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BioAssay: AID 485281

qHTS Assay for Identification of Novel General Anesthetics

Anesthetic development has remained a largely empirical process. There are growing concerns about the cognitive effects of known general anesthetics [1] and acceleration of the onset of neurodegenerative disease [2]. The compounds' toxicity [3] and multiple specific binding targets in the mammalian brain [4] has made them less attractive as drug leads. In this assay, a GABAergic mimetic model more ..
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 Tested Compounds
 Tested Compounds
All(341509)
 
 
Active(255)
 
 
Inactive(314539)
 
 
Inconclusive(27095)
 
 
 Tested Substances
 Tested Substances
All(346372)
 
 
Active(256)
 
 
Inactive(318898)
 
 
Inconclusive(27218)
 
 
AID: 485281
Data Source: NCGC (APOF102)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-09-27

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 255
Related Experiments
AIDNameTypeComment
2323qHTS Validation Assay for Identification of Novel General AnestheticsConfirmatorydepositor-specified cross reference: Validation apoferritin assay run on SigmaAldrich LOPAC1280 collection
2385Probe Development Summary for Identification of Novel General AnestheticsSummarydepositor-specified cross reference: Probe Development Summary for Identification of Novel General Anesthetics
489008Confirmation Assay for Identification of Novel General AnestheticsConfirmatorydepositor-specified cross reference
624098Assay for General Anesthetic Phenotype in Xenopus TadpolesScreeningdepositor-specified cross reference
624122Isothermal Titration Calorimetry Based Assay for Measurement of Binding Affinity to ApoferritinConfirmatorydepositor-specified cross reference
Description:
Source: NIH Chemical Genomics Center [NCGC]
Assay Submitter: Roderic Eckenhoff, University of Pennsylvania
Screening Center PI: Christopher P. Austin, NIH
Probe Development: NIH Chemical Genomics Center [NCGC]
NIH Grant Number: MH084836-01

Anesthetic development has remained a largely empirical process. There are growing concerns about the cognitive effects of known general anesthetics [1] and acceleration of the onset of neurodegenerative disease [2]. The compounds' toxicity [3] and multiple specific binding targets in the mammalian brain [4] has made them less attractive as drug leads. In this assay, a GABAergic mimetic model system, apoferritin and a profluorescent 1-aminoanthracene ligand (1-AMA), was used to construct a competitive binding assay for identification of novel general anesthetics. The assay was validated for 1-AMA using other known anesthetics, such as isoflurane (CID:3763) and propofol (CID: 4943) [5], [6].

References:

[1] S.L. Bianchi, T. Tran, C. Liu, S. Lin, Y. Li, J.M. Keller, R.G. Eckenhoff, and M.F. Eckenhoff, Brain and behavior changes in 12-month-old Tg2576 and nontransgenic mice exposed to anesthetics. Neurobiol Aging 29 (2008) 1002-10.

[2] Z. Xie, Y. Dong, U. Maeda, R.D. Moir, W. Xia, D.J. Culley, G. Crosby, and R.E. Tanzi, The inhalation anesthetic isoflurane induces a vicious cycle of apoptosis and amyloid beta-protein accumulation. J Neurosci 27 (2007) 1247-54.

[3] P. Honegger, and J.M. Matthieu, Selective toxicity of the general anesthetic propofol for GABAergic neurons in rat brain cell cultures. J Neurosci Res 45 (1996) 631-6.

[4] M.F. Eckenhoff, K. Chan, and R.G. Eckenhoff, Multiple specific binding targets for inhaled anesthetics in the mammalian brain. J Pharmacol Exp Ther 300 (2002) 172-9.

[5] C.A. Butts, J. Xi, G. Brannigan, A.A. Saad, S.P. Venkatachalan, R.A. Pearce, M.L. Klein, R.G. Eckenhoff, and I.J. Dmochowski, Identification of a fluorescent general anesthetic, 1-aminoanthracene. Proc Natl Acad Sci U S A 106 (2009) 6501-6.

[6] W.A. Lea, J. Xi, A. Jadhav, L. Lu, C.P. Austin, A. Simeonov, and R.G. Eckenhoff, A high-throughput approach for identification of novel general anesthetics. PLoS One 4 (2009) e7150.
Protocol
Three uL of reagents (free 1-AMA (50% saturated solution of 1-aminoanthracene
in PBS) in columns 3, 4 as negative control and 1-AMA/apoferritin mixture (10 uM horse-spleen apoferritin in 50% saturated 1-aminoanthracene in PBS) in columns 1, 2, 5-48) was dispensed into 1536-well Greiner black assay plates. Compounds (23 nL) were transferred via Kalypsys pintool equipped with 1,536-pin array (10 nL slotted pins, V&P Scientific, San Diego, CA). Titration of the positive control, propofol, were delivered via pin transfer from separate control plates to the 2nd column of each assay plate. The starting concentration of the control, dissolved in DMSO, was at 160 mM, followed by two-fold dilution points in duplicate, for a total of 16 concentrations. The plates were incubated for 10 min at room temperature, and then read on an EnVision HTS multilabel reader (Perkin-Elmer, Waltham, MA) (end point read, 3 min/plate) using Fura2 BFP excitation filter (380 nm, bandwidth 10 nm) and fluorescein emission filter (535 nm, bandwidth 25 nm) set. A LANCE/DELFIA dichroic mirror with a cutoff wavelength at 400 nm was used to maximize signal accuracy and strength. Vehicle-only plates, with DMSO being pin-transferred to the entire column 5-48 compound area, were inserted uniformly at the beginning and the end of each library in order to monitor for and record any shifts in the background. Activity was computed as the normalized fluorescence response relative to free 1-AMA and 1-AMA/apoferritin complex values. Concentration-effect relationships were derived by using curve fitting algorithms developed in-house (http://ncgc.nih.gov/pub/openhts/). A four parameter Hill equation was fitted to the concentration-response data by minimizing the residual error between the modeled and observed responses.
Comment
Compound Ranking:
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Binding
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
13Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Activity at 0.011 uM (0.011μM**)% Activity at given concentration.Float%
15Activity at 0.025 uM (0.0246μM**)% Activity at given concentration.Float%
16Activity at 0.055 uM (0.0551μM**)% Activity at given concentration.Float%
17Activity at 0.123 uM (0.123μM**)% Activity at given concentration.Float%
18Activity at 0.275 uM (0.275μM**)% Activity at given concentration.Float%
19Activity at 0.639 uM (0.638521μM**)% Activity at given concentration.Float%
20Activity at 0.962 uM (0.961566μM**)% Activity at given concentration.Float%
21Activity at 1.424 uM (1.42364μM**)% Activity at given concentration.Float%
22Activity at 2.758 uM (2.75838μM**)% Activity at given concentration.Float%
23Activity at 3.337 uM (3.33678μM**)% Activity at given concentration.Float%
24Activity at 5.328 uM (5.32752μM**)% Activity at given concentration.Float%
25Activity at 7.591 uM (7.59106μM**)% Activity at given concentration.Float%
26Activity at 15.30 uM (15.2952μM**)% Activity at given concentration.Float%
27Activity at 19.93 uM (19.9309μM**)% Activity at given concentration.Float%
28Activity at 29.26 uM (29.2614μM**)% Activity at given concentration.Float%
29Activity at 43.01 uM (43.0122μM**)% Activity at given concentration.Float%
30Activity at 77.53 uM (77.5311μM**)% Activity at given concentration.Float%
31Activity at 109.6 uM (109.569μM**)% Activity at given concentration.Float%
32Activity at 163.3 uM (163.313μM**)% Activity at given concentration.Float%
33Activity at 253.3 uM (253.333μM**)% Activity at given concentration.Float%
34Activity at 368.6 uM (368.649μM**)% Activity at given concentration.Float%
35Activity at 502.0 uM (502μM**)% Activity at given concentration.Float%
36Compound QCSource of compound QCString

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH084836-01

Data Table (Concise)
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