qHTS Assay for NPC1 Promoter Activators
Niemann Pick Type C (NPC) is a rare neurodegenerative lipidosis that is characterized by lipid storage in the endosomal/lysosomal system. Treatment modalities for this devastating disease are currently non-existent due to the severe obstacles associated with accessing the central nervous system with proteins or genes. The majority of mutations causing NPC disease are missense mutations that are more ..
BioActive Compounds: 7576
Depositor Specified Assays
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Production centers Network [MLPCN]
MLPCN Grant: MH089375-01A1
Assay Submitter (PI): Yiannis A. Ioannou
NCGC Assay Overview:
Niemann Pick Type C (NPC) is a rare neurodegenerative lipidosis that is characterized by lipid storage in the endosomal/lysosomal system. Treatment modalities for this devastating disease are currently non-existent due to the severe obstacles associated with accessing the central nervous system with proteins or genes. The majority of mutations causing NPC disease are missense mutations that are distributed throughout the length of the NPC1 protein, with the most prevalent being the I1061T allele. Although NPC1 mutant proteins may be functional, they are typically trapped in the ER due to misfolding. Furthermore, over-expression of some mutant NPC1 proteins can rescue the disease phenotype, suggesting that up-regulation of the endogenous NPC1 mutant protein is a new drug treatment modality of the disorder.
We have developed and optimized a cell based luciferase reporter assay in 1536 well format for the identification of up-regulators of the mutant NPC1 promoter.
The NPC1 promoter is cloned in front of the luciferase reporter gene, therefore NPC promoter activation results in luciferase expression. Frozen stock of the NPC1 promoter transfected Huh7 cells were obtained from the laboratory of Dr. Yiannis Ioannou (The Mount Sinai School of Medicine, New York, NY). Cells were propagated and maintained at NCGC in medium containing RPMI-1640 supplemented with 10% FBS, 2mM L-glutamine, and 50ug/ml penicillin/streptomycin. Cells were harvested in and plated for assays in OP-MEM I reduced-serum medium supplemented with 10% FBS. The Amplite luciferase reporter gene assay kit was purchased from ABD Bioquest and prepared/stored according to manufacturers recommendations.
qHTS Assay Setup:
1.#Dispensed 5ul of Huh7 cells stably expressing NPC1-luciferase at a density of 1500 cells per well 1536-well plates.
2.#Incubate cells at 37C, 5% CO2, and 95% humidity for 2 hours.
3.#Transferred 23nl per well of compound in DMSO solution.
4.#Incubated at 37C, 5% CO2, and 95% humidity for 24 hours.
5.#Dispensed 3ul per well of Amplite reagent(ABD Bioquest) per well.
6.#Incubate at ambient for 10 minutes.
7.#Read using the luminescence protocol on the ViewLux plate reader (Perkin Elmer).
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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)