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 ..
BioActive Compounds: 823
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.
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
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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)