S16 Schwann cell viability assay (CellTiter-Glo assay)
Charcot-Marie-Tooth (CMT) disease was first characterized by Jean-Martin Charcot and Pierre Marie in France and, independently, by Howard Henry Tooth in England in 1886. CMT is one of the most common inherited neurological diseases, affecting approximately 1 in 2,500 Americans. CMT patients typically exhibit muscle atrophy in the extremities and sensory loss. The most prevalent type of CMT is more ..
BioActive Compounds: 105
National Center for Advancing Translational Sciences [NCATS]
Charcot-Marie-Tooth (CMT) disease was first characterized by Jean-Martin Charcot and Pierre Marie in France and, independently, by Howard Henry Tooth in England in 1886. CMT is one of the most common inherited neurological diseases, affecting approximately 1 in 2,500 Americans. CMT patients typically exhibit muscle atrophy in the extremities and sensory loss. The most prevalent type of CMT is known as CMT1A and caused by a duplication of the PMP22 gene which leads to over-expression of this protein. PMP22 is a glycosylated intrinsic membrane protein accounting for 2-5% of the myelin protein content, and its over-expression results in demyelination and subsequently axonal loss.
A pair of cellular transcription-based assays was developed in the S16 rat Schwann cell lines (Hai, M., et al., 2002) stably transfected with orthogonal reporters - firefly luciferase (Fluc) and beta-lactamase (beta-Lac) - whose expression is driven by an intronic regulatory element of the PMP22 gene (Jones, E.A., et al., 2011) to recapitulate endogenous expression of PMP22. A comprehensive library of approved drugs, the NCGC Pharmaceutical Collection (Huang, R., et al., 2011), was screened in the cross-validating platform of the orthogonal reporter assays to identify drug modulators inhibitory towards PMP22 expression.
A set of counter-screens was devised to address non-specific activity of the library. An ATP-dependent CellTiter-Glo assay (developed by Promega Corp) was utilized to control cell viability. In addition, biochemical Fluc enzyme assay was performed to assess direct inhibition of the Fluc enzymatic activity.
Hai, M., Muja, N., DeVries, G.H., Quarles, R.H. and Patel, P.I. Comparative analysis of Schwann cell lines as model systems for myelin gene transcription studies. J Neurosci Res, 2002. 69(4): pp 497-508
Huang, R., Southall, N., Wang, Y., Yasgar A., Shinn P., Jadhav, A. , Nguyen, D.-T. and Austin, C.P., The NCGC pharmaceutical collection: a comprehensive resource of clinically approved drugs enabling repurposing and chemical genomics. Sci Transl Med, 2011. 3(80): p. 80ps16.
Native S16 cells were seeded in white solid-bottom 1536 well plates at between 500-750 cells/6uL in DMEM medium containing 10% FBS, w/o phenol red. After overnight incubation at 37 degree Celsius/5% C02, 23 nL of compounds or DMSO were delivered to each well using a pin tool, followed by 24h-incubation at 37 degree Celsius/5% C02. Then one volume of CellTiter-Glo assay reagent (Promega) was added to each well for cell viability measured by a ViewLux plate reader (PerkinElmer). The % activity was determined by normalizing to the average readings from wells containing no cells relative to DMSO-treated cells (0% Activity). The concentration-effect curves were then classified based on curve quality (r2), response magnitude and degree of measured activity.
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, toxic compounds are ranked higher than non-toxic compounds.
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. Toxic 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.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)