMFloat
33Conc11 Rep3 (30.2μM**)Replicate 3 at 30.2 uMFloat
34Conc1 Rep4 (0.000513μM**)Replicate 4 at 0.000513 uMFloat
35Conc2 Rep4 (0.00151μM**)Replicate 4 at 0.00151 uMFloat
36Conc3 Rep4 (0.00457μM**)Replicate 4 at 0.00457 uMFloat
37Conc4 Rep4 (0.0138μM**)Replicate 4 at 0.0138 uMFloat
38Conc5 Rep4 (0.0407μM**)Replicate 4 at 0.0407 uMFloat
39Conc6 Rep4 (0.123μM**)Replicate 4 at 0.123 uMFloat
40Conc7 Rep4 (0.372μM**)Replicate 4 at 0.372 uMFloat
41Conc8 Rep4 (1.12μM**)Replicate 4 at 1.12 uMFloat
42Conc9 Rep4 (3.31μM**)Replicate 4 at 3.31 uMFloat
43Conc10 Rep4 (10μM**)Replicate 4 at 10 uMFloat
44Conc11 Rep4 (30.2μM**)Replicate 4 at 30.2 uMFloat
45AtropineConc1 Rep1 (1.7e-05μM**)Atropine Replicate 1 at 0.000017 uMFloat
46AtropineConc2 Rep1 (5.13e-05μM**)Atropine Replicate 1 at 0.0000513 uMFloat
47AtropineConc3 Rep1 (0.000151μM**)Atropine Replicate 1 at 0.000151 uMFloat
48AtropineConc4 Rep1 (0.000457μM**)Atropine Replicate 1 at 0.000457 uMFloat
49AtropineConc5 Rep1 (0.00138μM**)Atropine Replicate 1 at 0.00138 uMFloat
50AtropineConc6 Rep1 (0.00407μM**)Atropine Replicate 1 at 0.00407 uMFloat
51AtropineConc7 Rep1 (0.0123μM**)Atropine Replicate 1 at 0.0123 uMFloat
52AtropineConc8 Rep1 (0.0372μM**)Atropine Replicate 1 at 0.0372 uMFloat
53AtropineConc9 Rep1 (0.112μM**)Atropine Replicate 1 at 0.112 uMFloat
54AtropineConc10 Rep1 (0.331μM**)Atropine Replicate 1 at 0.331 uMFloat
55AtropineConc11 Rep1 (1μM**)Atropine Replicate 1 at 1 uMFloat
56AtropineConc1 Rep2 (1.7e-05μM**)Atropine Replicate 2 at 0.000017 uMFloat
57AtropineConc2 Rep2 (5.13e-05μM**)Atropine Replicate 2 at 0.0000513 uMFloat
58AtropineConc3 Rep2 (0.000151μM**)Atropine Replicate 2 at 0.000151 uMFloat
59AtropineConc4 Rep2 (0.000457μM**)Atropine Replicate 2 at 0.000457 uMFloat
60AtropineConc5 Rep2 (0.00138μM**)Atropine Replicate 2 at 0.00138 uMFloat
61AtropineConc6 Rep2 (0.00407μM**)Atropine Replicate 2 at 0.00407 uMFloat
62AtropineConc7 Rep2 (0.0123μM**)Atropine Replicate 2 at 0.0123 uMFloat
63AtropineConc8 Rep2 (0.0372μM**)Atropine Replicate 2 at 0.0372 uMFloat
64AtropineConc9 Rep2 (0.112μM**)Atropine Replicate 2 at 0.112 uMFloat
65AtropineConc10 Rep2 (0.331μM**)Atropine Replicate 2 at 0.331 uMFloat
66AtropineConc11 Rep2 (1μM**)Atropine Replicate 2 at 1 uMFloat
67AtropineConc1 Rep3 (1.7e-05μM**)Atropine Replicate 3 at 0.000017 uMFloat
68AtropineConc2 Rep3 (5.13e-05μM**)Atropine Replicate 3 at 0.0000513 uMFloat
69AtropineConc3 Rep3 (0.000151μM**)Atropine Replicate 3 at 0.000151 uMFloat
70AtropineConc4 Rep3 (0.000457μM**)Atropine Replicate 3 at 0.000457 uMFloat
71AtropineConc5 Rep3 (0.00138μM**)Atropine Replicate 3 at 0.00138 uMFloat
72AtropineConc6 Rep3 (0.00407μM**)Atropine Replicate 3 at 0.00407 uMFloat
73AtropineConc7 Rep3 (0.0123μM**)Atropine Replicate 3 at 0.0123 uMFloat
74AtropineConc8 Rep3 (0.0372μM**)Atropine Replicate 3 at 0.0372 uMFloat
75AtropineConc9 Rep3 (0.112μM**)Atropine Replicate 3 at 0.112 uMFloat
76AtropineConc10 Rep3 (0.331μM**)Atropine Replicate 3 at 0.331 uMFloat
77AtropineConc11 Rep3 (1μM**)Atropine Replicate 3 at 1 uMFloat
78BottomCalculated bottom parameter for compoundFloat
79TopCalculated top parameter for compoundFloat
80LogEC50calculated LogEC50 value for compoundFloatμM
81EC50_uM*Calculated EC50 value for compoundFloatμM
82KICalculated KI for compoundFloatμM
83Std. Error BottomStd Error for bottom for compoundFloat
84Std. ErrorTopStd Error for top for compoundFloat
85Std. Error LogEC50Std Error for LogEC50 for compoundFloat
8695% CI Bottom95% confidence intervals for calculated bottom for compoundString
8795% CI Top95% confidence intervals for calculated top for compoundString
8895% CI LogEC5095% confidence intervals for calculated LogEC50 for compoundString
8995% CI EC5095% confidence intervals for calculated EC50 for compoundString
9095% CI KI95% confidence intervals for calculated KI for compoundString
91Degrees of FreedomDegrees of freedom in calculation for compoundInteger
92RsquaredRsquared in calculation of EC50 for compoundFloat
93AbsSumOfSquaresAbsolute sum of squares in calculation for compoundFloat
94Atropine BottomCalculated bottom parameter for atropineFloat
95Atropine TopCalculated top parameter for atropineFloat
96Atropine LogEC50calculated LogEC50 value for atropineFloat
97Atropine EC50_uMCalculated EC50 value for atropineFloatμM
98Atropine KICalculated KI for atropineFloatμM
99Atropine Std. Error BottomStd Error for bottom for atropineFloat
100Atropine Std. ErrorTopStd Error for top for atropineFloat
101Atropine Std. Error LogEC50Std Error for LogEC50 for atropineFloat
102Atropine 95% CI Bottom95% confidence intervals for calculated bottom for atropineString
103Atropine 95% CI Top95% confidence intervals for calculated top for atropineString
104Atropine 95% CI LogEC5095% confidence intervals for calculated LogEC50 for atropineString
105Atropine 95% CI EC5095% confidence intervals for calculated EC50 for atropineString
106Atropine 95% CI KI95% confidence intervals for calculated KI for atropineString
107Atropine Degrees of FreedomDegrees of freedom in calculation for atropineInteger
108Atropine RsquaredRsquared in calculation of EC50 for atropineFloat
109Atropine AbsSumOfSquaresAbsolute sum of squares in calculation for atropineFloat

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

Data Table (Concise)
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