SAR analysis for the identification of translation initiation inhibitors (PABP) via an Electrophoretic Mobility Shift Dose Response Assay
Translation is an essential cellular process whose deregulation is associated with alterations in cell growth, cell cycle progression, and cell death responses. The initiation phase of translation is a key target for regulation when cells are exposed to various environmental cues (e.g. insulin, amino acid starvation, mitogenic stimulation, hypoxia, etc). As well, translation initiation control is more ..
BioActive Compounds: 2
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: 1R03MH084835-01
Assay Provider: Jerry Pelletier, Ph.D, McGill University, Montreal, Canada
Translation is an essential cellular process whose deregulation is associated with alterations in cell growth, cell cycle progression, and cell death responses. The initiation phase of translation is a key target for regulation when cells are exposed to various environmental cues (e.g. insulin, amino acid starvation, mitogenic stimulation, hypoxia, etc). As well, translation initiation control is usurped upon viral infection and is deregulated in many human cancers. Over-expression of certain translation factors can lead to malignant transformation and many of the components of the translational apparatus are over-expressed in human cancers. Several tumor suppressor genes directly influence the translation process and recently, chemoresistance in vivo has been linked to deregulated translation initiation. In a transformed setting, where translation can be inhibited by a small molecule modulator (e.g. rapamycin), decreased translation rates are associated with reversal of chemoresistance, possibly by inhibition of pro-survival pathways or resetting of pro-apoptotic program. These results validate translation initiation as a potential chemotherapeutic target.
This fluorescence polarization assay has been developed and performed to confirm hits originally identified in "uHTS fluorescence polarization assay for the identification of translation initiation inhibitors (PABP)" (AID 2014) and to study the structure-activity relationship on analogs of the confirmed hits. Compounds are either acquired from commercial sources or synthesized internally.
This assay identifies small molecules that block translation initiation by targeting an important components of this pathway, poly(A) binding protein (PABP). This protein requires RNA binding to mediate its effects on translation initiation and the screen is designed to find inhibitors that block this process.
The compounds were evaluated using electrophoretic mobility shift assays (EMSA) to visualize PABP RRM1/2:RNA interactions (Galicia-Vazquez et al. 2009). EMSA was performed by incubating PABP GST-RRM1/2 and 32P-labeled polyA15 RNA in the presence of vehicle, cold RNA or 50 uM compound for single point analysis. Dose dependency of the compounds were tested at 100, 50, 10, 5 and 1 uM. Complexes were resolved on 4% polyacrylamide 1% TBE gels and viewed by autoradiography.
Compounds that exhibited dose dependent inhibition inhibited PABP:RNA interactions with an IC50 <= 100 uM are considered active.
IC50 values were estimated from the blot images
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.
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 and is not applicable in this assay.
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues
a. Inactive compounds of the confirmatory stage are assigned a score value equal 81.
b. Active compounds of the confirmatory stage are assigned a score value equal 95.
Categorized Comment - additional comments and annotations
* Activity Concentration.
Data Table (Concise)