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BioAssay: AID 2434

Discovery of novel allosteric modulators of the M1 muscarinic receptor: Acetylcholine Fold-shift Activity of PAMS

Selective M1 activation is an attractive therapeutic approach for the treatment of cognitive impairment, Alzheimer's disease, schizophrenia and a number of other CNS disorders. Until recently, no highly selective M1 activators existed, and those that claimed to be highly M1 selective were either not centrally penetrant or possessed significant ancillary pharmacology which prohibited their use as probes to study M1 receptor function. We have identified that different M1 PAM chemotypes display different modes of activity on downstream receptor signaling. Thus, all ..more
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 Tested Compounds
 Tested Compounds
All(5)
 
 
Active(3)
 
 
Inactive(2)
 
 
 Tested Substances
 Tested Substances
All(5)
 
 
Active(3)
 
 
Inactive(2)
 
 
AID: 2434
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (M1 PAM Ach FS)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2010-02-26
Modify Date: 2010-03-21

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 3
Related Experiments
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AIDNameTypeProbeComment
626Discovery of Novel Allosteric Modulators of the M1 Muscarinic Receptor: Agonist Primary ScreenScreening depositor-specified cross reference: Discovery of Novel Allosteric Modulators of the M1 Muscarinic Receptor: Agonist Primary Screen [Prim
2543Discovery of novel allosteric modulators of the M1 muscarinic receptor: PAM SummarySummary2 depositor-specified cross reference
2626Discovery of novel allosteric modulators of the M1 Muscarinic receptor: PAM Activity with AcetylcholineScreening depositor-specified cross reference
2651Discovery of Novel Allosteric Modulators of the M1 Muscarinic Receptor: PAM Calcium Assay SARConfirmatory depositor-specified cross reference
2425Discovery of novel allosteric modulators of the M1 muscarinic receptor: PAM Calcium Assay Dose-Response with M1Confirmatory same project related to Summary assay
2428Discovery of novel allosteric modulators of the M1 muscarinic receptor: PAM Calcium Assay Dose-Response with M3Confirmatory same project related to Summary assay
2430Discovery of novel allosteric modulators of the M1 muscarinic receptor: PAM Calcium Assay Dose-response with M2Other same project related to Summary assay
2433Discovery of novel allosteric modulators of the M1 muscarinic receptor: PAM Calcium Assay Dose-Response with M5Other same project related to Summary assay
2438Discovery of novel allosteric modulators of the M1 muscarinic receptor: PAM Calcium Assay Dose-Response with M4Confirmatory same project related to Summary assay
Description:
Selective M1 activation is an attractive therapeutic approach for the treatment of cognitive impairment, Alzheimer's disease, schizophrenia and a number of other CNS disorders. Until recently, no highly selective M1 activators existed, and those that claimed to be highly M1 selective were either not centrally penetrant or possessed significant ancillary pharmacology which prohibited their use as probes to study M1 receptor function. We have identified that different M1 PAM chemotypes display different modes of activity on downstream receptor signaling. Thus, all
allosteric M1 activation is not equivalent, and additional tool compounds representing diverse chemotypes are required to truly dissect and study M1 function in the CNS.
Protocol
Experimental Protocol:
CHO-K1 cells stably expressing rat M1 were loaded with calcium indicator dye (2mM Fluo-4 AM) for 45-60 min at 37C. 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 2X stock concentration in 0.6 percent DMSO. This stock was then added to the assay plate for a final DMSO concentration of 0.3 percent. 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 25C 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 concentration of test compound were determined using GraphPad Prism (4.0c), which fit curves using standard non-linear regression (variable slope). Compounds with a calculated EC50 at 10uM were assigned as 'Active' and given a score of '100'. All other compounds were assigned as 'Inactive' and given a score of '0'.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Rep1 Conc1 10uM (1μM**)replicate 1 at 1uM ACh concentration and 10uM compound concentrationFloat%
2Rep1 Conc2 10uM (0.1μM**)replicate 1 at 0.1uM ACh concentration and 10uM compound concentrationFloat%
3Rep1 Conc3 10uM (0.01μM**)replicate 1 at 0.01uM ACh concentration and 10uM compound concentrationFloat%
4Rep1 Conc4 10uM (0.001μM**)replicate 1 at 0.001uM ACh concentration and 10uM compound concentrationFloat%
5Rep1 Conc5 10uM (0.0001μM**)replicate 1 at 0.0001uM ACh concentration and 10uM compound concentrationFloat%
6Rep1 Conc6 10uM (1e-05μM**)replicate 1 at 0.00001uM ACh concentration and 10uM compound concentrationFloat%
7Rep1 Conc7 10uM (1e-06μM**)replicate 1 at 0.000001uM ACh concentration and 10uM compound concentrationFloat%
8Rep1 Conc8 10uM (1e-07μM**)replicate 1 at 0.0000001uM ACh concentration and 10uM compound concentrationFloat%
9Rep2 Conc1 10uM (1μM**)replicate 2 at 1uM ACh concentration and 10uM compound concentrationFloat%
10Rep2 Conc2 10uM (0.1μM**)replicate 2 at 0.1uM ACh concentration and 10uM compound concentrationFloat%
11Rep2 Conc3 10uM (0.01μM**)replicate 2 at 0.01uM ACh concentration and 10uM compound concentrationFloat%
12Rep2 Conc4 10uM (0.001μM**)replicate 2 at 0.001uM ACh concentration and 10uM compound concentrationFloat%
13Rep2 Conc5 10uM (0.0001μM**)replicate 2 at 0.0001uM ACh concentration and 10uM compound concentrationFloat%
14Rep2 Conc6 10uM (1e-05μM**)replicate 2 at 0.00001uM ACh concentration and 10uM compound concentrationFloat%
15Rep2 Conc7 10uM (1e-06μM**)replicate 2 at 0.000001uM ACh concentration and 10uM compound concentrationFloat%
16Rep2 Conc8 10uM (1e-07μM**)replicate 2 at 0.0000001uM ACh concentration and 10uM compound concentrationFloat%
17Rep3 Conc1 10uM (1μM**)replicate 3 at 1uM ACh concentration and 10uM compound concentrationFloat%
18Rep3 Conc2 10uM (0.1μM**)replicate 3 at 0.1uM ACh concentration and 10uM compound concentrationFloat%
19Rep3 Conc3 10uM (0.01μM**)replicate 3 at 0.01uM ACh concentration and 10uM compound concentrationFloat%
20Rep3 Conc4 10uM (0.001μM**)replicate 3 at 0.001uM ACh concentration and 10uM compound concentrationFloat%
21Rep3 Conc5 10uM (0.0001μM**)replicate 3 at 0.0001uM ACh concentration and 10uM compound concentrationFloat%
22Rep3 Conc6 10uM (1e-05μM**)replicate 3 at 0.00001uM ACh concentration and 10uM compound concentrationFloat%
23Rep3 Conc7 10uM (1e-06μM**)replicate 3 at 0.000001uM ACh concentration and 10uM compound concentrationFloat%
24Rep3 Conc8 10uM (1e-07μM**)replicate 3 at 0.0000001uM ACh concentration and 10uM compound concentrationFloat%
25Rep1 Conc1 30uM (1μM**)replicate 1 at 1uM ACh concentration and 30uM compound concentrationFloat%
26Rep1 Conc2 30uM (0.1μM**)replicate 1 at 0.1uM ACh concentration and 30uM compound concentrationFloat%
27Rep1 Conc3 30uM (0.01μM**)replicate 1 at 0.01uM ACh concentration and 30uM compound concentrationFloat%
28Rep1 Conc4 30uM (0.001μM**)replicate 1 at 0.001uM ACh concentration and 30uM compound concentrationFloat%
29Rep1 Conc5 30uM (0.0001μM**)replicate 1 at 0.0001uM ACh concentration and 30uM compound concentrationFloat%
30Rep1 Conc6 30uM (1e-05μM**)replicate 1 at 0.00001uM ACh concentration and 30uM compound concentrationFloat%
31Rep1 Conc7 30uM (1e-06μM**)replicate 1 at 0.000001uM ACh concentration and 30uM compound concentrationFloat%
32Rep1 Conc8 30uM (1e-07μM**)replicate 1 at 0.0000001uM ACh concentration and 30uM compound concentrationFloat%
33Rep2 Conc1 30uM (1μM**)replicate 2 at 1uM ACh concentration and 30uM compound concentrationFloat%
34Rep2 Conc2 30uM (0.1μM**)replicate 2 at 0.1uM ACh concentration and 30uM compound concentrationFloat%
35Rep2 Conc3 30uM (0.01μM**)replicate 2 at 0.01uM ACh concentration and 30uM compound concentrationFloat%
36Rep2 Conc4 30uM (0.001μM**)replicate 2 at 0.001uM ACh concentration and 30uM compound concentrationFloat%
37Rep2 Conc5 30uM (0.0001μM**)replicate 2 at 0.0001uM ACh concentration and 30uM compound concentrationFloat%
38Rep2 Conc6 30uM (1e-05μM**)replicate 2 at 0.00001uM ACh concentration and 30uM compound concentrationFloat%
39Rep2 Conc7 30uM (1e-06μM**)replicate 2 at 0.000001uM ACh concentration and 30uM compound concentrationFloat%
40Rep2 Conc8 30uM (1e-07μM**)replicate 2 at 0.0000001uM ACh concentration and 30uM compound concentrationFloat%
41Rep3 Conc1 30uM (1μM**)replicate 3 at 1uM ACh concentration and 30uM compound concentrationFloat%
42Rep3 Conc2 30uM (0.1μM**)replicate 3 at 0.1uM ACh concentration and 30uM compound concentrationFloat%
43Rep3 Conc3 30uM (0.01μM**)replicate 3 at 0.01uM ACh concentration and 30uM compound concentrationFloat%
44Rep3 Conc4 30uM (0.001μM**)replicate 3 at 0.001uM ACh concentration and 30uM compound concentrationFloat%
45Rep3 Conc5 30uM (0.0001μM**)replicate 3 at 0.0001uM ACh concentration and 30uM compound concentrationFloat%
46Rep3 Conc6 30uM (1e-05μM**)replicate 3 at 0.00001uM ACh concentration and 30uM compound concentrationFloat%
47Rep3 Conc7 30uM (1e-06μM**)replicate 3 at 0.000001uM ACh concentration and 30uM compound concentrationFloat%
48Rep3 Conc8 30uM (1e-07μM**)replicate 3 at 0.0000001uM ACh concentration and 30uM compound concentrationFloat%
49Rep1 Conc1 Veh (1μM**)replicate 1 at 1uM ACh concentration and vehicle onlyFloat%
50Rep1 Conc2 Veh (0.1μM**)replicate 1 at 0.1uM ACh concentration and vehicle onlyFloat%
51Rep1 Conc3 Veh (0.01μM**)replicate 1 at 0.01uM ACh concentration and vehicle onlyFloat%
52Rep1 Conc4 Veh (0.001μM**)replicate 1 at 0.001uM ACh concentration and vehicle onlyFloat%
53Rep1 Conc5 Veh (0.0001μM**)replicate 1 at 0.0001uM ACh concentration and vehicle onlyFloat%
54Rep1 Conc6 Veh (1e-05μM**)replicate 1 at 0.00001uM ACh concentration and vehicle onlyFloat%
55Rep1 Conc7 Veh (1e-06μM**)replicate 1 at 0.000001uM ACh concentration and vehicle onlyFloat%
56Rep1 Conc8 Veh (1e-07μM**)replicate 1 at 0.0000001uM ACh concentration and vehicle onlyFloat%
57Rep2 Conc1 Veh (1μM**)replicate 2 at 1uM ACh concentration and vehicle onlyFloat%
58Rep2 Conc2 Veh (0.1μM**)replicate 2 at 0.1uM ACh concentration and vehicle onlyFloat%
59Rep2 Conc3 Veh (0.01μM**)replicate 2 at 0.01uM ACh concentration and vehicle onlyFloat%
60Rep2 Conc4 Veh (0.001μM**)replicate 2 at 0.001uM ACh concentration and vehicle onlyFloat%
61Rep2 Conc5 Veh (0.0001μM**)replicate 2 at 0.0001uM ACh concentration and vehicle onlyFloat%
62Rep2 Conc6 Veh (1e-05μM**)replicate 2 at 0.00001uM ACh concentration and vehicle onlyFloat%
63Rep2 Conc7 Veh (1e-06μM**)replicate 2 at 0.000001uM ACh concentration and vehicle onlyFloat%
64Rep2 Conc8 Veh (1e-07μM**)replicate 2 at 0.0000001uM ACh concentration and vehicle onlyFloat%
65Rep3 Conc1 Veh (1μM**)replicate 3 at 1uM ACh concentration and vehicle onlyFloat%
66Rep3 Conc2 Veh (0.1μM**)replicate 3 at 0.1uM ACh concentration and vehicle onlyFloat%
67Rep3 Conc3 Veh (0.01μM**)replicate 3 at 0.01uM ACh concentration and vehicle onlyFloat%
68Rep3 Conc4 Veh (0.001μM**)replicate 3 at 0.001uM ACh concentration and vehicle onlyFloat%
69Rep3 Conc5 Veh (0.0001μM**)replicate 3 at 0.0001uM ACh concentration and vehicle onlyFloat%
70Rep3 Conc6 Veh (1e-05μM**)replicate 3 at 0.00001uM ACh concentration and vehicle onlyFloat%
71Rep3 Conc7 Veh (1e-06μM**)replicate 3 at 0.000001uM ACh concentration and vehicle onlyFloat%
72Rep3 Conc8 Veh (1e-07μM**)replicate 3 at 0.0000001uM ACh concentration and vehicle onlyFloat%
73Rep4 Conc1 Veh (1μM**)replicate 4 at 1uM ACh concentration and vehicle onlyFloat%
74Rep4 Conc2 Veh (0.1μM**)replicate 4 at 0.1uM ACh concentration and vehicle onlyFloat%
75Rep4 Conc3 Veh (0.01μM**)replicate 4 at 0.01uM ACh concentration and vehicle onlyFloat%
76Rep4 Conc4 Veh (0.001μM**)replicate 4 at 0.001uM ACh concentration and vehicle onlyFloat%
77Rep4 Conc5 Veh (0.0001μM**)replicate 4 at 0.0001uM ACh concentration and vehicle onlyFloat%
78Rep4 Conc6 Veh (1e-05μM**)replicate 4 at 0.00001uM ACh concentration and vehicle onlyFloat%
79Rep4 Conc7 Veh (1e-06μM**)replicate 4 at 0.000001uM ACh concentration and vehicle onlyFloat%
80Rep4 Conc8 Veh (1e-07μM**)replicate 4 at 0.0000001uM ACh concentration and vehicle onlyFloat%
81Bottom 10uMBottom of curve calculated for ACh CRC with 10uM compound concentrationFloat
82Top 10uMTop of curve calculated for ACh CRC with 10uM compound concentrationFloat
83LogEC50 10uMLog of EC50 value (uM) calculated for ACh CRC with 10uM compound concentrationFloat
84Hillslope 10uMHillslope calculated for ACh CRC with 10uM compound concentrationFloat
85EC50 10uM*EC50 value (uM) calculated for ACh CRC with 10uM compound concentrationFloatμM
86Std Error Bottom 10uMStandard Error of Bottom of curve calculated for ACh CRC with 10uM compound concentrationFloat
87Std Error Top 10uMStandard Error of Top of curve calculated for ACh CRC with 10uM compound concentrationFloat
88Std Error LogEC50 10uMStandard Error of LogEC50 calculated for ACh CRC with 10uM compound concentrationFloat
89Std Error Hillslope 10uMStandard Error of Hillslope calculated for ACh CRC with 10uM compound concentrationFloat
9095%CI Bottom 10uM95% Confidence Interval for Bottom of curve calculated for ACh CRC with 10uM compound concentrationString
9195%CI Top 10uM95% Confidence Interval for Top of curve calculated for ACh CRC with 10uM compound concentrationString
9295%CI LogEC50 10uM95% Confidence Interval for LogEC50 calculated for ACh CRC with 10uM compound concentrationString
9395%CI Hillslope 10uM95% Confidence Interval for Hillslope calculated for ACh CRC with 10uM compound concentrationString
9495%CI EC50 10uM95% Confidence Interval for EC50 calculated for ACh CRC with 10uM compound concentrationString
95Deg Freedom 10uMDegrees of Freedom calculated for ACh CRC with 10uM compound concentrationFloat
96R squared 10uMR squared calculated for ACh CRC with 10uM compound concentrationFloat
97Abs Sum Squares 10uMAbsolute Sum of Squares calculated for ACh CRC with 10uM compound concentrationFloat
98Bottom 30uMBottom of curve calculated for ACh CRC with 30uM compound concentrationFloat
99Top 30uMTop of curve calculated for ACh CRC with 30uM compound concentrationFloat
100LogEC50 30uMLog of EC50 value (uM) calculated for ACh CRC with 30uM compound concentrationFloat
101Hillslope 30uMHillslope calculated for ACh CRC with 30uM compound concentrationFloat
102EC50 30uMEC50 value (uM) calculated for ACh CRC with 30uM compound concentrationFloatμM
103Std Error Bottom 30uMStandard Error of Bottom of curve calculated for ACh CRC with 30uM compound concentrationFloat
104Std Error Top 30uMStandard Error of Top of curve calculated for ACh CRC with 30uM compound concentrationFloat
105Std Error LogEC50 30uMStandard Error of LogEC50 calculated for ACh CRC with 30uM compound concentrationFloat
106Std Error Hillslope 30uMStandard Error of Hillslope calculated for ACh CRC with 30uM compound concentrationFloat
10795%CI Bottom 30uM95% Confidence Interval for Bottom of curve calculated for ACh CRC with 30uM compound concentrationString
10895%CI Top 30uM95% Confidence Interval for Top of curve calculated for ACh CRC with 30uM compound concentrationString
10995%CI LogEC50 30uM95% Confidence Interval for LogEC50 calculated for ACh CRC with 30uM compound concentrationString
11095%CI Hillslope 30uM95% Confidence Interval for Hillslope calculated for ACh CRC with 30uM compound concentrationString
11195%CI EC50 30uM95% Confidence Interval for EC50 calculated for ACh CRC with 30uM compound concentrationString
112Deg Freedom 30uMDegrees of Freedom calculated for ACh CRC with 30uM compound concentrationInteger
113R squared 30uMR squared calculated for ACh CRC with 30uM compound concentrationFloat
114Abs Sum Squares 30uMAbsolute Sum of Squares calculated for ACh CRC with 30uM compound concentrationFloat
115Bottom VehBottom of curve calculated for ACh CRC with Vehicle onlyFloat
116Top VehTop of curve calculated for ACh CRC with Vehicle onlyFloat
117LogEC50 VehLog of EC50 value (uM) calculated for ACh CRC with Vehicle onlyFloat
118Hillslope VehHillslope calculated for ACh CRC with Vehicle onlyFloat
119EC50 VehEC50 value (uM) calculated for ACh CRC with Vehicle onlyFloatμM
120Std Error Bottom VehStandard Error of Bottom of curve calculated for ACh CRC with Vehicle onlyFloat
121Std Error Top VehStandard Error of Top of curve calculated for ACh CRC with Vehicle onlyFloat
122Std Error LogEC50 VehStandard Error of LogEC50 calculated for ACh CRC with Vehicle onlyFloat
123Std Error Hillslope VehStandard Error of Hillslope calculated for ACh CRC with Vehicle onlyFloat
12495%CI Bottom Veh95% Confidence Interval for Bottom of curve calculated for ACh CRC with Vehicle onlyString
12595%CI Top Veh95% Confidence Interval for Top of curve calculated for ACh CRC with Vehicle onlyString
12695%CI LogEC50 Veh95% Confidence Interval for LogEC50 calculated for ACh CRC with Vehicle onlyString
12795%CI Hillslope Veh95% Confidence Interval for Hillslope calculated for ACh CRC with Vehicle onlyString
12895%CI EC50 Veh95% Confidence Interval for EC50 calculated for ACh CRC with Vehicle onlyString
129Deg Freedom VehDegrees of Freedom calculated for ACh CRC with Vehicle onlyInteger
130R squared VehR squared calculated for ACh CRC with Vehicle onlyFloat
131Abs Sum Squares VehAbsolute Sum of Squares calculated for ACh CRC with Vehicle onlyFloat
132VUIDVanderbilt University Internal Registration NumberInteger

* 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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