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BioAssay: AID 602259

Dose-response confirmation of microRNA-mediated mRNA deadenylation inhibitors by fluoresence polarization assay

The regulation of gene expression is a mechanism that allows cells to respond to growth and proliferation stimuli, stress, and nutrient availability. It is managed at multiple levels: mRNA expression, mRNA translation initiation and mRNA decay (1). MicroRNAs (miRNAs) are endogenous small RNAs that post-transcriptionally regulate gene expression to control a wide range of biological processes including cell growth, division and differentiation, as well as metabolism and development. ..more
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 Tested Compounds
 Tested Compounds
All(1186)
 
 
Active(11)
 
 
Inactive(1175)
 
 
 Tested Substances
 Tested Substances
All(1186)
 
 
Active(11)
 
 
Inactive(1175)
 
 
AID: 602259
Data Source: Burnham Center for Chemical Genomics (SBCCG-A779-MLLE-PAM2-DMSO-DR-Assay)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-02-14

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 11
Related Experiments
AIDNameTypeComment
588489uHTS identification of microRNA-mediated mRNA deadenylation inhibitors by fluoresence polarization assayScreeningdepositor-specified cross reference: Primary Screen
588500Summary assay for small molecule inhibitors of microRNA-mediated mRNA deadenylationSummarydepositor-specified cross reference: Summary AID
602260Dose-response secondary confirmation of microRNA-mediated mRNA deadenylation inhibitors by fluoresence polarization assay using Cy5 labeled peptideConfirmatorysame project related to Summary assay
Description:
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Production Centers Network(MLPCN)
Grant Number: R03 MH094198-01
Assay Provider: Kalle Gehring, Ph.D., McGill University

The regulation of gene expression is a mechanism that allows cells to respond to growth and proliferation stimuli, stress, and nutrient availability. It is managed at multiple levels: mRNA expression, mRNA translation initiation and mRNA decay (1). MicroRNAs (miRNAs) are endogenous small RNAs that post-transcriptionally regulate gene expression to control a wide range of biological processes including cell growth, division and differentiation, as well as metabolism and development.

The poly(A) tail of mRNA is bound by several molecules of the poly(A)-binding protein (PABP), an abundant cytoplasmic protein in eukaryotes that promotes translation (2). PABPC1 is a multi-domain protein that contains four phylogenetically conserved RNA recognition motifs (RRM1-4) (3,4) a proline-rich unstructured region and a 70-residue C-terminal domain termed Mlle or PABC (5). Mlle (Mademoiselle) refers to the conserved KITGMLLE signature motif of this domain. The Mlle domain binds proteins with a 12-15 amino acid motif termed PAM2 to regulate PABPC1 involvement in mRNA decay. The goal of this grant is to screen for small molecule modulators of Mlle interactions as chemical probes for the study of PABP regulation in translation and mRNA silencing. This will open new therapeutic opportunities for altering gene expression in cancer, neurodegeneration, and other diseases.

The purpose of this assay is to confirm hits from "uHTS identification of microRNA-mediated mRNA deadenylation inhibitors by fluoresence polarization assay", AID 588489.

REFERENCES
1. Sonenberg N, Hinnebusch AG. (2007) Regulation of Translation Initiation in Eukaryotes: Mechanisms and Biological Targets. Cell 136: 731-745.
2. Kahvejian A, Roy G, Sonenberg N. (2001) The mRNA closed-loop model: the function of PABP and PABP-interacting proteins in mRNA translation. Cold Spring Harbor Symp. Quant. Biol. 66: 293-300.
3. Adam SA, Nakagawa T, Swanson MS, Woodruff TK, Dreyfuss G. (1986) mRNA polyadenylate-binding protein: gene isolation and sequencing and identification of a ribonucleoprotein consensus sequence. Mol. Cell. Biol. 6: 2932-2943.
4. Sachs AB, Davis RW, Kornberg RD. (1987) A single domain of yeast poly(A)-binding protein is necessary and sufficient for RNA binding and cell viability. Mol. Cell. Biol. 7: 3268-3276.
5. Kozlov G, Trempe JF, Khaleghpour K, Kahvejian A, I. Ekiel I, Gehring K. (2001) Structure and function of the C-terminal PABC domain of human poly(A)-binding protein. Proc. Natl Acad. Sci. USA 98: 4409-4413.
Protocol
Assay Materials:
Hepes (Sigma)
NaCl (Sigma)
EDTA (Gibco 15575)
10% Tween 20 (MP biomedicals ICN10316890)
GST-Mlle protein (Assay Provider)
FITC Pam2 peptide (Assay Provider)
Molecular grade water (CellGro 46-000-CM)
Assay plate: Aurora 1536 Black Solid Bottom (00019120BX)

I. Prepare Solutions:
1- Prepare 1Xassay buffer in molecular grade water (20mM Hepes pH 7.4, 150 mM NaCl, 0.5mM EDTA, 0.05% Tween-20)
2- Prepare 2x protein solution (1.35uM) in assay buffer
3- Prepare 2x substrate solution (0.01uM) in assay buffer
II. Compound Addition:
4- Using LabCyte Echo 555, transfer 40 to 5 nL from 10 mM compound source plate for 5-point high dose-response test or transfer 40 to 2.5 nL from 10 mM and 40 nl to 2.5 nl from 0.3125 mM source plate for 10-point full dose-response test into assay plate Columns 5-44 (final concentration of test compounds is 66 - 4.125 uM or 66 - 0.129 uM and 0.67% DMSO), and 40 nL of DMSO to control wells in Columns 1-4 and 45-48.

III. Reagent Addition
5- Dispense 3 uL/well of assay buffer in columns 1-4 and 3ul/well protein solution in columns 5-48 using Bioteck dispenser.
6- Dispense 3 ul/well of FITC-PAM2 substrate into all wells using Biotek dispenser.
7- Spin down plates without lids on Vspin at 1000 rpm for 1 min
8- incubate plates at room temp for 2 hours.

IV. Reading plates:
9- Read the plate on PerkinElmer-EnVision plate reader FP protocol with dual mirror
Comment
Compounds that demonstrated IC50_Mean <= 20 uM are defined as active in this assay.

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

a. Inactive compounds of the confirmatory stage are assigned a score value equal 41.
b. The score is linearly correlated with a compound's 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- or pathway-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 the 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 all the items discussed above is
Score = 44 + 6*(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 50 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.

3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
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. If the 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 a sigmoidal dose response equationFloatμM
3IC50_Qualifier_1_Range1This qualifier is to be used with the next TID, . If the 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 a sigmoidal dose response equationFloatμM
5Std.Err(IC50)_1_Range1Standard Error of the IC50 valueFloatμM
6nH_1_Range1Hill coefficient determined using sigmoidal dose response equationFloat
7Excluded_Points_first_point_Range1Flags to indicate which of the dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
8% Activity at 66 uM_first_point_Range1 (66μM**)% Activity at the test concentrationFloat%
9% Activity at 33 uM_first_point_Range1 (33μM**)% Activity at the test concentrationFloat%
10% Activity at 16.5 uM_first_point_Range1 (16.5μM**)% Activity at the test concentrationFloat%
11% Activity at 8.25 uM_first_point_Range1 (8.25μM**)% Activity at the test concentrationFloat%
12% Activity at 4.125 uM_first_point_Range1 (4.125μM**)% Activity at the test concentrationFloat%
13IC50_Qualifier_2_Range1This qualifier is to be used with the next TID, . If the 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
14IC50_2_Range1IC50 value determined using a sigmoidal dose response equationFloatμM
15Std.Err(IC50)_2_Range1Standard Error of the IC50 valueFloatμM
16nH_2_Range1Hill coefficient determined using sigmoidal dose response equationFloat
17Excluded_Points_second_point_Range1Flags to indicate which of the dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
18% Activity at 66 uM_second_point_Range1 (66μM**)% Activity at the test concentrationFloat%
19% Activity at 33 uM_second_point_Range1 (33μM**)% Activity at the test concentrationFloat%
20% Activity at 16.5 uM_second_point_Range1 (16.5μM**)% Activity at the test concentrationFloat%
21% Activity at 8.25 uM_second_point_Range1 (8.25μM**)% Activity at the test concentrationFloat%
22% Activity at 4.125 uM_second_point_Range1 (4.125μM**)% Activity at the test concentrationFloat%
23IC50_Qualifier_1_Range2This qualifier is to be used with the next TID, . If the 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
24IC50_1_Range2IC50 value determined using a sigmoidal dose response equationFloatμM
25Std.Err(IC50)_1_Range2Standard Error of the IC50 valueFloatμM
26nH_1_Range2Hill coefficient determined using sigmoidal dose response equationFloat
27Excluded_Points_first_point_Range2Flags to indicate which of the dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
28% Activity at 66 uM_first_point_Range2 (66μM**)% Activity at the test concentrationFloat%
29% Activity at 33 uM_first_point_Range2 (33μM**)% Activity at the test concentrationFloat%
30% Activity at 16.5 uM_first_point_Range2 (16.5μM**)% Activity at the test concentrationFloat%
31% Activity at 8.25 uM_first_point_Range2 (8.25μM**)% Activity at the test concentrationFloat%
32% Activity at 4.125 uM_first_point_Range2 (4.125μM**)% Activity at the test concentrationFloat%
33% Activity at 2.0625 uM_first_point_Range2 (2.0625μM**)% Activity at the test concentrationFloat%
34% Activity at 1.03125 uM_first_point_Range2 (1.03125μM**)% Activity at the test concentrationFloat%
35% Activity at 0.515625 uM_first_point_Range2 (0.515625μM**)% Activity at the test concentrationFloat%
36% Activity at 0.2578125 uM_first_point_Range2 (0.257812μM**)% Activity at the test concentrationFloat%
37% Activity at 0.1289062 uM_first_point_Range2 (0.128906μM**)% Activity at the test concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: R03 MH094198-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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