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BioAssay: AID 624263

A Quantitative High throughput Screen to Identify Chemical Modulators of PINK1 Expression

The overall goal of this project is to develop new tools for understanding and treating the mitochondrial basis for neurodegenerative disease such as Parkinson's disease (PD). The PINK1 campaign was developed from recent data pointing towards mitochondrial dysfunction as a major contributor to dopaminergic neuron loss. The discovery of PINK1/Parkin mediated mitophagy has highlighted the more ..
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 Tested Compounds
 Tested Compounds
All(406275)
 
 
Active(823)
 
 
Inactive(396196)
 
 
Inconclusive(9362)
 
 
 Tested Substances
 Tested Substances
All(410116)
 
 
Active(825)
 
 
Inactive(399906)
 
 
Inconclusive(9385)
 
 
AID: 624263
Data Source: NCGC (PINK1PRI-Acumen)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-06-05

Data Table ( Complete ):           Active    All
Targets
BioActive Compounds: 823
Depositor Specified Assays
AIDNameTypeComment
624272A Quantitative High throughput Screen to Identify Chemical Modulators of PINK1 Expression: Summarysummary
Description:
The overall goal of this project is to develop new tools for understanding and treating the mitochondrial basis for neurodegenerative disease such as Parkinson's disease (PD). The PINK1 campaign was developed from recent data pointing towards mitochondrial dysfunction as a major contributor to dopaminergic neuron loss. The discovery of PINK1/Parkin mediated mitophagy has highlighted the potential for mitochondrial quality control as a novel target for therapeutic intervention. Mitophagy has been shown to selectively eliminate damaged mitochondria from the cell and enhancing mitophagy on a genetic level is correlated with better outcomes in fly/rodent models of PD. A second aim of this project is the development of chemical probes to study the mechanics of mitochondrial quality control. As the PINK1/Parkin mitophagy pathway is a relative recent discovery, no specific chemical probes are available that modulate the process. Presently, only crude tools that cause nonspecific mitochondrial damage are available. To advance the understanding of mitophagy, specific, well characterized chemicals that target various steps of mitophagy regulation need to be developed. New chemical probes may have the ability to dynamically manipulate mitophagy in model organisms such as rodents and zebrafish, advancing our understanding of the process in development and disease. This HTS campaign seeks to discover an activator of PINK1 expression by modulation at the level of post-translational processing.

The PINK1 assay determines the amount and localization of PINK1 protein in a cell using a combination of immunofluorescence and cell staining. The goal of the assay is to determine small molecules that can increase the expression of Pink1 on or inside the cell's mitochondria (ideally without mitochondrial depolarization) using a stable cell line. Since PINK1 protein is rapidly degraded in un-perturbed cells, compounds that induce the enhancement of PINK1 protein expression are identified by determining PINK1 immunofluorescence in conjunction with a mitochondrial, cell, and nuclear marker post fixation. To facilitate rapid high content screening, the primary assay is run in a decision-based HTS mode where compound titration data collected by laser-scanning microplate cytometers is processed and only promising compound series are passed off for high content drill down.
Protocol
1 Add cells to black, clear-bottom, low-base 1536-well plates 6 uL (250 cells/well)
2 Incubate in TC incubator (12 hours)
3 Pin compounds or DMSO controls 23 nL (5 concentrations per compound set)
4 Dispense CCCP to control columns
5 Incubate in TC incubator 24 hours
6 Aspirate media and fix cells (5 uL of fixative containing nuclear dye)
7 Incubate 15 minutes at RT
8 Wash in PBSTween (12 uL/well)
9 Wash in PBSTriton (12 uL/well)
10 Aspirate wash and add Blocking buffer (4 uL/well)
11 Incubate 1 hour at RT
12 Aspirate block and add primary antibodies 3.5 (uL/well)
13 Incubate 12 hours at 10 degrees C
14 Wash in PBSTween-20 two times (12 uL/well)
15 Aspirate wash and add secondary antibodies (12 uL/well)
16 Incubate 1 hour at RT
17 Wash in PBSTween (12 uL/well)
18 Wash in PBSTween with cell stain (12 uL/well)
19 Read plates on Acumen EX3 and determine Wells2Cells coordinates
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 activators are ranked higher than compounds that showed apparent inhibition.
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.
Result Definitions
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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
5Activity_ScoreActivity score.Integer
6Curve_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
7Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
8Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
9Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
10Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
11Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
12Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
13Excluded_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
14Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
15Activity at 0.0004900000 uM (0.00049μM**)% Activity at given concentration.Float%
16Activity at 0.00245 uM (0.00245μM**)% Activity at given concentration.Float%
17Activity at 0.012 uM (0.0123μM**)% Activity at given concentration.Float%
18Activity at 0.062 uM (0.0619386μM**)% Activity at given concentration.Float%
19Activity at 0.153 uM (0.153μM**)% Activity at given concentration.Float%
20Activity at 0.307 uM (0.306912μM**)% Activity at given concentration.Float%
21Activity at 0.422 uM (0.42192μM**)% Activity at given concentration.Float%
22Activity at 0.623 uM (0.623076μM**)% Activity at given concentration.Float%
23Activity at 1.530 uM (1.52953μM**)% Activity at given concentration.Float%
24Activity at 2.033 uM (2.03269μM**)% Activity at given concentration.Float%
25Activity at 3.120 uM (3.12009μM**)% Activity at given concentration.Float%
26Activity at 6.692 uM (6.69153μM**)% Activity at given concentration.Float%
27Activity at 7.670 uM (7.66977μM**)% Activity at given concentration.Float%
28Activity at 15.71 uM (15.7093μM**)% Activity at given concentration.Float%
29Activity at 30.63 uM (30.6299μM**)% Activity at given concentration.Float%
30Activity at 38.34 uM (38.3432μM**)% Activity at given concentration.Float%
31Activity at 76.59 uM (76.5875μM**)% Activity at given concentration.Float%
32Activity at 107.9 uM (107.894μM**)% Activity at given concentration.Float%
33Activity at 153.0 uM (153μM**)% Activity at given concentration.Float%
34Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH095599-01

Data Table (Concise)
Classification
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