qHTS Assay for Rab9 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 (Ioannou, 2000). We have developed a new paradigm, termed "Orphan Receptor more ..
BioActive Compounds: 9134
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Production centers Network [MLPCN]
MLPCN Grant: MH089537-01
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 (Ioannou, 2000). We have developed a new paradigm, termed "Orphan Receptor Bypass Therapy" (ORByT), to address these disorders. This paradigm posits the existence of endogenous "suppressor" proteins whose expression can dramatically improve LSD pathogenesis. The goal is the identification/discovery of small chemical compounds that can modulate the expression of these proteins and provide a new treatment modality for these devastating disorders. One such protein whose overexpression reverses aspects of the NPC phenotype is Rab9.
We have developed and optimized a cell based luciferase reporter assay in 1536 well format for the identification of up-regulators of the Rab9 promoter.
The Rab9 promoter is cloned in front of the luciferase reporter gene, therefore NPC promoter activation results in luciferase expression. Frozen stock of the Rab9 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 Rab9-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 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)