In Vitro Hsp70 Dose Response Fluorescence Polarization Assay for SAR Study
This Hsp70 dose response assay is developed and performed for the purpose of SAR study on analogs of hits originally identified in the fluorescence polarization HTS assay for Hsp70 Inhibitors (AID 583). ..more
BioActive Compounds: 31
Sanford-Burnham Center for Chemical Genomics (SBCCG)
Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
NIH Molecular Libraries Screening Centers Network (MLSCN)
MLSCN Grant: XO1 MH079863-01
Assay Provider: Dr. John C. Reed, Sanford-Burnham Medical Research Institute
This Hsp70 dose response assay is developed and performed for the purpose of SAR study on analogs of hits originally identified in the fluorescence polarization HTS assay for Hsp70 Inhibitors (AID 583).
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.
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 dose response 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.
A positive of the assay is defined as a compound with IC50 value in the range of tested concentrations, i.e. below 100 uM.
Activity scoring rules developed at Sanford-Burnham Center for Chemical Genomics were devised to take into consideration compound efficacy, the screening stage of the data and apparent compound behavior in the assay. 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 and therefore is not applicable in this assay.
2) Second tier (41-80 range) is reserved for dose-response confirmation data of the primary hits and therefore is not applicable in this assay.
3) Third tier (81-100 range) is reserved for dry-powder compounds that represent purchased and resynthesized positives and their analogues and utilized for SAR studies.
a. Compounds that failed to reproduce from dry powder or have IC50 > 100 uM are assigned inactive and a score value of 81.
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. 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 a target- or a pathway-specific compound effect vs. its non-specific behavior in the assay. This factor is based on the expectation that a compound with a single mode of action that achieved an equilibrium in the assay would demonstrate the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their divergence.
c. The score is calculated using the following equation:
Score = 82+3*(pIC50-4)*QC,
where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units, and QC is calculated using Hill coefficient as above. This equation results in the Score values above 85 for compounds that demonstrate high potency and predictable behavior in the assays.
Categorized Comment - additional comments and annotations
* Activity Concentration.
Data Table (Concise)