AID	Name	Type	Comment
2012	uHTS fluorescence polarization assay for the identification of translation initiation inhibitors (eIF4H)	confirmatory
2014	uHTS fluorescence polarization assay for the identification of translation initiation inhibitors (PABP)	confirmatory
2028	Summary assay for the identification of translation initiation inhibitors (eIF4H)	summary

Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBIMR, 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.

A fluorescence polarization HTS assay has been developed to identify small molecules that block translation initiation by targeting an important components of this pathway the eukaryotic initiation factor 4H (eIF4H). This protein requires RNA binding to mediate its effects on translation initiation and the screen is designed to find inhibitors that block this process.

This dose response 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.

HTS Assay Protocol

Assay materials:
1) His6-eIF4H (Obtained from Assay Provider)
2) 5'-fluorescein-labeled poly(U)10 (Purchased from Dharmacon)
3) Assay Buffer: 5 mM Hepes pH 7.5, 0.005 % Tween-20

Procedure:
1. Dispense 2 ul of assay buffer into columns 1 and 2 of a black, Corning (#2725) 1536 well assay plate.
2. Add 2 ul of eIF4H protein, 30 nM final concentration into columns 3-48.
3. Add 2 ul of 5'-fluorescein-labeled poly(A)10 2.5 nM final concentration into columns 1-48.
4. Using a HighRes biosolutions pintool dispense 70 nl of 2 mM compounds in DMSO to columns 5-48.
5. Using a HighRes biosolutions pintool dispense 70 nl of DMSO to columns 1-4.
6. Read plate on a BMG PHERAstar at 485/520/520nm in Fluorescence Polarization mode.
i. Positioning delay = 0.0
ii. Flashes/well = 10
7. Data analysis was performed using CBIS software (ChemInnovations, Inc).
8. Fluorescence intensity of each sample was normalized to the average fluorescence intensity value of the plate negative control wells to calculate F_ratio parameter.

Dose Response Protocol

Assay materials:
1) His6-eIF4H (Obtained from Assay Provider)
2) 5'-fluorescein-labeled poly(U)10 (Purchased from Dharmacon)
3) Assay Buffer: 5 mM Hepes pH 7.5, 0.005 % Tween-20

Procedure:
1. Using a Labcyte Echo, DMSO and test compounds are transferred to wells of a black, Corning 1536 well assay plate. DMSO only is transferred to columns 1-4 (Control wells), while varying volumes of test compounds are transferred to columns 5-48 to achieve the desired test concentrations. Compounds are transferred from a 2 mM stock to give the stated final concentration. Test compound wells in the assay plate are back-filled with DMSO to equalize final assay concentrations.
2. After compounds have been added, dispense 2 ul of assay buffer into columns 1 and 2 of a black, Corning (#2725) 1536 well assay plate.
3. Add 2 ul of eIF4H protein, 30 nM final concentration into columns 3-48.
4. Add 2 ul of 5'-fluorescein-labeled poly(A)10 2.5 nM final concentration into columns 1-48.
5. Read plate on a BMG PHERAstar at 485/520/520nm in Fluorescence Polarization mode.
i. Positioning delay = 0.0
ii. Flashes/well = 10
6. Data analysis was performed using CBIS software (ChemInnovations, Inc).
7. Fluorescence intensity of each sample was normalized to the average fluorescence intensity value of the plate negative control wells to calculate F_ratio parameter.

Compounds with an IC50 < 100 uM are considered 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.

Activity Scoring
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 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. 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 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-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 this 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 the items discussed above is
Score = 82 + 3*(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 85 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.

Grant Number: 1R03MH084835-01