qHTS Validation 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  and acceleration of the onset of neurodegenerative disease . The compounds' toxicity  and multiple specific binding targets in the mammalian brain  has made them less attractive as drug leads. In this assay, a GABAergic mimetic model more ..
BioActive Compounds: 5
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  and acceleration of the onset of neurodegenerative disease . The compounds' toxicity  and multiple specific binding targets in the mammalian brain  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) , .
 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.
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 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.
 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.
 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.
 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.
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.
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
* Activity Concentration. ** Test Concentration.
Data Table (Concise)