|uHTS for Small Molecule Inhibitors of Eukaryotic Translation Initiation - BioAssay Summary
complex) to the mRNA during translation initiation is highly regulated by eukaryotic initiation factor (eIF) 4F. This complex consists of three subunits: (i) eIF4E, the cap-binding protein responsible for binding of the complex to the mRNA cap structure; (ii) eIF4A, an RNA helicase thought to be required to unwind 5' proximal local mRNA secondary structure to facilitate access of the 43S more ..
BioActive Compounds: 799
Depositor Specified Assays
Screening for Small Molecule Inhibitors of Eukaryotic Translation Initiation
NIH Molecular Libraries Screening Centers Network [MLSCN]
Emory Chemical Biology Discovery Center in MLSCN
Assay provider: Dr. Jerry Pelletier, McGill UNIVERSITY
MLSCN Grant: 1 R03 MH081216-01
Title: uHTS for Small Molecule Inhibitors of Eukaryotic Translation Initiation
The recruitment of the 40S ribosomal subunit and associated factors (43S pre-initiation
complex) to the mRNA during translation initiation is highly regulated by eukaryotic initiation factor (eIF) 4F. This complex consists of three subunits: (i) eIF4E, the cap-binding protein responsible for binding of the complex to the mRNA cap structure; (ii) eIF4A, an RNA helicase thought to be required to unwind 5' proximal local mRNA secondary structure to facilitate access of the 43S ribosomal complex to the mRNA template; and (iii) eIF4G, a modular scaffold that mediates mRNA binding to the 43S pre-initiation complex. eIF4E is thought to be the least abundant translation initiation factor and consequently the mRNA recruitment step is generally the rate-limiting
step of translation (1). This phase of translation is the subject of regulation by signaling pathways, including the Akt/TOR axis, and often becomes deregulated during transformation. The aim of this screening is to identify small molecules that impair the ribosome recruitment phase of eukaryotic translation initiation by targeting the interaction between eIF4E and eIF4G. Compounds that directly target eIF4E:eIF4G association are likely to be more selective in downregulating translation initiation than rapamycin, since TORC1 (TOR Complex I) is implicated in a number of cellular processes, including transcriptional and translational regulation, ribosome biogenesis, and autophagy.
To screen small molecules that selectively inhibit the ribosome recruitment phase of cap-dependent protein synthesis, a time resolved fluorescence resonance energy transfer (TR -FRET) based HTS assay has been developed to monitor the association between the eIF4E and eIF4G subunits of eIF4F. The assay is in a homogenous format and has been optimized for ultra-high throughput screening (uHTS) in a 1536-well format. eIF4E with a 6xHis-tag was indirectly labeled with a europium chelate through a europium conjugated anti-6XHis antibody. GST-tagged eIF4G was indirectly labeled with a SureLight(tm) Allophycocyanin (APC)-anti-GST antibody. Europium and APC comprise a fluorescence energy transfer pair. Interaction of eIF4E and eIF4G brings the two conjugated fluorophores into proximity, leading to an energy transfer from europium to APC and the generation of FRET signal. The FRET signal is detected in an Envision plate reader (Ex 340 nM, Em615 nM and Em665 nM) and expressed as FRET signal ratio (F665nm / F615nm * 10000). The assay is robust with a consistent Z' factor of 0.6-0.8 in a 1536-well plate format and is used for the screening.
1. Assay buffer: 20 mM Tris buffer, pH 7.5, 50 mM NaCl, and 0.01% NP40
2. A GST-tagged fragment of eIF4GI (GST-eIF4GI (517-606))
3. 6xHis- tagged eIF4E protein
4. Europium Chelate Anti-6xHis-Antibody: LANCE Eu-W1024 (His-Eu) from Perkin Elmer Life Sciences.
5. SureLight(tm) Allophycocyanin (APC)- anti-GST antibody (GST-APC) from Perkin Elmer Life Sciences.
1. Make assay reaction buffer for HTS that contains recombinant GST- eIF4GI (517-606) protein (100 nM, final concentration), His-eIF4E (4.8 nM, final concentration), His-Eu (1 nM, final concentration) and GST-APC (50 nM, final concentration).
2. Dispense 4.5 uL of assay reaction buffer to 1536-well black assay plate (Costar 3724).
3. Add 0.1 uL of library compound (1 mM in DMSO) to each well and incubate plates at room temperature for 2 hr. Final concentration of each compound in each well is 21.7 uM.
4. Record FRET signal with an Envision plate reader (Perkin Elmer Life Sciences). An excitation filter at 340 nm and emission filters at 615 nm and 665 nm are used with a LANCE/DELFIA Dual mirror.
1. FRET signals are expressed as FRET ratios:
FRET = F665 nm / F615 nm * 10000
F665 nm: Fluorescence counts at 665 nm emission (units: cps)
F620 nm: Fluorescence counts at 615 nm emission (units: cps)
2. Assay data are analyzed using BioAssay software from CambridgeSoft. Percentage of inhibition is calculated with the following equation based on normalized data from each plate.
Normalized FRET signal = ((FRET compound well - FRET background) (compound added)) / ((FRET compound well - FRET background) (no compound added))
% of activity = (Normalized FRET signal from compound well / average normalized FRET signal from control wells) * 100
% of inhibition = 100 - % of activity
Where FRET compound well is the FRET ratio from a well with a test compound, FRET background is an average FRET signal from wells with His-eIF4E, His-Eu and GST-APC only; FRET control is an average FRET ratio from wells containing GST- eIF4GI517-606 protein, His-eIF4E, His-Eu and GST-APC that defines maximum FRET.
3. Compounds that cause > 30% inhibition are defined as active.
The PubChem Activity Score is calculated by rounding the Pct Inhibition to 0 decimal places. Negative scores are changed to 0, and scores higher than 100 are changed to 100.
1. Artifacts of this assay could result from, but are not limited to, intrinsic fluorescence of some compounds, compounds that can quench europium or APC fluorescence, dust or lint. These can be easily eliminated by looking at the raw counts at Em615nm and Em665nm.
2. "Possible fluorescence artifact" compounds were labeled in the data submitted to PubChem.
3. All data reported were normalized on a per-plate basis.
4. One human and one mouse protein were used. The mouse and human eIF4E proteins are 99% identical and the human eIF4GI and mouse eIF4GI fragments are 93% identical. As well, the interaction domain between the two proteins is 100% conserved.
Data Table (Concise)