Dose Response confirmation of uHTS hits for a small molecule Caspase-8 TRAIL sensitizers in a luminescence panel assay
Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) is of great potential use as an anticancer therapeutic; however its use as a potential therapeutic agent has been limited by resistance. TRAIL selectively induces apoptosis in cancer cells whilst normal cells are refractory . TRAIL is safe when administered in vivo and shows none of the toxicity usually associated with other more ..
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego CA)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Number: 1 R03 MH095562-01A1
Assay Provider: Kristiina Vuori, M.D., Ph.D. Sanford Burnham Medical Research Institute
Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) is of great potential use as an anticancer therapeutic; however its use as a potential therapeutic agent has been limited by resistance. TRAIL selectively induces apoptosis in cancer cells whilst normal cells are refractory . TRAIL is safe when administered in vivo and shows none of the toxicity usually associated with other members of the TNF superfamily . TRAIL binds to Death Receptor (DR)-4 or -5 resulting in recruitment of adapter molecules and caspase-8. This recruitment and clustering results in procaspase-8 dimerization, activation, processing and release of active caspase-8 from the complex. Caspase-8, then, activates the effector caspases resulting in classical apoptotic cell death (Fig. 1; ) DR4 and DR5 are widely expressed on cancer cells and it has been suggested that this may be one reason for TRAIL's selective anti-tumor properties . However, a full understanding of the mechanisms of TRAIL selectivity remains elusive.
As TRAIL resistance is the major preclusion to its clinical use, we envisaged a novel high-throughput screen (HTS) to identify sensitizing agents. As non-specificity and toxicity are a major drawback with conventional chemotherapeutics, we propose to discover novel agents acting specifically through the caspase-8 axis as opposed to generalized toxicity. Caspase-8 is an apical protease involved in the "extrinsic" or death receptor mediated form of cell death and, as such, would seem to be an ideal candidate for silencing or deletion in cancers. Surprisingly, however, the protein is rarely silenced or absent in cancers, making it an extremely attractive therapeutic intervention point [1, 5, 6]. Caspase-8 has been shown to be involved in various non-apoptotic and potentially pro-tumorigenic processes, such as increased cell motility and metastasis, promotion of growth factor signaling and associations with Rab5 and focal adhesions [1, 6, 7, 8, 9]. Utilization of the caspase-8 null neuroblastoma cell line, NB7, and the same line reconstituted with endogenous levels of wild-type caspase-8  in parallel allows the identification of agents that act specifically via the caspase-8 apoptotic pathway, as the ability to directly compare TRAIL killing ability in cells with the CASP8 gene deleted (NB7) as opposed to those engineered to re-express the protein (NB7+Caspase-8). Thus generally toxic agents can easily be dismissed whilst probes sensitizing to TRAIL induced apoptosis via the caspase-8 axis should be readily apparent. The system is particularly powerful in that it specifically detects agents acting on any molecule or process, known or unknown that relies on the caspase-8 specific apoptotic axis. Additionally, the use of the two cell lines in parallel will allow easy identification of false positives that induce nonspecific cell death.
This assay is a follow-up to "uHTS identification of Caspase-8 TRAIL sensitizers in a luminesence assay BioAssay Summary", AID 624354. Compounds were tested in the primary Caspase-8 assay with the TRAIL ligand for 24hrs. Compounds considered active in this assay were further tested in the same assay system but without the TRAIL ligand. Both assays were employed to test selectivity of compounds for the TRAIL ligand. Compounds active in the assay without TRAIL are considered non-selective or artifacts of the system and are not desired.
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§ Panel component ID.
NB7+C8 Cells (Assay Provider Lab)
TRAIL peptide 100 ug (Calbiochem 616374)
RPMI 1640 medium (with Phenol Red) (Cellgro/Mediatech)
RPMI 1640 medium (without Phenol Red) (Cellgro/Mediatech)
Fetal Bovine Serum (Hyclone)
Penicillin/Streptomycin solution (Invitrogen)
Primocin (50 mg/mL stock) (Invivogen)
ATPlite 1Step 1000mL Kit (Perkin Elmer)
T225 Tissue Culture flasks (Corning)
Trypsin/EDTA 0.25% (Invitrogen)
Sterile Cell Strainer, 40 um Nylon Mesh (Fisherbrand)
1536-well white solid bottom TC-treated plate (Nexus Biosystems)
1 - Using Labcyte Echo, predispense MG-132 at a final concentration of 10 uM in assay plates column 2
2 -Using Thermo Combi, seed 500 cells/well in 4 uL/well to columns 2-48 plate to white 1536 TC-treated white solid bottom plate (Aurora #00019846).
3 -Using Combi, Add 4 uL of Assay media in column 1.
4 -Spin plates @ 500 RPM for 1 min, lid plate, and then lid plates with Kalypsys metal lids.
5 -Incubate plate overnight in 37 oC 5% CO2 incubator.
6 - After overnight incubation, dispense 5 and 2.5 nL of 10 mM, 2.5 mM and 0.625 mM source plates for 6 point dose-response of resupplied liquid stock and backfill with 100% DMSO to plates with Echo. Add 5 nL of DMSO controls (col 1-4). (final concentration Is 12.5 uM and 0.1% DMSO Final)
7 - Lid plates with Kalypsys metal lids, and incubate them for 4 hours in 37 oC 5% CO2 incubator.
8 - Using Beckman Coulter Bioraptr, dispense 1 uL of 100 ng/mL (5X) TRAIL (or assay media only for assay without TRAIL)in assay media (20 ng/mL final) to entire plate.
Spin plates @ 500 RPM for 1 min, lid plate, and then lid plates with Kalypsys metal lids.
9 - Incubate plate overnight in 37 oC 5% CO2 incubator.
10 - Add 3 ul/well PerkinElmer ATPlite to all wells with Bioraptor.
11 - Spin plates @ 2000 RPM for 2 min, lid plate and incubate for 10 min at RT.
12 - Read luminescence on PerkinElmer Viewlux Luminescent protocol.
Compounds that demonstrated EC50 activity of < 12.5 uM in the assay with TRAIL and demonstrated EC50 > 12.5 uM in the assay without TRAIL are defined as sensitizers of the reaction.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the assay is described below.
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data and is not applicable in this assay.
2) Second tier (41-80 range) is reserved for dose-response confirmation data
a. Inactive compounds of the confirmatory stage are assigned a score value equal 41.
b. The score is linearly correlated with a compound's potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. The Hill coefficient is taken as a measure of compound behavior in the assay via an additional scaling factor QC:
QC = 2.6*[exp(-0.5*nH^2) - exp(-1.5*nH^2)]
This empirical factor prorates the likelihood of target- or pathway-specific compound effect vs. its non-specific behavior in the assay. This factor is based on expectation that a compound with a single mode of action that achieved equilibrium in the assay demonstrates the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their deviation.
d. Summary equation that takes into account all the items discussed above is
Score = 44 + 6*(pIC50-3)*QC,
Where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units. This equation results in the Score values above 50 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior are likely to be an artifact of that assay will generally have lower Score values.
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay.
* Activity Concentration. ** Test Concentration. § Panel component ID.