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BioAssay: AID 595

qHTS Assay for Disrupters of an Hsp90 Co-Chaperone Interaction

Hsp90 (heat shock protein 90) is the essential molecular chaperone and it accounts for 1-2% of all cytosolic proteins and is critical for the activity of diverse cellular proteins that are involved in a variety of cellular processes, including development, cell cycle, and steroid hormone signaling. Its client proteins include signaling kinases such as IGF1R, Akt, v-Src, Raf-1; regulators of cell more ..
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 Tested Compounds
 Tested Compounds
All(69666)
 
 
Active(300)
 
 
Inactive(66220)
 
 
Inconclusive(3224)
 
 
 Tested Substances
 Tested Substances
All(70699)
 
 
Active(309)
 
 
Inactive(67150)
 
 
Inconclusive(3240)
 
 
 Related BioAssays
 Related BioAssays
AID: 595
Data Source: NCGC (HSPT775)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-02-15
Modify Date: 2007-10-19

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 300
Related Experiments
AIDNameTypeComment
1400Quantitative High-Throughput Screen for Disrupters of an Hsp90 Co-Chaperone Interaction: SummarySummarydepositor-specified cross reference
632Confirmation Concentration-Response Assay and Counterscreen for Disrupters of an Hsp90 Co-Chaperone InteractionConfirmatorysame project related to Summary assay
Description:
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Screening Centers Network [MLSCN]

NCGC Assay Overview:
Hsp90 (heat shock protein 90) is the essential molecular chaperone and it accounts for 1-2% of all cytosolic proteins and is critical for the activity of diverse cellular proteins that are involved in a variety of cellular processes, including development, cell cycle, and steroid hormone signaling. Its client proteins include signaling kinases such as IGF1R, Akt, v-Src, Raf-1; regulators of cell survival such as Bcr-Abl; and transcription factors such as mutated p53, estrogen and progesterone receptors. Hsp90 has emerged as an important target for cancer treatment due to its essential role in maintaining transformation and regulating the function of oncogenic signaling proteins.
The in vivo function of Hsp90 is regulated by the binding and hydrolysis of ATP at its N-terminal. Geldanamycin binds to ATP site and inhibit Hsp90 function. Geldanamycin and its analogues are under clinical trials in cancer patients.
Recent data indicate the C terminal domain of Hsp90 as an important regulator as the N terminus and may represent a second site for pharmacological intervention in chaperone function. This domain contains the binding site for tetratricopeptide repeat (TPR) domains on HOP (Hsp Organizing Protein). TPR is a structural motif used by a significant number of cochaperones by which the in vivo function of Hsp90 is regulated. TPR2A on HOP binds specifically to MEEVD motif of the C-terminal of Hsp90.
A homogeneous AlphaScreen is developed to assess the interaction between TPR2A and MEEVD motif of the C-terminal of Hsp90 by using the isolated TPR2A domain and a synthesized 20mer Hsp90 C terminal peptide. AlphaScreen is two bead-based proximity-dependent chemical energy transfer luminescent assay platform. TPR2A-His-6 and biotinylated Hsp90 C terminal peptide will bring streptavidin donor beads and nickel chelate acceptor beads to a proximity range (less than 200 nm), and subsequently lead a chemical luminescent reaction upon exciting donor beads with 680 nm laser. Inhibition of TPR2A and MEEVD motif interaction by small molecules could attenuate or abolish this chemical luminescent reaction.
Counter screening: Biotinylated-His-6 (PerkinElmer) can be utilized as a counter screen, as it serves as a linker to bring streptavidin beads and nickel chelate beads together. The counter screen can exclude alpha screen platform false positives due to small molecules being metal chelators, biotin analogues and imidazole analogues, or due to interfering with oxygen release from donor beads. Such an assay should be utilized to qualify any activity observed in this screen.
Protocol
NCGC Assay Protocol Summary:
The assay was performed on 1536-well white plate (untreated solid bottom). Liquids were dispensed by 1536-well plate washer (Kalypsys, San Diego, CA). Plates were read on EnVision (PerkinElmer, Boston, MA).TPR2A-His6 was produced using a bacteria expression system and purified using a well developed method (Ghosh et al., 2000). Hsp90 C terminal 20-mer peptide with or without an N-terminal biotin group are synthesized using automated solid phase synthesis by the Yale Keck facility. The composition of assay buffer was (in mM) 25 HEPES, 100 KCl, 2 MgCl2 and 2 DTT; 0.1% BSA and 0.01% Tween-20. TPR2A-His6 binding to biotin-Hsp90 will bring streptavidin donor beads and nickel chelate acceptor beads (PerkinElmer) together and can be measured by Alpha reading by EnVision (PerkinElmer). Free Hsp90 blocks biotin-Hsp90 binding to TPR2 and serves as a positive control in the current asssay.
The assay was performed at room temperature. All compounds were screened as titrations from 0.6 nM to 46 uM final concentration. The assay was performed in 1536-well format plates (untreated white solid bottom, Corning Inc.) as follows:
Sequence,Parameter,Value,Description
1,Reagent,3 uL TPR2A-His6,Final concentration was 5nM
2,Compound,23 nL,Final titration was from 0.7 nM to 46 uM
3,Time,15 minutes,Room temperature
4,Reagent,1 uL biotin-Hsp90,With and without free Hsp90. Final concentration was 10 nM for biotin-Hsp90 and 10uM for free Hsp90.
5,Time,1 hour,Room temperature
6,Reagent,1 uL bead mixture,10 ng/well for each bead
7,Time,2 hour,Room temperature
8,Detector,Alpha count,EnVision
Keywords: Hsp90, TPR2A, AlphaScreen, MLSMR, MLSCN, NIH Roadmap, qHTS and NCGC
Comment
Compound Ranking:
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Classification". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For inconclusive compounds, PUBCHEM_ACTIVITY_SCORE is 10.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: activation of assay signal, inhibition of assay signal, inactive, or inconclusive.String
2PotencyConcentration (in molar units) at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.String
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis Comment>Annotation/notes on a particular compound's data or its analysis.String
5Curve DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically signficant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the activity data to the Hill equation.Float
7Fit_HillSlopeThe Hill slope from a fit of the activity data to the Hill equation.Float
8Fit_R2R^2 fit value of curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivity>The asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Activity at 0.7 nM% Activity at given concentration.Float%
13Activity at 0.7 nM is validWhether measured activity was utilized during Hill curve fitting (true) or treated as an outlier (false) for data analysis purposes.Boolean
14Activity at 1.6 nM% Activity at given concentration.Float%
15Activity at 1.6 nM is validWhether measured activity was utilized during Hill curve fitting (true) or treated as an outlier (false) for data analysis purposes.Boolean
16Activity at 3.7 nM% Activity at given concentration.Float%
17Activity at 3.7 nM is validWhether measured activity was utilized during Hill curve fitting (true) or treated as an outlier (false) for data analysis purposes.Boolean
18Activity at 8.2 nM% Activity at given concentration.Float%
19Activity at 8.2 nM is validWhether measured activity was utilized during Hill curve fitting (true) or treated as an outlier (false) for data analysis purposes.Boolean
20Activity at 18 nM% Activity at given concentration.Float%
21Activity at 18 nM is validWhether measured activity was utilized during Hill curve fitting (true) or treated as an outlier (false) for data analysis purposes.Boolean
22Activity at 41 nM% Activity at given concentration.Float%
23Activity at 41 nM is validWhether measured activity was utilized during Hill curve fitting (true) or treated as an outlier (false) for data analysis purposes.Boolean
24Activity at 92 nM% Activity at given concentration.Float%
25Activity at 92 nM is validWhether measured activity was utilized during Hill curve fitting (true) or treated as an outlier (false) for data analysis purposes.Boolean
26Activity at 200 nM% Activity at given concentration.Float%
27Activity at 200 nM is validWhether measured activity was utilized during Hill curve fitting (true) or treated as an outlier (false) for data analysis purposes.Boolean
28Activity at 460 nM% Activity at given concentration.Float%
29Activity at 460 nM is validWhether measured activity was utilized during Hill curve fitting (true) or treated as an outlier (false) for data analysis purposes.Boolean
30Activity at 1.0 uM% Activity at given concentration.Float%
31Activity at 1.0 uM is validWhether measured activity was utilized during Hill curve fitting (true) or treated as an outlier (false) for data analysis purposes.Boolean
32Activity at 2.3 uM% Activity at given concentration.Float%
33Activity at 2.3 uM is validWhether measured activity was utilized during Hill curve fitting (true) or treated as an outlier (false) for data analysis purposes.Boolean
34Activity at 5.1 uM% Activity at given concentration.Float%
35Activity at 5.1 uM is validWhether measured activity was utilized during Hill curve fitting (true) or treated as an outlier (false) for data analysis purposes.Boolean
36Activity at 11 uM% Activity at given concentration.Float%
37Activity at 11 uM is validWhether measured activity was utilized during Hill curve fitting (true) or treated as an outlier (false) for data analysis purposes.Boolean
38Activity at 26 uM% Activity at given concentration.Float%
39Activity at 26 uM is validWhether measured activity was utilized during Hill curve fitting (true) or treated as an outlier (false) for data analysis purposes.Boolean
40Activity at 57 uM% Activity at given concentration.Float%
41Activity at 57 uM is validWhether measured activity was utilized during Hill curve fitting (true) or treated as an outlier (false) for data analysis purposes.Boolean
42Compound TypeNCGC designation for compound stage: 'qHTS Exploratory', 'NIHSMR', 'Compound Followup', 'Compound Verification', 'Probe Optimization'String
43Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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