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BioAssay: AID 623948

Chemical optimization of in vitro pharmacology and DMPK properties of the highly selective mAChR 4 (M4) Positive Allosteric Modulator (PAM) Series with Greatly Improved Human Receptor Activity (hM4 Fold Shift)

To date, five muscarinic acetylcholine receptor (mAChR) subtypes have been identified (M1-M5) and play important roles in mediating the actions of ACh in the peripheral and central nervous systems (Langmead et al. 2008). Of these, M1 and M4 are the most heavily expressed in the CNS and represent attractive therapeutic targets for cognition, Alzheimer's disease, and schizophrenia (Felder et al. more ..
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 Tested Compounds
 Tested Compounds
All(15)
 
 
Probe(1)
 
 
Active(15)
 
 
 Tested Substances
 Tested Substances
All(15)
 
 
Probe(1)
 
 
Active(15)
 
 
AID: 623948
Data Source: Vanderbilt Specialized Chemistry Center (hM4_PAM_EC2_Fold_Shift)
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-03-29
Hold-until Date: 2013-03-27
Modify Date: 2013-03-27

Data Table ( Complete ):           View Active Data    View All Data
Target
Sequence: muscarinic acetylcholine receptor M4 [Homo sapiens]
Description ..   

Gene:CHRM4     Conserved Domain     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: Chemical Probe: 1    Active: 15
Related Experiments
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AIDNameTypeProbeComment
2616Chemical optimization of in vitro pharmacology and DMPK properties of the highly selective mAChR 4 (M4) Positive Allosteric Modulator (PAM) Series with Greatly Improved Human Receptor ActivitySummary depositor-specified cross reference: Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator (PAM)
625Discovery of Novel Allosteric Agonists of the M4 Muscarinic Receptor: Primary ScreenScreening same project related to Summary assay
626Discovery of Novel Allosteric Modulators of the M1 Muscarinic Receptor: Agonist Primary ScreenScreening same project related to Summary assay
643Discovery of Novel Allosteric Agonists of the M4 Muscarinic Receptor: Confirmation ScreenOther same project related to Summary assay
1921Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator: Ancillary ActivityOther same project related to Summary assay
1923Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Analog PotencyConfirmatory same project related to Summary assay
1928Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Analog Fold-shift Selectivity at hM5Confirmatory same project related to Summary assay
1929Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Analog Foldshift Selectivity with hM3Confirmatory same project related to Summary assay
1930Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Fold-shift Selectivity with hM2Confirmatory same project related to Summary assay
1932Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Analog Fold-shift Selectivity at rM1Confirmatory same project related to Summary assay
1938Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator (PAM): Analog Dose Response with rM4Confirmatory same project related to Summary assay
1939Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator (PAM): NMS Competition at rM4Confirmatory same project related to Summary assay
449767Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Foldshift of AcetylcholineOther same project related to Summary assay
449769Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Analog Fold-shift Selectivity at rM4Confirmatory same project related to Summary assay
449770Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Foldshift of Acetylcholine with human M4Confirmatory same project related to Summary assay
588743Chemical optimization of the highly selective mAChR 4 (M4) Positive Allosteric Modulator (PAM) SeriesConfirmatory same project related to Summary assay
588744Human M1 PAM Extended Characterization CounterScreen (CRC)Confirmatory same project related to Summary assay
588745Human M3 PAM Extended Characterization CounterScreen (CRC)Confirmatory same project related to Summary assay
588746Human M2 PAM Extended Characterization CounterScreen (CRC)Confirmatory same project related to Summary assay
588747Human M5 PAM Extended Characterization CounterScreen (CRC)Confirmatory same project related to Summary assay
588748Rat M2 PAM Extended Characterization CounterScreen (CRC)Confirmatory same project related to Summary assay
588749Rat M1 PAM Extended Characterization CounterScreen (CRC)Confirmatory same project related to Summary assay
588750Rat M3 PAM Extended Characterization CounterScreen (CRC)Confirmatory same project related to Summary assay
588751Rat M4 PAM Extended Characterization CounterScreen (CRC)Confirmatory same project related to Summary assay
588755Human M4 PAM Extended Characterization Fold ShiftOther same project related to Summary assay
588758ML253 Competition in Radioligand Binding assays (Riserca)Other same project related to Summary assay
623924Chemical optimization of in vitro pharmacology and DMPK properties of the highly selective mAChR 4 (M4) Positive Allosteric Modulator (PAM) Series with Greatly Improved Human Receptor Activity (hM1 CounterScreen)Confirmatory same project related to Summary assay
623925Chemical optimization of in vitro pharmacology and DMPK properties of the highly selective mAChR 4 (M4) Positive Allosteric Modulator (PAM) Series with Greatly Improved Human Receptor Activity (hM2 CounterScreen)Confirmatory same project related to Summary assay
623926Chemical optimization of in vitro pharmacology and DMPK properties of the highly selective mAChR 4 (M4) Positive Allosteric Modulator (PAM) Series with Greatly Improved Human Receptor Activity (hM3 CounterScreen)Confirmatory same project related to Summary assay
623938Chemical optimization of in vitro pharmacology and DMPK properties of the highly selective mAChR 4 (M4) Positive Allosteric Modulator (PAM) Series with Greatly Improved Human Receptor Activity (hM4 Calcium Potency)Confirmatory1 same project related to Summary assay
623939Chemical optimization of in vitro pharmacology and DMPK properties of the highly selective mAChR 4 (M4) Positive Allosteric Modulator (PAM) Series with Greatly Improved Human Receptor Activity (hM5 CounterScreen)Confirmatory same project related to Summary assay
623940Chemical optimization of in vitro pharmacology and DMPK properties of the highly selective mAChR 4 (M4) Positive Allosteric Modulator (PAM) Series with Greatly Improved Human Receptor Activity (rM1 CounterScreen)Confirmatory same project related to Summary assay
623941Chemical optimization of in vitro pharmacology and DMPK properties of the highly selective mAChR 4 (M4) Positive Allosteric Modulator (PAM) Series with Greatly Improved Human Receptor Activity (rM2 CounterScreen)Confirmatory same project related to Summary assay
623943Chemical optimization of in vitro pharmacology and DMPK properties of the highly selective mAChR 4 (M4) Positive Allosteric Modulator (PAM) Series with Greatly Improved Human Receptor Activity (rM3 CounterScreen)Confirmatory same project related to Summary assay
623945Chemical optimization of in vitro pharmacology and DMPK properties of the highly selective mAChR 4 (M4) Positive Allosteric Modulator (PAM) Series with Greatly Improved Human Receptor Activity (rM4 CounterScreen)Confirmatory1 same project related to Summary assay
623946Chemical optimization of in vitro pharmacology and DMPK properties of the highly selective mAChR 4 (M4) Positive Allosteric Modulator (PAM) Series with Greatly Improved Human Receptor Activity (rM5 CounterScreen)Confirmatory same project related to Summary assay
Description:
To date, five muscarinic acetylcholine receptor (mAChR) subtypes have been identified (M1-M5) and play important roles in mediating the actions of ACh in the peripheral and central nervous systems (Langmead et al. 2008). Of these, M1 and M4 are the most heavily expressed in the CNS and represent attractive therapeutic targets for cognition, Alzheimer's disease, and schizophrenia (Felder et al. 2000; Clader and Wang 2005; Wess et al. 2007; Langmead et al. 2008). For example, clinical trials with xanomeline, a M1/M4-preferring orthosteric agonist, demonstrated efficacy as both a cognition-enhancing agent and an antipsychotic agent (Bodick et al. 1997a; Bodick et al. 1997b; Shekhar et al. 2008). However, a long standing question concerns whether or not the antipsychotic efficacy or antipsychotic-like activity in animal models is mediated by activation of M1, M4, or a combination of both receptors. Data from mAChR knockout (KO) mice led to the suggestion that a selective M1 agonist would be beneficial for cognition, whereas an M4 agonist would provide antipsychotic activity for the treatment of schizophrenia (Gomeza et al. 1999; Gomeza et al. 2001; Anagnostaras et al. 2003; Tzavara et al. 2004). This proposal is further supported by recent studies demonstrating that M4 receptors modulate the dynamics of cholinergic and dopaminergic neurotransmission and that loss of M4 function results in a state of dopamine hyperfunction (Tzavara et al. 2004). These data, coupled with findings that schizophrenic patients have altered hippocampal M4 but not M1 receptor expression (Raedler et al. 2007), suggest that selective activators of M4 may provide a novel treatment strategy for schizophrenia patients.

Unfortunately, xanomeline lacks true M1/M4 specificity and also has significant affinity and efficacy at M2 and M3 mAChRs. Thus xanomeline, like many other cholinergic agents including acetylcholinesterase (AChE) inhibitors, displays significant adverse effects including bradycardia, GI distress, excessive salivation, and sweating, which are thought to be primarily due to activation of peripheral M2 and M3 mAChRs (Bymaster et al. 2003; Wess et al. 2007; Langmead et al. 2008). Due to the high sequence homology and conservation of the orthosteric ACh binding site among the mAChR subtypes, development of chemical agents that are selective for a single mAChR subtype has been largely unsuccessful, and in the absence of highly selective activators of M4, it has been impossible to test the role of selective M4 activation. However, in recent years, we have been highly successful in the identification of multiple highly selective allosteric activators for M1 (Bridges et al. 2008; Jones et al. 2008; Ma et al. 2009; Marlo et al. 2009; Shirey et al. 2009; Bridges et al. 2010) and M4 (Brady et al. 2008; Shirey et al. 2008; Kennedy et al. 2009). Though we are encouraged by these results, unfortunately, highly selective centrally penetrant activators of either M1 or M4 remain unavailable, making it impossible to determine the in vivo effects of selective activation of these receptors. Future compounds developed should exhibit sufficient potency, efficacy, and pharmacokinetic properties, including brain penetration, to make useful probes to progress M4 biology, which will undoubtedly allow the intense study of M4 activation in multiple areas of neuroscience.

Potency and efficacy of compounds will be determined by performing concentration-response curves (CRCs, 10 points, ranging from approximately 30 uM-1 nM at 0.3% final DMSO concentration) at human M4 using a calcium assay in which the cells also express the chimeric G protein Gqi5 to couple M4 to calcium mobilization (Brady et al. 2008; Shirey et al. 2008; Kennedy et al. 2009). PAMs with EC50 values < 1 uM versus human M4 will next be evaluated for potency versus rat M4 in an analogous calcium assay utilizing Gqi5. Following potency evaluation at human and rat M4, PAMs with EC50 values less than 1 uM at rat M4 will be evaluated for their ability to left-shift the CRC of acetylcholine (ACh) for rat M4 in a calcium assay and will be examined for their selectivity for M4 relative to other mAChR subtypes (Brady et al. 2008; Shirey et al. 2008; Kennedy et al. 2009). PAMs with a fold-shift of the ACh CRC of > 5 will then be evaluated for Tier 1 DMPK assays including plasma protein binding (PPB), intrinsic clearance (Obach 1997; Obach 1999), and inhibition of cytochrome p450 enzymes (CYP) (Youdim et al. 2008; Engers et al. 2009; Lebois et al. 2011; Jones et al. 2012). Next, compounds demonstrating PPB > 0.01 fraction unbound (Fu), a clean CYP profile, and moderate clearance will be evaluated for CNS exposure and Plasma:Brain levels using an in vivo snapshot PK paradigm (Frick et al. 1998; Engers et al. 2009; Lebois et al. 2011; Jones et al. 2012). Novel M4 PAMs showing a CNS exposure > PAM EC50, a Brain:Plasma ratio of >0.5, and at least a 10-fold or greater selectivity versus other mAChR subtypes will next be evaluated in a preclinical model of antipsychotic drug action: amphetamine-induced hyperlocomotion (AHL) (Brady et al. 2008; Jones et al. 2008). M4 PAMs demonstrating activity in AHL will have their ancillary pharmacology fully evaluated and those compounds that show no significant off-target activity and suitable solubility will be declared an MLPCN probe. The ultimate goal of this project from the PI's perspective is to generate compounds which should exhibit sufficient potency, efficacy, and pharmacokinetic properties, including brain penetration, to make useful probes to progress M4 biology.

REFERERENCES
1. Anagnostaras, S. G., G. G. Murphy, et al. (2003). "Selective cognitive dysfunction in acetylcholine M1 muscarinic receptor mutant mice." Nat Neurosci 6(1): 51-8.
2. Bodick, N. C., W. W. Offen, et al. (1997a). "Effects of xanomeline, a selective muscarinic receptor agonist, on cognitive function and behavioral symptoms in Alzheimer disease." Arch Neurol 54(4): 465-73.
3. Bodick, N. C., W. W. Offen, et al. (1997b). "The selective muscarinic agonist xanomeline improves both the cognitive deficits and behavioral symptoms of Alzheimer disease." Alzheimer Dis Assoc Disord 11 Suppl 4: S16-22.
4. Brady, A. E., C. K. Jones, et al. (2008). "Centrally active allosteric potentiators of the M4 muscarinic acetylcholine receptor reverse amphetamine-induced hyperlocomotor activity in rats." J Pharmacol Exp Ther 327(3): 941-53.
5. Bridges, T. M., A. E. Brady, et al. (2008). "Synthesis and SAR of analogues of the M1 allosteric agonist TBPB. Part I: Exploration of alternative benzyl and privileged structure moieties." Bioorg Med Chem Lett 18(20): 5439-42.
6. Bridges, T. M., J. Phillip Kennedy, et al. (2010). "Chemical lead optimization of a pan Gq mAChR M1, M3, M5 positive allosteric modulator (PAM) lead. Part II: development of a potent and highly selective M1 PAM." Bioorg Med Chem Lett 20(6): 1972-5.
7. Bymaster, F. P., P. A. Carter, et al. (2003). "Role of specific muscarinic receptor subtypes in cholinergic parasympathomimetic responses, in vivo phosphoinositide hydrolysis, and pilocarpine-induced seizure activity." Eur J Neurosci 17(7): 1403-10.
8. Clader, J. W. and Y. Wang (2005). "Muscarinic receptor agonists and antagonists in the treatment of Alzheimer's disease." Curr Pharm Des 11(26): 3353-61.
9. Engers, D. W., C. M. Niswender, et al. (2009). "Synthesis and evaluation of a series of heterobiarylamides that are centrally penetrant metabotropic glutamate receptor 4 (mGluR4) positive allosteric modulators (PAMs)." J Med Chem 52(14): 4115-8.
10. Felder, C. C., F. P. Bymaster, et al. (2000). "Therapeutic opportunities for muscarinic receptors in the central nervous system." J Med Chem 43(23): 4333-53.
11. Frick, L. W., K. K. Adkison, et al. (1998). "Cassette dosing: rapid in vivo assessment of pharmacokinetics." Pharmaceutical Science & Technology Today 1(1): 12-18.
12. Gomeza, J., L. Zhang, et al. (1999). "Enhancement of D1 dopamine receptor-mediated locomotor stimulation in M(4) muscarinic acetylcholine receptor knockout mice." Proc Natl Acad Sci U S A 96(18): 10483-8.
13. Gomeza, J., L. Zhang, et al. (2001). "Generation and pharmacological analysis of M2 and M4 muscarinic receptor knockout mice." Life Sci 68(22-23): 2457-66.
14. Jones, C. K., A. E. Brady, et al. (2008). "Novel selective allosteric activator of the M1 muscarinic acetylcholine receptor regulates amyloid processing and produces antipsychotic-like activity in rats." J Neurosci 28(41): 10422-33.
15. Jones, C. K., M. Bubser, et al. (2012). "The metabotropic glutamate receptor 4-positive allosteric modulator VU0364770 produces efficacy alone and in combination with L-DOPA or an adenosine 2A antagonist in preclinical rodent models of Parkinson's disease." J Pharmacol Exp Ther 340(2): 404-21.
16. Kennedy, J. P., T. M. Bridges, et al. (2009). "Synthesis and structure-activity relationships of allosteric potentiators of the m(4) muscarinic acetylcholine receptor." ChemMedChem 4(10): 1600-7.
17. Langmead, C. J., J. Watson, et al. (2008). "Muscarinic acetylcholine receptors as CNS drug targets." Pharmacol Ther 117(2): 232-43.
18. Lebois, E. P., G. J. Digby, et al. (2011). "Development of a highly selective, orally bioavailable and CNS penetrant M1 agonist derived from the MLPCN probe ML071." Bioorg Med Chem Lett 21(21): 6451-5.
19. Ma, L., M. A. Seager, et al. (2009). "Selective activation of the M1 muscarinic acetylcholine receptor achieved by allosteric potentiation." Proc Natl Acad Sci U S A 106(37): 15950-5.
20. Marlo, J. E., C. M. Niswender, et al. (2009). "Discovery and characterization of novel allosteric potentiators of M1 muscarinic receptors reveals multiple modes of activity." Mol Pharmacol 75(3): 577-88.
21. Obach, R. S. (1997). "Nonspecific binding to microsomes: impact on scale-up of in vitro intrinsic clearance to hepatic clearance as assessed through examination of warfarin, imipramine, and propranolol." Drug Metab Dispos 25(12): 1359-69.
22. Obach, R. S. (1999). "Prediction of human clearance of twenty-nine drugs from hepatic microsomal intrinsic clearance data: An examination of in vitro half-life approach and nonspecific binding to microsomes." Drug Metab Dispos 27(11): 1350-9.
23. Raedler, T. J., F. P. Bymaster, et al. (2007). "Towards a muscarinic hypothesis of schizophrenia." Mol Psychiatry 12(3): 232-46.
24. Shekhar, A., W. Z. Potter, et al. (2008). "Selective muscarinic receptor agonist xanomeline as a novel treatment approach for schizophrenia." Am J Psychiatry 165(8): 1033-9.
25. Shirey, J. K., A. E. Brady, et al. (2009). "A selective allosteric potentiator of the M1 muscarinic acetylcholine receptor increases activity of medial prefrontal cortical neurons and restores impairments in reversal learning." J Neurosci 29(45): 14271-86.
26. Shirey, J. K., Z. Xiang, et al. (2008). "An allosteric potentiator of M4 mAChR modulates hippocampal synaptic transmission." Nat Chem Biol 4(1): 42-50.
27. Tzavara, E. T., F. P. Bymaster, et al. (2004). "M4 muscarinic receptors regulate the dynamics of cholinergic and dopaminergic neurotransmission: relevance to the pathophysiology and treatment of related CNS pathologies." FASEB J 18(12): 1410-2.
28. Wess, J., R. M. Eglen, et al. (2007). "Muscarinic acetylcholine receptors: mutant mice provide new insights for drug development." Nat Rev Drug Discov 6(9): 721-33.
29. Youdim, K. A., R. Lyons, et al. (2008). "An automated, high-throughput, 384 well Cytochrome P450 cocktail IC50 assay using a rapid resolution LC-MS/MS end-point." J Pharm Biomed Anal 48(1): 92-9.
Protocol
hM4 Muscarinic Acetylcholine Fold Shift Response

Cell line creation and culture of the human M4/Gqi5/CHO line
Human M4 (hM4) cDNA in pcDNA3.1(+) was purchased from www.cDNA.org and stably transfected into Chinese hamster ovary (CHO K1) cells purchased from the ATCC (www.atcc.org). To make a stable hM4 cell line for use in calcium mobilization assays, cells were cotransfected with the chimeric G protein Gqi5 (Conklin et al., 1993). All transfections utilized Lipofectamine 2000. hM4-Gqi5 cells were cultured in Ham's F-12; 10% heat-inactivated fetal bovine serum (FBS), 20mM HEPES, 500 ug/mL G418 (Mediatech, Inc., Herndon, VA), and 500 ug/mL hygromycin B. All cell culture reagents were purchased from Invitrogen Corp. (Carlsbad, CA) unless otherwise noted.

REFERENCES
Conklin, B.R., Farfel, Z., et al. (1993). "Substitution of three amino acids switches receptor specificity of Gq alpha to that of Gi alpha." Nature 20;363(6426):274-6.

Fold-Shift Assay
Assays were performed within the Vanderbilt Center for Neuroscience Drug Discovery's Screening Center. CHO cell lines expressing muscarinic acetylcholine receptors were plated (15,000 cells/20 uL/well) in black-walled, clear-bottomed, TC treated, 384 well plates (Greiner Bio-One, Monroe, North Carolina) in Ham's F-12, 10% FBS, 20 mM HEPES. The cells were grown overnight at 37 degrees C in the presence of 5% CO2. The following day, plated cells had their medium exchanged to Assay Buffer (Hank's balanced salt solution, 20 mM HEPES and 2.5 mM Probenecid (Sigma-Aldrich, St. Louis, MO)) using an ELX405 microplate washer (BioTek), leaving 20 uL/well, followed by addition of with 20 uL of 4.5 uM Fluo-4, AM (Invitrogen, Carlsbad, CA) prepared as a 2.3 mM stock in DMSO and mixed in a 1:1 ratio with 10% (w/v) pluronic acid F-127 and diluted in Assay Buffer for 45 minutes at 37 degrees C. The dye was then exchanged to Assay Buffer using an ELX405, leaving 20 uL/well and the plates were incubated at room temperature for 10 min prior to assay. Test compounds were prepared in Assay Buffer to generate a 2x stock in 0.6% DMSO (0.3% final). Acetylcholine (ACh) concentration responses were prepared at a 5X stock solution in assay buffer prior to addition to assay plates. Calcium mobilization was measured at 37 degrees C using a Functional Drug Screening System 6000 or 7000 (FDSS6000 or FDSS7000, Hamamatsu, Japan) kinetic plate reader according to the following protocol. Cells were preincubated with test compound (or vehicle) for 144 seconds prior to the addition of a concentration response of the agonist ACh and the fluorescence was monitored for a total of 5 min.

Data analysis
The signal amplitude was first normalized to baseline and then as a percentage of the maximal response to acetylcholine. Microsoft XLfit (IDBS, Bridgewater, NJ) was utilized for curve fitting and EC50 value determination using a four point logistical equation. Fold-Shift values were calculated by dividing the ACh EC50 in the presence of 30 uM compound by the ACh EC50 in the presence of vehicle.
Comment
Compounds showing greater than a 3-fold shift were assigned 'Outcome' = 'Active', Fold-Shift='Value', and % ACh max='Value'.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Test Type: In vitro
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Average_Fold_ShiftAverage Fold Shift from replicates 1-3Float
2SD_Average_Fold_ShiftStandard deviation for the average Fold Shift valueFloat
3SEM_Average_Fold_ShiftStandard error mean for the average Fold Shift valueFloat
4Average_%_ACh_MaxAverage Percent Maximum Acetylcholine response from replicates 1-3Float%
5SD_Average_%_ACh_MaxStandard deviation for the average percent maximum Acetylcholine response Float%
6SEM_Average_%_ACh_MaxStandard error mean for the average percent maximum Acetylcholine response Float%
7Average_%_ACh_MinAverage Percent Minimum Acetylcholine response from replicates 1-3Float%
8SD_Average_%_ACh_MinStandard deviation for the average Percent Minimum Acetylcholine response Float%
9SEM_Average_%_ACh_MinStandard error mean for the average Percent Minimum Acetylcholine response Float%
10Average_EC50_uMAverage of potency values from replicates 1-3FloatμM
11SD_Average_EC50_uMStandard deviation for the average potency valueFloatμM
12SEM_Average_EC50_uMStandard error mean for the average potency valueFloatμM
13Fold_Shift_1The EC50 of the acetylcholine response in the presence of compound divided by the EC50 of the acetylcholine response in the presence of vehicle (replicate 1)Float
14Compound_Concentration_uM_1Concentration of compound tested in assay in micromolar (replicate 1)FloatμM
15EC50_uM_1Potency value in micromolar (replicate 1)FloatμM
16EC50_uM_UCL_1Potency Upper Confidence Limit in micromolar (replicate 1)FloatμM
17EC50_uM_LCL_1Potency Lower Confidence Limit in micromolar (replicate 1)FloatμM
18%_ACh_Max_1Percent Maximum Acetylcholine Response (replicate 1)Float%
19%_ACh_Max_UCL_1Percent Maximum Acetylcholine Response Upper Confidence Limit (replicate 1)Float%
20%_ACh_Max_LCL_1Percent Maximum Acetylcholine Response Lower Confidence Limit (replicate 1)Float%
21%_ACh_Min_1Percent Minimum Acetylcholine Response (replicate 1)Float%
22%_ACh_Min_UCL_1Percent Minimum Acetylcholine Response Upper Confidence Limit (replicate 1)Float%
23%_ACh_Min_LCL_1Percent Minimum Acetylcholine Response Lower Confidence Limit (replicate 1)Float%
24Fold_Shift_2The EC50 of the acetylcholine response in the presence of compound divided by the EC50 of the acetylcholine response in the presence of vehicle (replicate 2)Float
25Compound_Concentration_uM_2Concentration of compound tested in assay in micromolar (replicate 2)FloatμM
26EC50_uM_2Potency value in micromolar (replicate 2)FloatμM
27EC50_uM_UCL_2Potency Upper Confidence Limit in micromolar (replicate 2)FloatμM
28EC50_uM_LCL_2Potency Lower Confidence Limit in micromolar (replicate 2)FloatμM
29%_ACh_Max_2Percent Maximum Acetylcholine Response (replicate 2)Float%
30%_ACh_Max_UCL_2Percent Maximum Acetylcholine Response Upper Confidence Limit (replicate 2)Float%
31%_ACh_Max_LCL_2Percent Maximum Acetylcholine Response Lower Confidence Limit (replicate 2)Float%
32%_ACh_Min_2Percent Minimum Acetylcholine Response (replicate 2)Float%
33%_ACh_Min_UCL_2Percent Minimum Acetylcholine Response Upper Confidence Limit (replicate 2)Float%
34%_ACh_Min_LCL_2Percent Minimum Acetylcholine Response Lower Confidence Limit (replicate 2)Float%
35Fold_Shift_3The EC50 of the acetylcholine response in the presence of compound divided by the EC50 of the acetylcholine response in the presence of vehicle (replicate 3)Float
36Compound_Concentration_uM_3Concentration of compound tested in assay in micromolar (replicate 3)FloatμM
37EC50_uM_3Potency value in micromolar (replicate 3)FloatμM
38EC50_uM_UCL_3Potency Upper Confidence Limit in micromolar (replicate 3)FloatμM
39EC50_uM_LCL_3Potency Lower Confidence Limit in micromolar (replicate 3)FloatμM
40%_ACh_Max_3Percent Maximum Acetylcholine Response (replicate 3)Float%
41%_ACh_Max_UCL_3Percent Maximum Acetylcholine Response Upper Confidence Limit (replicate 3)Float%
42%_ACh_Max_LCL_3Percent Maximum Acetylcholine Response Lower Confidence Limit (replicate 3)Float%
43%_ACh_Min_3Percent Minimum Acetylcholine Response (replicate 3)Float%
44%_ACh_Min_UCL_3Percent Minimum Acetylcholine Response Upper Confidence Limit (replicate 3)Float%
45%_ACh_Min_LCL_3Percent Minimum Acetylcholine Response Lower Confidence Limit (replicate 3)Float%
46CategoryWhether the compound is categorized as inactive, a potentiator, or an antagonistString
47Value_at_0.00000093_uM_1 (9.3e-07μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
48Value_at_0.0000075_uM_1 (7.5e-06μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
49Value_at_0.00006_uM_1 (6e-05μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
50Value_at_0.00048_uM_1 (0.00048μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
51Value_at_0.0038_uM_1 (0.0038μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
52Value_at_0.031_uM_1 (0.031μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
53Value_at_0.24_uM_1 (0.24μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
54Value_at_2_uM_1 (2μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
55Value_at_16_uM_1 (16μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
56Value_at_130_uM_1 (130μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
57Value_at_1000_uM_1 (1000μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
58Value_at_0.00000093_uM_2 (9.3e-07μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
59Value_at_0.0000075_uM_2 (7.5e-06μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
60Value_at_0.00006_uM_2 (6e-05μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
61Value_at_0.00048_uM_2 (0.00048μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
62Value_at_0.0038_uM_2 (0.0038μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
63Value_at_0.031_uM_2 (0.031μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
64Value_at_0.24_uM_2 (0.24μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
65Value_at_2_uM_2 (2μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
66Value_at_16_uM_2 (16μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
67Value_at_130_uM_2 (130μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
68Value_at_1000_uM_2 (1000μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
69Value_at_0.00000093_uM_3 (9.3e-07μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
70Value_at_0.0000075_uM_3 (7.5e-06μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
71Value_at_0.00006_uM_3 (6e-05μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
72Value_at_0.00048_uM_3 (0.00048μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
73Value_at_0.0038_uM_3 (0.0038μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
74Value_at_0.031_uM_3 (0.031μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
75Value_at_0.24_uM_3 (0.24μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
76Value_at_2_uM_3 (2μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
77Value_at_16_uM_3 (16μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
78Value_at_130_uM_3 (130μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
79Value_at_1000_uM_3 (1000μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
80Value_at_0.000102_uM_1 (0.000102μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
81Value_at_0.000512_uM_1 (0.000512μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
82Value_at_0.00256_uM_1 (0.00256μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
83Value_at_0.0128_uM_1 (0.0128μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
84Value_at_0.064_uM_1 (0.064μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
85Value_at_0.32_uM_1 (0.32μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
86Value_at_1.6_uM_1 (1.6μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
87Value_at_8_uM_1 (8μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
88Value_at_40_uM_1 (40μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
89Value_at_200_uM_1 (200μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
90Value_at_0.00001_uM_1 (1e-05μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
91Value_at_0.000051_uM_1 (5.1e-05μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
92Value_at_0.000256_uM_1 (0.000256μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
93Value_at_0.00128_uM_1 (0.00128μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
94Value_at_0.0064_uM_1 (0.0064μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
95Value_at_0.032_uM_1 (0.032μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
96Value_at_0.16_uM_1 (0.16μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
97Value_at_0.8_uM_1 (0.8μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
98Value_at_4_uM_1 (4μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
99Value_at_20_uM_1 (20μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
100Value_at_100_uM_1 (100μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float

** Test Concentration.
Additional Information
Grant Number: MH077607-1

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