HTS fluorescence polarization-based dose response confirmatory screen for the Siah-1 primary assay utilizing an alternative fluorophore, fluorescein-labeled plectin
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: 8
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 which utilized a rhodamine-labeled ligand. In this secondary assay, an alternatively labeled fluorophore, fluorescein-plectin, was utilized as a screen to further verify compound activity.
1) Fluorescein-Plectin and Siah provided by the assay provider
2) Assay buffer: 25mM Bis-Tris pH 7.0, 1mM TCEP and 0.005% Tween 20
3) Siah/Fl-Plectin Assay mix: 0.005uM Fl-Plectin, 0.016uM SIAH in Assay Buffer
Dose Response Protocol
1) Add 6 uL of Fl-Plectin (Positive Control) to Columns 1-2 of a black, opaque, Corning 1536-well plate (Cat#3724) using a Multidrop Combi.
2) Add 6 uL of Siah/ Fl-Plectin Assay Mix to Columns 3-48 using a Multidrop Combi.
3) 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 1 hr incubation at room temperature, in the dark, the plates are read on a BMG Pherastar using Fluorescein FP filters.
Compounds with EC50 < 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
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 applicable in this assay.
Categorized Comment - additional comments and annotations
* Activity Concentration.
Data Table (Concise)