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BioAssay: AID 435011

SAR analysis for the identification of translation initiation inhibitors (eIF4H)

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 ..
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 Tested Compounds
 Tested Compounds
All(86)
 
 
Active(43)
 
 
Inactive(43)
 
 
 Tested Substances
 Tested Substances
All(86)
 
 
Active(43)
 
 
Inactive(43)
 
 
AID: 435011
Data Source: Burnham Center for Chemical Genomics (BCCG-A380-eIF4H-DryPowder-Assay)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-06-22
Hold-until Date: 2010-10-26
Modify Date: 2011-01-14

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 43
Depositor Specified Assays
AIDNameTypeComment
2012uHTS fluorescence polarization assay for the identification of translation initiation inhibitors (eIF4H)confirmatory
2014uHTS fluorescence polarization assay for the identification of translation initiation inhibitors (PABP)confirmatory
2028Summary assay for the identification of translation initiation inhibitors (eIF4H)summary
Description:
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.
Protocol
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.
Comment
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.
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50_Mean_QualifierThis qualifier is to be used with the next TID, IC50_Mean. If qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
2IC50_Mean*IC50 value determined using sigmoidal dose response equationFloatμM
3IC50_Qualifier_1_Range1This qualifier is to be used with the next TID, IC50_1_Range1. If qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
4IC50_1_Range1IC50 value determined using sigmoidal dose response equationFloatμM
5Std.Err(IC50)_1_Range1Standard Error of IC50 valueFloatμM
6nH_1_Range1Hill coefficient determined using sigmoidal dose response equationFloat
7Excluded_Points_first_point_Range1Flags to indicate which of the first dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
8% inhibition at 100 uM_first_point_Range1 (100μM**)% Inhibition at a given concentrationFloat%
9% inhibition at 50 uM_first_point_Range1 (50μM**)% Inhibition at a given concentrationFloat%
10% inhibition at 25 uM_first_point_Range1 (25μM**)% Inhibition at a given concentrationFloat%
11% inhibition at 12.5 uM_first_point_Range1 (12.5μM**)% Inhibition at a given concentrationFloat%
12% inhibition at 6.25 uM_first_point_Range1 (6.25μM**)% Inhibition at a given concentrationFloat%
13% inhibition at 3.125 uM_first_point_Range1 (3.125μM**)% Inhibition at a given concentrationFloat%
14% inhibition at 1.5625 uM_first_point_Range1 (1.5625μM**)% Inhibition at a given concentrationFloat%
15% inhibition at 0.78125 uM_first_point_Range1 (0.78125μM**)% Inhibition at a given concentrationFloat%
16% inhibition at 0.390625 uM_first_point_Range1 (0.390625μM**)% Inhibition at a given concentrationFloat%
17% inhibition at 0.1953125 uM_first_point_Range1 (0.195312μM**)% Inhibition at a given concentrationFloat%
18% inhibition at 0.09765625 uM_first_point_Range1 (0.0976562μM**)% Inhibition at a given concentrationFloat%
19% inhibition at 0.04882812 uM_first_point_Range1 (0.0488281μM**)% Inhibition at a given concentrationFloat%
20Excluded_Points_second_point_Range1Flags to indicate which of the second dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
21% inhibition at 100 uM_second_point_Range1 (100μM**)% Inhibition at a given concentrationFloat%
22% inhibition at 50 uM_second_point_Range1 (50μM**)% Inhibition at a given concentrationFloat%
23% inhibition at 25 uM_second_point_Range1 (25μM**)% Inhibition at a given concentrationFloat%
24% inhibition at 12.5 uM_second_point_Range1 (12.5μM**)% Inhibition at a given concentrationFloat%
25% inhibition at 6.25 uM_second_point_Range1 (6.25μM**)% Inhibition at a given concentrationFloat%
26% inhibition at 3.125 uM_second_point_Range1 (3.125μM**)% Inhibition at a given concentrationFloat%
27% inhibition at 1.5625 uM_second_point_Range1 (1.5625μM**)% Inhibition at a given concentrationFloat%
28% inhibition at 0.78125 uM_second_point_Range1 (0.78125μM**)% Inhibition at a given concentrationFloat%
29% inhibition at 0.390625 uM_second_point_Range1 (0.390625μM**)% Inhibition at a given concentrationFloat%
30% inhibition at 0.1953125 uM_second_point_Range1 (0.195312μM**)% Inhibition at a given concentrationFloat%
31% inhibition at 0.09765625 uM_second_point_Range1 (0.0976562μM**)% Inhibition at a given concentrationFloat%
32% inhibition at 0.04882812 uM_second_point_Range1 (0.0488281μM**)% Inhibition at a given concentrationFloat%
33IC50_Qualifier_2_Range1This qualifier is to be used with the next TID, IC50_2_Range1. If qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
34IC50_2_Range1IC50 value determined using sigmoidal dose response equationFloatμM
35Std.Err(IC50)_2_Range1Standard Error of IC50 valueFloatμM
36nH_2_Range1Hill coefficient determined using sigmoidal dose response equationFloat
37Excluded_Points_third_point_Range1Flags to indicate which of the third dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
38% inhibition at 100 uM_third_point_Range1 (100μM**)% Inhibition at a given concentrationFloat%
39% inhibition at 50 uM_third_point_Range1 (50μM**)% Inhibition at a given concentrationFloat%
40% inhibition at 25 uM_third_point_Range1 (25μM**)% Inhibition at a given concentrationFloat%
41% inhibition at 12.5 uM_third_point_Range1 (12.5μM**)% Inhibition at a given concentrationFloat%
42% inhibition at 6.25 uM_third_point_Range1 (6.25μM**)% Inhibition at a given concentrationFloat%
43% inhibition at 3.125 uM_third_point_Range1 (3.125μM**)% Inhibition at a given concentrationFloat%
44% inhibition at 1.5625 uM_third_point_Range1 (1.5625μM**)% Inhibition at a given concentrationFloat%
45% inhibition at 0.78125 uM_third_point_Range1 (0.78125μM**)% Inhibition at a given concentrationFloat%
46% inhibition at 0.390625 uM_third_point_Range1 (0.390625μM**)% Inhibition at a given concentrationFloat%
47% inhibition at 0.1953125 uM_third_point_Range1 (0.195312μM**)% Inhibition at a given concentrationFloat%
48% inhibition at 0.09765625 uM_third_point_Range1 (0.0976562μM**)% Inhibition at a given concentrationFloat%
49% inhibition at 0.04882812 uM_third_point_Range1 (0.0488281μM**)% Inhibition at a given concentrationFloat%
50Excluded_Points_fourth_point_Range1Flags to indicate which of the fourth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
51% inhibition at 100 uM_fourth_point_Range1 (100μM**)% Inhibition at a given concentrationFloat%
52% inhibition at 50 uM_fourth_point_Range1 (50μM**)% Inhibition at a given concentrationFloat%
53% inhibition at 25 uM_fourth_point_Range1 (25μM**)% Inhibition at a given concentrationFloat%
54% inhibition at 12.5 uM_fourth_point_Range1 (12.5μM**)% Inhibition at a given concentrationFloat%
55% inhibition at 6.25 uM_fourth_point_Range1 (6.25μM**)% Inhibition at a given concentrationFloat%
56% inhibition at 3.125 uM_fourth_point_Range1 (3.125μM**)% Inhibition at a given concentrationFloat%
57% inhibition at 1.5625 uM_fourth_point_Range1 (1.5625μM**)% Inhibition at a given concentrationFloat%
58% inhibition at 0.78125 uM_fourth_point_Range1 (0.78125μM**)% Inhibition at a given concentrationFloat%
59% inhibition at 0.390625 uM_fourth_point_Range1 (0.390625μM**)% Inhibition at a given concentrationFloat%
60% inhibition at 0.1953125 uM_fourth_point_Range1 (0.195312μM**)% Inhibition at a given concentrationFloat%
61% inhibition at 0.09765625 uM_fourth_point_Range1 (0.0976562μM**)% Inhibition at a given concentrationFloat%
62% inhibition at 0.04882812 uM_fourth_point_Range1 (0.0488281μM**)% Inhibition at a given concentrationFloat%
63IC50_Qualifier_3_Range1This qualifier is to be used with the next TID, IC50_3_Range1. If qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
64IC50_3_Range1IC50 value determined using sigmoidal dose response equationFloatμM
65Std.Err(IC50)_3_Range1Standard Error of IC50 valueFloatμM
66nH_3_Range1Hill coefficient determined using sigmoidal dose response equationFloat
67Excluded_Points_fifth_point_Range1Flags to indicate which of the fifth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
68% inhibition at 100 uM_fifth_point_Range1 (100μM**)% Inhibition at a given concentrationFloat%
69% inhibition at 50 uM_fifth_point_Range1 (50μM**)% Inhibition at a given concentrationFloat%
70% inhibition at 25 uM_fifth_point_Range1 (25μM**)% Inhibition at a given concentrationFloat%
71% inhibition at 12.5 uM_fifth_point_Range1 (12.5μM**)% Inhibition at a given concentrationFloat%
72% inhibition at 6.25 uM_fifth_point_Range1 (6.25μM**)% Inhibition at a given concentrationFloat%
73% inhibition at 3.125 uM_fifth_point_Range1 (3.125μM**)% Inhibition at a given concentrationFloat%
74% inhibition at 1.5625 uM_fifth_point_Range1 (1.5625μM**)% Inhibition at a given concentrationFloat%
75% inhibition at 0.78125 uM_fifth_point_Range1 (0.78125μM**)% Inhibition at a given concentrationFloat%
76% inhibition at 0.390625 uM_fifth_point_Range1 (0.390625μM**)% Inhibition at a given concentrationFloat%
77% inhibition at 0.1953125 uM_fifth_point_Range1 (0.195312μM**)% Inhibition at a given concentrationFloat%
78% inhibition at 0.09765625 uM_fifth_point_Range1 (0.0976562μM**)% Inhibition at a given concentrationFloat%
79% inhibition at 0.04882812 uM_fifth_point_Range1 (0.0488281μM**)% Inhibition at a given concentrationFloat%
80Excluded_Points_sixth_point_Range1Flags to indicate which of the sixth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
81% inhibition at 100 uM_sixth_point_Range1 (100μM**)% Inhibition at a given concentrationFloat%
82% inhibition at 50 uM_sixth_point_Range1 (50μM**)% Inhibition at a given concentrationFloat%
83% inhibition at 25 uM_sixth_point_Range1 (25μM**)% Inhibition at a given concentrationFloat%
84% inhibition at 12.5 uM_sixth_point_Range1 (12.5μM**)% Inhibition at a given concentrationFloat%
85% inhibition at 6.25 uM_sixth_point_Range1 (6.25μM**)% Inhibition at a given concentrationFloat%
86% inhibition at 3.125 uM_sixth_point_Range1 (3.125μM**)% Inhibition at a given concentrationFloat%
87% inhibition at 1.5625 uM_sixth_point_Range1 (1.5625μM**)% Inhibition at a given concentrationFloat%
88% inhibition at 0.78125 uM_sixth_point_Range1 (0.78125μM**)% Inhibition at a given concentrationFloat%
89% inhibition at 0.390625 uM_sixth_point_Range1 (0.390625μM**)% Inhibition at a given concentrationFloat%
90% inhibition at 0.1953125 uM_sixth_point_Range1 (0.195312μM**)% Inhibition at a given concentrationFloat%
91% inhibition at 0.09765625 uM_sixth_point_Range1 (0.0976562μM**)% Inhibition at a given concentrationFloat%
92% inhibition at 0.04882812 uM_sixth_point_Range1 (0.0488281μM**)% Inhibition at a given concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1R03MH084835-01

Data Table (Concise)
Classification
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