Quantitative High-Throughput Multiplex Assay to Identify Dual Action Probes in a Cell Model of Huntington: Summary
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. ..more
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.
Categorized Comment - additional comments and annotations
Assay Cell Type: PC-12
Assay Type: Functional
From MLP Probe Report:
Probe count: 1
MLP Probe ML# for probe 1: ML168
PubChem Substance ID (SID) for probe 1: 89650053
PubChem Compound ID (CID) for probe 1: 2432214
Probe type for probe 1: Inhibitor
IC50/EC50 (nM) for probe 1: 7940
Target for probe 1: Q103HTT induced cytotoxicity (gi: 90903231)
Disease relevance for probe 1: Huntington's disease
Anti-target for probe 1: Q25HTT induced cytotoxicity
Fold selectivity for probe 1: >10
NCBI Book chapter link for probe 1: http://www.ncbi.nlm.nih.gov/books/NBK56233/ (ID: 2509616)
Grant number for probe 1: MH084839-01A1
NCBI Book chapter title for probe 1: Identification of compounds which inhibit cytotoxicity associated with mutant Huntingtin protein expression
* Activity Concentration.
Data Table (Concise)