AID	Name	Type	Probe	Comment
1482	Quantitative High-Throughput Multiplex Assay to Identify Dual Action Probes in a Cell Model of Huntington: Summary	summary	2
2672	qHTS Assay Multiplex Screening to Identify Dual Action Probes in a Cell Model of Huntington: Cytoprotection in HTT Q25 expressing cells (ATP)	confirmatory
2669	qHTS Assay Multiplex Screening to Identify Dual Action Probes in a Cell Model of Huntington: Cytoprotection (Protease relase)	confirmatory
1688	qHTS Multiplex Assay to Identify Dual Action Probes in a Cell Model of Huntington: Aggregate Formation (GFP)	confirmatory
2713	qHTS Multiplex Assay to Identify Dual Action Probes in a Cell Model of Huntington: Cytotoxicity in HTT Striatal Cells	other
2673	qHTS Multiplex Assay to Identify Dual Action Probes in a Cell Model of Huntington: Cytoprotection confirmation (ATP)	confirmatory

NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Production centers Network [MLPCN]

MLPCN Grant: R03MH084839-01A1
Assay Submitter (PI): Wei Zheng

NCGC Assay Overview:

Huntington's disease is a neurodegenerative disorder caused by a trinucleotide repeat expansion in Exon 1 of the Huntingtin gene. The CAG trinucleotide encodes glutamine and polyglutamine expansions cause cell death in selective areas of the brain. Huntington polyglutamine repeats have the tendency to form aggregates which are observable in later stages of the disease. The exact nature of the aggregates, whether toxic, protective, or insignificant, is unclear.

A stable PC12 cell line containing a gene fusion of Exon 1 of the Huntingtin gene linked to GFP under the control of the inducible ecdysone promoter were used as the cell-based model of Huntington Disease for high throughput screening. Exon 1 of the Huntingtin gene contained an expansion of 103 polyglutamines (Q103) which, when expressed, caused cell death and distinct, bright GFP aggregates. The amount of cell death and the size and intensity of GFP aggregates increased over time and induction level. The cell death were quantified with a measurement of ATP content in the cells. A maximal 40-50% of cell death was observed when the Huntington gene was induced in this cell line.

We optimized this assay in a homogeneous 1536 well format. A quantitative high throughput screen (qHTS) was conducted on the PC12 cells which were induced to express the toxic fusion construct. Small molecules which prevented cell death were identified by using a luminescent ATP quantification method (Perkin Elmer's ATPlite).

PC12 cells stably containing a construct with an ecdysone inducible promoter expressing GFP fused to the exon 1 of HTTQ103 (Huntington protein which containing a 103 ployglutamine expansion) were used.

NCGC Assay Protocol Summary:
Sequence, Parameter, Description
1, Reagent, 5 uL 1500 cells/well 1536 TC treated White clear bottom plate
2, Compound, 23 nL Tebufenozide inducer (100 nM final) to all columns
3, Compound, 23 nL 40 uM to 0.5 nM Libraries
4, Time, 40 hours at 37C
5, Reagent, 5 ul ATPLite cell death assay reagent
6, Detector, 4 sec integration with a Viewlux Luminescent

Media consisted of phenol red free DMEM + 5% equine serum (Hyclone # SH30072.03 ), 5% supplemented Bovine Calf Serum (Hyclone # SH30074.03 ), 1x pen/strep, no G418
Control columns: 1 = DMSO, 2= 2 uM teb dilns (1:1), 4 = 100 nM Teb inducer. Sample columns received 100 nM Teb.

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.

Grant Number: qHTS Multiplex Assay to Identify Dual Action Probes in a Cell Model of Huntington: Cytoprotection (ATP)