SAR analysis for the identification of translation initiation inhibitors (eIF4H) via a crosslinking 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 ..
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 cross-linking assay is developed and performed to confirm hits originally identified in "uHTS fluorescence polarization assay for the identification of translation initiation inhibitors (eIF4H)" (AID 2012) and to study the structure-activity relationship on analogs of the confirmed hits. Compounds are either acquired from commercial sources or synthesized internally.
The secondary screen to visualize eIF4H:RNA interaction was based on a UV approach in which translation initiation factors were incubated with mRNA that was specifically radiolabelled 2 nucleotides from the cap structure (Lindqvist et al. 2008). RNA-protein complexes are covalently crosslinked with UV light, the mRNA degraded, and the proteins resolved by SDS-PAGE. Proteins crosslinked in the vicinity of the cap structure are visualized by autoradiography, the canonical initiation factor, eIF4H is easily visualized by this approach. Specificity of crosslinking to the cap structure is assessed by performing parallel incubations in the presence of the cap analogue, m7GDP, the non-specific nucleotide GDP (lanes 3), and omitting ATP. The behavior of eIF4H under these conditions has been previously reported (Lindqvist et al. 2008) and allows us to confidently assign the radiolabelled band to the appropriate eIF.
Compounds that inhibited cap-dependent eIF4H:RNA interactions at 100 uM compared to the DMSO control.
None of the compounds were found to be active
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.
** Test Concentration.
Data Table (Concise)