HTS TR-FRET-based dose response confirmatory assay for Siah-1
Proteasomal degradation typically requires post-translational modification of target proteins with K48-linked polyubiquitin chains. This process of protein proteolysis plays a key role in normal cellular function. The E3 ubiquitin ligase, Siah-1, facilitates the transfer of ubiquitin to its substrate proteins destined for degradation by way of its RING domain. Siah-1 is a member of a family of more ..
BioActive Compounds: 61
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 MH086475-01
Assay Provider: Dr. John C. Reed, Sanford-Burnham Medical Research Institute, San Diego CA
Proteasomal degradation typically requires post-translational modification of target proteins with K48-linked polyubiquitin chains. This process of protein proteolysis plays a key role in normal cellular function. The E3 ubiquitin ligase, Siah-1, facilitates the transfer of ubiquitin to its substrate proteins destined for degradation by way of its RING domain. Siah-1 is a member of a family of highly conserved RING domain proteins, which regulate a variety of cellular functions, including cell cycle arrest, tumor suppression, and apoptosis through the beta-catenin degradation pathway. Siah-1 has also been identified as a p53-inducible gene, functionally linking it to an important tumor suppressor. Chemical modulators of the Siah-1 pathway would provide powerful research tools for elucidating the roles of this signaling pathway in cancer development and progression.
This assay serves as a confirmatory screen for the fluorescence polarization-based primary assay. In this secondary assay, an alternative method of detection, time-resolved fluorescence resonance energy transfer, was utilized as a screen to further verify compound activity.
1) Fluorescein-Plectin and Siah provided by the assay provider
2) Terbium His Antibody was obtained from Invitrogen
3) Assay buffer: 25mM Bis-Tris pH 7.0, 1mM TCEP and 0.005% Tween 20
4) Siah - TB His Antibody Mix: 10 nM Siah, 5 nM Tb His Antibody in Assay Buffer
5) Positive Control: 2.5 nM Tb His Antibody, 5 nM Fl-Plectin in Assay Buffer
6) Fl-Plectin Solution: 10 nM Fl-Plectin
Dose Response Protocol
1) Add 6 uL of Positive Control to Columns 1-2 of a black, opaque, Corning 1536-well plate (Cat#3724) using a Multidrop Combi.
2) Combine in equal parts the Siah - Tb His Antibody Mix with the Fl-Plectin solution.
3) Add 6 uL of Siah - Tb His Antibody/Fl-Plectin solution Mix to Columns 3-48 using a Multidrop Combi.
4) Using a Labcyte Echo, DMSO and test compounds are transferred to wells. DMSO only is transferred to columns 1-3 and 46-48(Control wells), while varying volumes of test compounds are transferred to columns 4-45 to achieve the desired test concentrations. Test compound wells in the assay plate are back-filled with DMSO to equalize final assay concentrations. A total of 60 nL is added to each well of the assay plate.
4) Following a 90 minute incubation at room temperature, in the dark, the plates are read on a BMG Pherastar using an appropriate HTRF protocol.
5) Data analysis was performed using a sigmoidal dose-response equation through non-linear regression.
Compounds with IC50 < 20 uM are considered "active" in this assay. Due to the confirmatory goal of the assay, activity in the assay provides additional evidence that these compounds are true hits and decreases the likelihood that these compounds appear to be active in the primary FP assay(AID 1817) due to fluorescent interference
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 to this assay.
2) Second tier (41-80 range) is reserved for dose-response confirmation data in the primary assay and also does not apply to this assay.
a. Inactive compounds of the confirmatory stage are assigned a score value equal 41.
b. The score is linearly correlated with a compound potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. The score is calculated as:
Score = 44 + 6*(pIC50 - 3),
where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units.
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is applicable in this assay.
Categorized Comment - additional comments and annotations
* Activity Concentration.
Data Table (Concise)