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BioAssay: AID 568

High Throughput Screening Assay for Hsc70 Inhibitors

Over-expression of molecular chaperones occurs commonly in cancers and provides protection from a wide variety of cellular stresses, both endogenous and iatrogenic. Molecular chaperones also play important roles in maintaining the activity of several signal-transducing proteins and transcriptions factors involved in malignant transformation. The human genome contains nine Hsp70-family genes. These chaperones include Hsp70 and Hsc70, which are commonly over-expressed in cancers and which confer resistance to myriad cellular stresses, including cytotoxic chemotherapy. ..more
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 Tested Compounds
 Tested Compounds
All(82549)
 
 
Active(2)
 
 
Inactive(82547)
 
 
 Tested Substances
 Tested Substances
All(82559)
 
 
Active(2)
 
 
Inactive(82557)
 
 
 Related BioAssays
 Related BioAssays
AID: 568
Data Source: Burnham Center for Chemical Genomics (SDCCG-A012-Hsc70)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2006-12-21
Modify Date: 2010-08-30

Data Table ( Complete ):           View Active Data    View All Data
Targets
BioActive Compounds: 2
Description:
Sanford-Burnham Center for Chemical Genomics (SBCCG)
Sanford-Burnham Medical Research Institute (San Diego, CA)
NIH Molecular Libraries Screening Centers Network (MLSCN)

Over-expression of molecular chaperones occurs commonly in cancers and provides protection from a wide variety of cellular stresses, both endogenous and iatrogenic. Molecular chaperones also play important roles in maintaining the activity of several signal-transducing proteins and transcriptions factors involved in malignant transformation. The human genome contains nine Hsp70-family genes. These chaperones include Hsp70 and Hsc70, which are commonly over-expressed in cancers and which confer resistance to myriad cellular stresses, including cytotoxic chemotherapy.

This work's aim is to identify chemical probes of Hsc70 through a fluorescence polarization assay (FPA) using Fluorescein-labeled ATP.
Hsc70 screening was performed at the Sanford-Burnham Center for Chemical Genomics (SBCCG) as part of the Molecular Library Screening Center Network (MLSCN). XO1 submission MH079863-01, High Throughput Screening Assay for Hsp70 Inhibitors, Assay Provider Dr. John C. Reed, Sanford-Burnham Medical Research Institute, San Diego, CA


Protocol
Hsc70 fluorescence polarization assay protocol.
Hsc70 assay materials:
1) Hsc70 protein (ATPase domain) was provided by Prof. John Reed laboratory (Sanford-Burnham Medical Research Institute, San Diego, CA).
2) Fluorescein-12-ATP (catalogue NEL439001EA) was purchased from PerkinElmer.
3) Assay Buffer: 25 mM Bis-Tris, pH 7.0, 12.5 mM MgCl2, 1 mM DTT, 0.00625% Tween 20.
4) Hsc70/Fluorescein-ATP working solution contained 81.25 nM Hsc70 and 12.5 nM fluorescein-12-ATP in assay buffer. Solution was prepared fresh and kept on ice prior to use.
5) ADP working solution contained 25 uM ADP prepared fresh from 20 mM stock solution that was kept frozen.
Hsc70 HTS protocol:
1) 4 uL of 100 uM compounds in 10% DMSO were dispensed in columns 1-22 of Greiner 384-well black small-volume plates (784076).
2) Column 23 was utilized for negative control samples and added with 4 uL of 10% DMSO using WellMate bulk dispenser (Matrix).
3) Column 24 was utilized for positive control samples and added with 4 uL of ADP working solution using WellMate bulk dispenser (Matrix).
4) 16 uL of Hsc70/Fluorescein-ATP working solution was added to columns 1-24 using WellMate bulk dispenser (Matrix).
5) Final concentrations of the components in the assay were as follows:
a. 20 mM Bis-Tris-HCl, pH 7.0, 10 mM MgCl2, 0.8 mM DTT, 0.005% Tween 20.
b. 10 nM Fluorescein-ATP (columns 1-24)
c. 65 nM Hsc70 (columns 1-24)
d. 2 % DMSO (columns 1-24)
e. 20 uM compounds (columns 1-22)
f. 5 uM ADP (column 24)
6) Plates were incubated for 1h at room temperature protected from direct light.
7) Fluorescence polarization was measured on an Analyst HT plate reader (Molecular Devices, Inc) using fluorescein filters: excitation filter - 485 nM, emission filter 530 nM, dichroic mirror 505 nM. The signal for each well was acquired for 100 ms.
8) Data analysis was performed using CBIS software (ChemInnovations, Inc).
9) Fluorescence intensity of each sample was normalized to the average fluorescence intensity value of the plate negative control wells to calculate F_ratio parameter.
Hsc70 concentration-dependent confirmation protocol:
1) Dose-response curves contained 10 concentrations of compounds obtained using 2-fold serial dilution. Compounds were serially diluted in 100% DMSO, and then diluted with water to 10% final DMSO concentration. 4 uL compounds in 10% DMSO were transferred into columns 3-22 of Greiner 384-well black small-volume plates (784076). Columns 1-2 and 23-24 were added with 4 uL of 25 uM ADP (in 10% DMSO) and 10% DMSO, respectively, using WellMate bulk dispenser (Matrix).
2) 16 uL of Hsc70 working solution was added to columns 1-24 using WellMate bulk dispenser (Matrix).
3) Plates were incubated for 1h at room temperature protected from direct light.
4) Fluorescence polarization was measured on an Analyst HT plate reader (Molecular Devices, Inc) using fluorescein filters: excitation filter - 485 nM, emission filter 530 nM, dichroic mirror 505 nM. The signal for each well was acquired for 100 ms.
5) Data analysis was performed using sigmoidal dose-response equation through non-linear regression.
Comment
Comments:
Compounds with greater than 50% displacement of Fluorescein-ATP in Hsc70 assay at 20 uM concentration and F_ratio parameter less than 1.5 are defined as actives of the primary screening.
The compounds identified as primary screening actives proceed to the dose-response confirmation stage. Concentrations of compounds that demonstrate a total fluorescence increase above the threshold, F_ratio equal 1.5, are not included in the data analysis. Compounds that demonstrate IC50 values in the range of analyzed concentrations remain 'active' in the outcome column. Compounds that failed dose-response confirmation are assigned IC50 values equal to 999 (uM) and downgraded to 'inactive' in the outcome field.
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 Hsp70 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 Hsp70 assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data the score is correlated with % displacement in the assay demonstrated by a compound at 20 uM concentration:
a. If primary % displacement is less than 0%, then the assigned score is 0
b. If primary % displacement is greater than 100%, then the assigned score is 40/(1+(F_ratio - 1)^2)
c. If primary % displacement is between 0% and 100%, then the calculated score is (% Displacement)*0.4/(1+(F_ratio-1)^2).
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 through the following equation:
Score = 44 + 6*(pIC50-3)* 2.6*[exp(-0.5*nH^2) * exp(-1.5*nH^2)]

where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units and nH is Hill coefficient value. Utilization of the Hill coefficient values ensures that compounds with extremely high or low values, that are likely a result of abnormal behavior in the assay, receive lower scores. In general, this equation results in the Score values above 50 for compounds that demonstrate predictable behavior and IC50 < 100 uM in the confirmatory assays.
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Binding
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50*IC50 value determined using sigmoidal dose response equationFloatμM
2St.Err(IC50)Standard Error of IC50 valueFloatμM
3nHHill coefficient determined using sigmoidal dose response equationFloat
4%displacement at 20 uM% displacement of Fluorescein-ATP relative to the controlsFloat
5F_ratioFluorescence intensity normalized to the average fluorescence intensity value of the plate negative contirolsFloat
6Mean HighMean fluorescence polarization signal (ex/em: 485/530) of negative controls in the corresponding plateFloatmP
7STD Deviation HighStandard deviation (n=16) of fluorescence polarization of negative controls in the corresponding plateFloatmP
8Mean LowMean fluorescence polarization signal (ex/em:485/530) of positive controls in the corresponding plateFloatmP
9STD Deviation LowStandard deviation (n=16) of fluorescence polarization of positive controls in the corresponding plateFloatmP

* Activity Concentration.
Additional Information
Grant Number: XO1 MH079863-01

Data Table (Concise)
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