Dose-response secondary confirmation of microRNA-mediated mRNA deadenylation inhibitors by fluoresence polarization assay using Cy5 labeled peptide
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 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 Production Centers Network(MLPCN)
Grant Number: R03 MH094198-01
Assay Provider: Kalle Gehring, Ph.D., McGill University
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. This Cy5 assay is an orthogonal assay to the FITC assay and done to eliminate false positives due to the interference at the FITC wavelength
The purpose of this assay is to confirm hits from "uHTS identification of microRNA-mediated mRNA deadenylation inhibitors by fluoresence polarization assay." AID 588489
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.
EDTA (Gibco 15575)
10% Tween 20 (MP biomedicals ICN10316890)
GST-Mlle protein (Assay Provider)
Cy5 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 (20 mM Hepes pH 7.4, 150 mM NaCl, 0.5 mM EDTA, 0.05% Tween-20)
2- Prepare 2x protein solution (1.35 uM) in assay buffer
3- Prepare 2x substrate solution (0.01 uM) in assay buffer
II. Compound Addition:
4- Using LabCyte Echo 555, transfer 40-5 nL from 10 mM compound source plate for 5-point high dose-response test or transfer 40-2.5 nL from 10 mM and 40 nl -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 66.6 - 4.2 uM or 66.6 - 0.130 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 3 ul/well protein solution in columns 5-48 using Bioteck dispenser.
6- Dispense 3 ul/well of Cy5-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 (620/688S/688P) with dual mirror
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 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
* Activity Concentration. ** Test Concentration.
Data Table (Concise)