AID	Name	Type	Comment
786	TR-FRET secondary assay for HTS discovery of chemical inhibitors of Hsp70	confirmatory
1072	In Vitro Hsp70 Dose Response Fluorescence Polarization Assay for SAR Study	confirmatory

Sanford-Burnham Center for Chemical Genomics (SBCCG)
Sanford-Burnham Medical Research Institute (San Diego, CA)
NIH Molecular Libraries Screening Centers Network (MLSCN)
MLSCN Grant: XO1 MH079863-01

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 Hsp70 through a fluorescence polarization (FP) assay using Fluorescein-labeled ATP. Additional TR-FRET-based assay was developed and utilized as secondary assay in hit confirmation.

Hsp70 fluorescence polarization assay protocol.

Hsp70 assay materials:
1)Hsp70 protein (GST-tagged 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)Hsp70/Fluorescein-ATP working solution contained 12.5 nM Hsp70 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 10 uM ADP prepared fresh from 20 mM stock solution that was kept frozen.

Hsp70 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 Hsp70/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. 10 nM Hsp70 (columns 1-24)
d. 2 % DMSO (columns 1-24)
e. 20 uM compounds (columns 1-22)
f. 1 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.

Hsp70 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 10 uM ADP (in 10% DMSO) and 10% DMSO, respectively, using WellMate bulk dispenser (Matrix).
2)16 uL of Hsp70 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.

Comments:

This assay was screened through two compound library collections. The first library was the NIH collection, which is supplied to all of the Molecular Libraries Screening Centers. The second library consisted of 50,000 compounds from ChemBridge Corp. The compounds in the ChemBridge collection were screened in mg/ml, which is different from the NIH library which was screened in uM. To designate this difference the comment line in the results table will state the following 'Primary testing at 3.333 ug/ml' for data from the Chembridge set.
Compounds with greater than 50% displacement of Fluorescein-ATP in Hsp70 assay at testing 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 downgraded to 'inactive' in the outcome field. Potential issues related to compound behavior in the assay, e.g. signal interference or precipitation, are stated in the Comments 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 of the two (FP and TR-FRET) assays.
a.Compounds inactive in both assays of the confirmatory stage are assigned a score value equal 41.
b.The score is linearly correlated with a compound's potency in both assays through the following equation:

Score = 44 + 3*(pIC50_FP - 3)* 2.6*[exp(-0.5*nH_FP^2) * exp(-1.5*nH_FP^2)] + 3*(pIC50_TR - 3)* 2.6*[exp(-0.5*nH_TR^2) * exp(-1.5*nH_TR^2)],

where pIC50_FP and pIC50_TR are a negative log(10) of the IC50 (mole/L) values and nH_FP and nH_TR are Hill coefficient values in FP and TR-FRET assays, respectively. 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 both assays.
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues

Grant Number: XO1 MH079863-01