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BioAssay: AID 504842

Inhibitors of TCP-1 ring complex (TRiC) of Methanococcus maripaludis (MmCpn): qHTS

The eukaryotic chaperonin TRiC (for TCP-1 ring complex) is an essential and conserved chaperone required for the folding and activation of 10-15% of the proteins encoded by the human genome. TRiC is indispensable for cell survival, as folding of an essential subset of cytosolic proteins, including cytoskeletal components, cell cycle regulators and tumor suppressor proteins, exhibits a strict more ..
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 Tested Compounds
 Tested Compounds
All(329620)
 
 
Active(101)
 
 
Inactive(324624)
 
 
Inconclusive(4910)
 
 
 Tested Substances
 Tested Substances
All(330046)
 
 
Active(101)
 
 
Inactive(325034)
 
 
Inconclusive(4911)
 
 
AID: 504842
Data Source: NCGC (TRiC001)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-06-23

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 101
Related Experiments
AIDNameTypeComment
488991High-Throughput Screening for Modulators of Cytosolic Chaperonin Activity: SummarySummarydepositor-specified cross reference
488978High-Throughput Screening for Modulators of Cytosolic Chaperonin Activity: MmCpn Primary ScreenConfirmatorysame project related to Summary assay
651819High-Throughput Screening for Modulators of Cytosolic Chaperonin ActivityScreeningsame project related to Summary assay
Description:
The eukaryotic chaperonin TRiC (for TCP-1 ring complex) is an essential and conserved chaperone required for the folding and activation of 10-15% of the proteins encoded by the human genome. TRiC is indispensable for cell survival, as folding of an essential subset of cytosolic proteins, including cytoskeletal components, cell cycle regulators and tumor suppressor proteins, exhibits a strict requirement for TRiC which cannot be substituted by other cytosolic chaperones. The TRiC complex consist of two stacked 7-9 membered rings, each containing a central cavity. Substrates are folded within the cavity in an ATP-dependent manner. TRiC is a complex of eight different, essential proteins. As each of the genes encoding the eight subunits is essential for cell viability and no protocol has been developed to over-express this enzyme to high levels. That is why several groups studying the TRiC chaperonin have turned to the archaeal homolog of TRiC, Methanococcus maripaludis (Mm-CPN), and have used this enzyme as a model system for the study of the type II chaperonin family. Small molecule inhibitors of the Mm-CPN enzyme would be extremely useful to study the mechanism of chaperonin activity. Importantly, TRiC and Mm-CPN both require ATP hydrolysis for function. Furthermore, the ATP binding sites are highly conserved among these type II chaperonins. This provides us with an easy, well tested and straightforward assay to identify inhibitors of chaperonin activity.

The Mm-CPN assay was developed in 1536-well plate format using the HTRF Transcreener ADP assay kit (Cisbio, Bedford, MA). This assay applied the time resolved fluorescence resonance energy transfer (TR-FRET) principle. ATP in the presence of Mm-CPN is converted to ADP + Pi. An antibody labeled with Eu3+-Cryptate specific and bound to ADP labeled with deuterium (d2) competes with the unlabeled ADP generated by Mm-CPN. The resulting TR-FRET signal is inversely proportional to the concentration of ADP in the sample. In this assay a reduction in TR-FRET signal is observed with ATP turnover.

NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]

MLPCN Grant: DA030554-01A1
Assay Submitter (PI): Judith Frydman, Stanford University
Protocol
Purified Mm-CPN was supplied by the laboratory of Dr. Judith Frydman. Protein samples were stored at -80C as single use aliquots. Protein was thawed in ambient temperature water bath and placed on ice once thawed. Buffer was prepared fresh day of screen. The buffer composition below was used to prepare the working solution of the Mm-CPN protein and ATP substrate. The buffer is comprised of 30 mM TRIS pH 7.4, 100mM KCl, 5mM MgCl2, and 1mM DTT. A final concentration of 25nM Mm-CPN and 10uM ATP per well were used in the screen. Instructions according to the manufacturer were followed for the ADP Transcreener kit. Used 1536 well Greiner white, medium binding (MB), solid bottom plates. Plates were read on the PerkinElmer EnVision plate reader using Excitation = 320 nm and Emission = 590, 665 nm.
Protocol-
1.Dispense 1.5ul per well of working [Mm-CPN] = 50nM (or 75 ng / well)
2.Spin plate at 1000 rpm for 30 seconds
3.23nl of compound transferred per well
4.Incubate at 37degC for 10 minutes
5.Dispense 1.5ul per well of working [ATP] = 20uM
6.Incubate at 37degC for 30 minutes
7.Dispense 3ul per well of Anti-ADP Cryptate / ADP-d2 mix (prepared as instructed by Cisbio)
8.Incubate at ambient for 30 minutes
9.Read using EnVision
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 Description". 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 all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*Concentration 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.FloatμM
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 CommentAnnotation/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 significant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe 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
12Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
13Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Activity at 0.123 uM (0.123μM**)% Activity at given concentration.Float%
15Activity at 0.615 uM (0.615μM**)% Activity at given concentration.Float%
16Activity at 3.080 uM (3.08μM**)% Activity at given concentration.Float%
17Activity at 15.40 uM (15.4μM**)% Activity at given concentration.Float%
18Activity at 76.90 uM (76.9μM**)% Activity at given concentration.Float%
19Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: DA030554

Data Table (Concise)
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