AID	Name	Type	Comment
1471	qHTS Multiplex Assay to Identify Dual Action Probes in a Cell Model of Huntington: Cytoprotection (ATP)	confirmatory	Primary screen for cytoprotection (ATP).
1688	qHTS Multiplex Assay to Identify Dual Action Probes in a Cell Model of Huntington: Aggregate Formation (GFP)	confirmatory	Primary screen for aggregate formation (GFP).
2669	qHTS Assay Multiplex Screening to Identify Dual Action Probes in a Cell Model of Huntington: Cytoprotection (Protease relase)	confirmatory	Alternate primary screen for cytoprotection (protease release).
2672	qHTS Assay Multiplex Screening to Identify Dual Action Probes in a Cell Model of Huntington: Cytoprotection in HTT Q25 expressing cells (ATP)	confirmatory	Counterscreen in q25 cells (ATP).
2673	qHTS Multiplex Assay to Identify Dual Action Probes in a Cell Model of Huntington: Cytoprotection confirmation (ATP)	confirmatory	Confirmation screen for cytoprotection (ATP).
2713	qHTS Multiplex Assay to Identify Dual Action Probes in a Cell Model of Huntington: Cytotoxicity in HTT Striatal Cells	other	Cytotoxicity in HTT striatal cells.

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).

Please refer to other AIDs (1471, 1688, 2669, 2672, 2673, 2713) for detailed assay protocols.

This summary is written for the purposes of summarizing the probe activities from the project. MLSCN probes are given a score of 100. Molecules in the prior art are given a score of 80. Other, less active molecules in the same chemical series as the probe molecules are given a score of 50. Inactive analogues from these series are given a score of 0. ML168 (SID 26752274) has been declared as a probe on this project.

Grant Number: R03MH084839-01A1