Name: High Throughput Screen to Identify Compounds that increase expression of NF-kB in Human Neuronal Cells - Dose Response
Award: R03 MH082367-01, Submitted by Dr. Maurizio Grimaldi (Neuropharmacology Laboratory, Southern Research Institute) ..more
BioActive Compounds: 842
Southern Research Molecular Libraries Screening Center (SRMLSC)
Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Screening Centers Network (MLSCN)
Award: R03 MH082367-01, Submitted by Dr. Maurizio Grimaldi (Neuropharmacology Laboratory, Southern Research Institute)
The pharmacological treatment of neurodegenerative disorders has been a disappointment when compared to the successes obtained in stroke, other neurological diseases like seizures, and in mental health diseases. It has to be said that the pathogenesis of neurodegenerative disorders and their early diagnosis represent a definite obstacle to effective intervention.
Nuclear factor kB (NF-kB) is a key cellular signaling factor in the central nervous system. Although NF-kB signaling pathways have been extensively investigated in cancer and in immunological diseases, NF-kB role in the central nervous system physiology and pathology in non inflammatory disorders of the brain is still unclear. NF-kB has an important role as an inhibitor of neuronal apoptosis and at least in this capacity it represents an interesting target to pursue. Recent evidence has also pointed out that this signaling system is involved in the resilience of neurons and in their ability to survive disparate insults. Functional knock out models of NF-kB, achieved via over-expression of its natural repressor I-kB, have shown that neurons from these animals are more sensitive to insults including trauma, Beta-amyloid toxicity and excitotoxicity. Also a direct neuroprotective effect from both non apoptotic and apoptotic models of neurodegeneration has been attributed to direct or indirect activation of NF-kB signaling. In addition, to the direct neuroprotective effect NF-kB up-regulation could also be of value in improving learning and memory. In fact, NF-kB signaling has been shown to improve long term potentiation and long term depression, two models of learning at cellular level. This background is of great appeal because up regulation of NF-kB in neurons could be useful in attacking, at the same time, neuronal degeneration and deterioration of neuronal functions associated with loss of learning and memory as seen in Alzheimer's disease. To pursue this scope, we have set up a novel human cell-based assay to identify small molecules able to up-regulate NF-kB expression for high throughput screening.
The cell line developed for this assay, which is used to identify pharmacological probes for the activation of NF-kB, was prepared as follows. The SH-5YSY human neuroblastoma cell line was doubly stably transfected with a reporter construct coding the human NF-kB promoter driving the expression of the firefly luciferase and a reporter coding the resistance to the antibiotic blasticidin. TNF-alpha was used as a positive control as it a potent enhancer of NF-kB expression. Negative control wells were treated with vehicle only.
Cell Culture: SH-5YSY (ATCC) cells stably transfected with a reporter construct containing the human NF-kB promoter fused upstream of a firefly luciferase reporter and blasticidin resistance (clone C1) were subcultured every 3-4 days in Dulbecco's Modified Eagle Medium (D-MEM) high glucose (1X) with L-glutamine and sodium pyruvate + 10% FBS + 3 ug/mL of blasticidin, and incubated at 37C in 5% CO2, and maintained at a subconfluent density of no more than 240,000 cells/cm2 for less than 30 passages.
Cell Culture Reagents used:
1. D-MEM high glucose (1X), liquid, with L-glutamine and sodium pyruvate, Invitrogen: 11995-065
2. Blasticidin S HCl Invitrogen: R210-01
Compound and Control Preparation:
Prior to cell addition, control drugs were diluted in assay media and added to each 384 well plate at a final concentration of 5 ng/mL for TNF-alpha (positive control). DMSO concentration of 0.3% was maintained for all control wells. Test compounds were serially diluted in a plate-to-plate matrix or "stacked plate" dose response format. All 320 compounds in a source plate were diluted together through the 10-plate matrix resulting in a 10-point dose response dilution series. It can be visualized as a serial dilution series proceeding vertically through a stack of plates with the high dose plate on top and the low dose plate on the bottom with the final compound concentration ranging from 0.0586 to 30 uM and a final DMSO concentration of 0.3% for all concentrations).
1. Add 5,000 cells/well in 20 uL with a Matrix WellMate
2. Centrifuge briefly at 1,000 RPM.
3. Incubate at RT for 30 min
4. Incubate at 37C, 5% CO2 for at least 20-26 h
5. Equilibrate Bright Glo (Promega, Inc) to room temp
6. Equilibrate assay plates to room temperature for 30 min.
7. Add 25 uL of Bright Glo into each well with a ThermoFisher MultiDrop
8. Incubate for 5 min at room temperature
9. Read plates Luminescence 0.1 s integration time on a PerkinElmer Envision
Data Analysis: Thirty two control wells containing cells only and 32 wells containing cells and control drug were included on each assay plate and used to calculate Z' factor for each plate and to normalize the data on a per plate basis.
Concentration response curves were calculated for each substance, each assessing fold induction of cell growth over the cell control (FI = compound well luminescence/median cell control luminescence).
An EC50 were calculated for each substance using the 4 parameter Levenburg-Marquardt algorithm with parameter A locked at 0. Standard deviation, normalized chi2 and hill slope were used to evaluate the curves. Values were extrapolated to assist in identification of weakly active compounds.
Possible artifacts in this assay include, but are not limited to, compounds that interfere with the luciferase reaction, absorb luminescence, or precipitate.
The criteria for determining compound activity are based on EC50 values. Compounds with EC50 values returned in the tested dose range (up to 30 uM) were labeled as "Active", others were considered "Inactive". Substances demonstrating activity in the tested range have been scored using a tiered range of 40-80 based on their relative activity. Substances that did not confirm activity received a score of 0
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)