Nuclear factor kappa-B (NF-kappa-B) plays an important role in normal B cell development and survival. Diffuse large B cell lymphoma (DLBCL) is the most commonly observed type of non-Hodgkin's lymphoma. Gene expression analysis has identified an activated B cell-like subtype of DLBCL (ABC-DLBCL) which expresses known NF-kappa-B target genes. In ABC-DLBCL cell lines this is due to high more ..
BioActive Compounds: 40
NIH Molecular Libraries Screening Centers Network [MLSCN]
NIH Chemical Genomics Center [NCGC]
NCGC Assay Overview:
Nuclear factor kappa-B (NF-kappa-B) plays an important role in normal B cell development and survival. Diffuse large B cell lymphoma (DLBCL) is the most commonly observed type of non-Hodgkin's lymphoma. Gene expression analysis has identified an activated B cell-like subtype of DLBCL (ABC-DLBCL) which expresses known NF-kappa-B target genes. In ABC-DLBCL cell lines this is due to high constitutive activity of I-kappa-B kinase (IKK), a key regulator of NF-kappa-B. Low molecular weight molecules that inhibit IKK have been shown to be selectively toxic for ABC-DLBCL cell lines. However, targets upstream of IKK have been largely unexplored and so nodes upstream of IKK in the NF-kappa-B pathway remain untested due to the lack of chemical probes.
A cell-sensor assay for I-kappa-B-alpha stabilization was developed with NIH investigator Dr. R. Eric Davis in Dr. Louis M. Staudt's laboratory. The assay uses green (CBG68) and red (CBR) emitting beetle luciferases [Chroma-Glo(TM) developed by Promega Corp.] where the green luciferase is fused to I-kappa-B-alpha and the red luciferase is present in its native state. Both luciferase genes are stably present in the OCI-Ly3 human ABC DLBCL cell line, but their expression is under the control of tetracycline-inducible promoters. Following the addition of doxycycline to induce expression of the luciferases, the green luminescence in control cultures rises only slowly because the CBG fusion partner I-kappa-B-alpha is targeted by IKK activity for rapid degradation. In contrast, green luminescence rises rapidly in the presence of proteasome inhibitors or IKK inhibitors. The rise in red luminescence of CBR is not affected by proteasome or IKK inhibitors, and serves as normalization for cell number and nonspecific effects. Compounds were screened as a concentration-titration series that ranged from 57 uM to 0.7 nM.
NCGC Assay Protocol Summary:
Cells were seeded in 1536-well plates at 5000 cells/3ul in IMDM medium containing 0.5% FBS, w/o phenol red, L-glutamine, 25 mM HEPES, 3 mg/ml sodium bicarbonate. After centrifugation for 1 min. at 1000 rpm, 23 nl of compounds or DMSO were delivered to each well using a pin tool. Then 1ul 80ng/ml doxycycline was dispensed into white solid 1536-well plates and the plates were incubated at 37C/5% C02 in a cell incubator for 4 hrs. Then 4 ul Chroma-Glo luminescent substrate mix (Promega) was added to each well. The plate was incubated at R.T. for 10-15 min. The plates were measured on a ViewLux plate reader for green luminescence (540/20 nm filter) and red luminescence (618/8 nm filter). The green and red luminescent signals were corrected for red and green luminescence filter overlap using previously determined filter calibration constants (determined using the procedure outlined in the Promega Technical Manual). The %Activity was determined from the ratio of corrected green/red luminescent values. Green luminescent and ratio %Activity was determined by normalizing to the difference in signal between basal cells (0% Activity) and cells incubated with 10 uM of the proteasome inhibitor MG-132 (100% Activity). Red luminescent %Activity was normalized to the difference between red luminesence in basal cells (0%Activity) and zero luminescence (-100% Activity).
Concentration-response curves were fitted to the signals arising from the green luminescence (I-kappa-B-alpha-luciferase fusion; IkB-luc) and the red luminescence (unfused luciferase; luc) as well as the calculated ratio. The concentration-effect curves were then classified based on curve quality (r2), response magnitude and degree of measured activity. Compounds were then categorized based on their concentration-response curves for all three datasets (Ikb-luc, luc and ratio). Active compounds showed concentration-dependent increases in the Ikb-luc and ratio data with little or no effect on the luc data. Signal Activators showed concentration-dependent increases in both the Ikb-luc and luc data with either no effect or a modest effect on the ratio data. Signal Inhibitors showed concentration-dependent decreases in either the Ikb-luc or luc datasets with either no effect or a concentration-dependent increase in the ratio data. Inactive compounds showed no effect in all three datasets.
Keywords: IKK, I-kappa-B, IkB, IkBa, cytotoxicity, luminescence, MLSMR, MLSCN, NIH Roadmap, qHTS, NCGC
1. Compounds are first classified as being probable IkB actives (99-50), signal activators (10), signal inhibitors (10) or inactive (0).
2. Within the IkB actives, compounds were ranked by efficacy and potency.
Data Table (Concise)