TR-FRET secondary assay for HTS discovery of chemical inhibitors of Hsp70
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
BioActive Compounds: 7
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.
The current assay was developed at the Sanford-Burnham Center for Chemical Genomics (SBCCG), based on Fluorescen-12-ATP binding to GST-Hsp70 in the presence of Terbium-labeled anti-GST antibody. The assay is aimed to support Hsp70 chemical probe identification through the TR-FRET assay confirmation of the results obtained in the primary fluorescence polarization (FP) assay (PubChem AID 583) performed at the BCCG.
Hsp70 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
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) Terbium-labeled anti-GST antibody (Tb-Ab) was purchased from Invitrogen.
4) Assay Buffer: 25 mM Bis-Tris, pH 7.0, 12.5 mM MgCl2, 1 mM DTT, 0.00625% Tween 20.
5) Hsp70/Fluorescein-ATP/Tb-Ab working solution contained 5 nM Hsp70, 12.5 nM fluorescein-12-ATP and 2.5 nM Tb-Ab in assay buffer. Solution was prepared fresh and kept on ice prior to use. Solution was incubated for 1 h at room temperature before dispensing into plates.
6) ADP working solution contained 25 uM ADP prepared fresh from 20 mM stock solution that was kept frozen.
Hsp70 TR-FRET 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 of compounds in 10% DMSO were transferred into columns 3-22 of Greiner 384-well white small-volume plates (784075). Each compound concentration was assayed in duplicate wells.
2) Columns 1-2 contained 4 uL of 25 uM ADP (positive controls). Columns 23-24 contained 4 uL of 10% DMSO (negative controls).
3) 16 uL of Hsp70/Fluorescein-ATP/Tb-Ab working solution was added to the entire plates using WellMate bulk dispenser (Matrix).
4) Plates were incubated for 30 min at room temperature.
5) Fluorescence was measured on an M5 plate reader, Molecular Devices (excitation: 340 nm, emission: 490 and 520 nm, cutoff: 475 and 515 nm, respectively) in Time Resolved (TR) mode with signal integrated for 1 ms after initial delay 0.1 ms and averaged from 5 readings. The TR-FRET signal was calculated as the ratio of TR-Fluorescence (TRF) at 520 nm to TRF at 490 nm.
6) Data analysis was performed using a sigmoidal dose-response equation through non-linear regression.
Compounds interfering with an assay often appear as positives in screening, thus resulting in false positives. False positives, e.g., artifacts are frequently assay-specific. Therefore, reconfirmation of the positive compounds with an independent assay provides an easy way to separate the true positives hits from assay-specific hits.
The compounds identified as primary screening actives in the Hsp70 fluorescence polarization assay (PubChem AID 583) proceed to the dose-response confirmation stage in both FP and TR-FRET assays performed in parallel from the same compound dilution plate. For the purposes of the TR-FRET assay, compounds that demonstrate IC50 values in the range of analyzed concentrations in the assay are defined as 'active' in the outcome column. Concentrations of compounds that demonstrate >2-fold decrease in TRF at 490 nm, corresponding to Terbium emission and Fluorescein excitation, are not included in the data analysis. Potential issues related to compound behavior in the assay, e.g. signal interference or precipitation, are stated in the Comments field.
Compounds that failed dose-response confirmation are defined as 'inactive' in the outcome field.
To take the advantage of the results of the two assays available to us, a special activity ranking system was implemented. This system takes into account IC50 values in both assays to provide clear differentiation between the compounds that are active in a single vs. both assays. Details of the scoring system applied to the Hsp70 project is described below.
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 the correlation of the results between primary and secondary assays. Details of the Scoring System will be published elsewhere.
Briefly, the outline of the scoring system utilized for the Hsp70 TR-FRET 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
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 (nH) 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 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 deviation.
d. The score is correlated with the compound potency in both the FP and the TR-FRET assays through the following function that takes into account the information obtained in both assays:
Score=44 +3*(pIC50(FP)-3)*QC(FP) + 3*(pIC50(TR-FRET)-3)*QC(TR-FRET),
where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units and indexes (FP) and (TR-FRET) refer to the primary and the secondary assays, respectively. This equation results in the Score values above 50 for compounds that demonstrate high potency and predictable behavior in both assays. Compounds that are inactive in either of the assays or whose concentration-dependent behavior is likely to be an artifact of their behavior in the assay will generally have lower score values.
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues)
Categorized Comment - additional comments and annotations
* Activity Concentration.
Data Table (Concise)