Bookmark and Share
BioAssay: AID 651965

qHTS Assay for Activators of ClpP

The Clp protease proteolytic subunit (ClpP) is the core unit of the Clp complex belonging to the peptidase family S14 that hydrolyzes protein into small peptides in the presence of ATP and magnesium. It is ubiquitously expressed and is the major protease in bacteria. Studies showed that ClpP is a target for a novel class of acyldepsipeptides (ADEP) in bacteria that are resistant to current more ..
_
   
 Tested Compounds
 Tested Compounds
All(338379)
 
 
Active(6438)
 
 
Inactive(320022)
 
 
Inconclusive(12139)
 
 
 Tested Substances
 Tested Substances
All(342035)
 
 
Active(6440)
 
 
Inactive(323426)
 
 
Inconclusive(12169)
 
 
AID: 651965
Data Source: NCGC (CLPP001)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-01-03

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 6438
Related Experiments
AIDNameTypeComment
651966qHTS Assay for Activators of ClpP: SummarySummarydepositor-specified cross reference
743245qHTS Assay for Activators of ClpP: Hit Validation of InhibitorsConfirmatorysame project related to Summary assay
Description:
The Clp protease proteolytic subunit (ClpP) is the core unit of the Clp complex belonging to the peptidase family S14 that hydrolyzes protein into small peptides in the presence of ATP and magnesium. It is ubiquitously expressed and is the major protease in bacteria. Studies showed that ClpP is a target for a novel class of acyldepsipeptides (ADEP) in bacteria that are resistant to current antibiotic treatment. Unlike common antibiotics that simply interfere with the essential enzyme activity, ADEP binding activates the ClpP core in the absence of the regulatory Clp-ATPases leading to uncontrolled proteolysis and eventually cell death. Activators binding to the docking site on ClpP can also block formation of the ClpXP and ClpA/C-P holoenzyme complexes interfering with the normal biological functions of ClpP and thus have the potential to become new anti-microbials.

To identify activators of ClpP, a FRET based quenching reversal assay was developed using a purified B. subtilis ClpP and a decapeptide substrate (FRET-FV). The FRET-FV is tagged with a fluorescent molecule amino benzoic acid (donor) at the N-terminus and a nitrotyrosine quencher (acceptor) at the C-terminus. FRET-FV cleaves upon activation of ClpP with ADEP treatement and reverses the quenching effect of the nitrotyrosine causing an increase in fluorescence emission signal at 430nm. This assay was used to screen the Molecular Libraries Small Molecule Repository (MLSMR).

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

MLPCN Grant: MH095569
Assay Submitter (PI): Michael Maurizi, National Cancer Institute
Protocol
Two and a half microL/well of purified enzyme solution (final concentrations 10 microg/mL B. subtilis ClpP in 100 mM Tris/HCl pH8, 100 mM KCl, 0.05% low protease BSA, 2.5% glycerol, 0.01% Tween-20 buffer) was dispensed into 1536-well assay plates (Greiner, black solid, medium-binding plates) with Aurora Discovery BioRAPTR Flying Reagent Dispenser (FRD, Beckton-Dickenson). Compound solution (23 nL) was transferred to the assay plate using Kalypsys pin tool equipped with a 1536-pin tool. One and a half microL/well substrate solution (final concentrations 3 microM FRET-FV substrate in 100 mM Tris/HCl pH8, 100 mM KCl, 0.05% low protease BSA, 2.5% glycerol, 0.01% Tween-20 buffer) was then added for a total of 4 microL final reaction volume. FRET signals at time 0 and after 20 min room temperature incubation were obtained using the PerkinElmer Envision plate reader with 320nm excitation and 430nm emission filters. Delta FRET value (Time 20 - Time 0) was calculated and normalized to the delta FRET value of positive control ADEP (5.8 microM final concentration) and negative control DMSO and no enzyme wells.
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
5Activity_ScoreActivity score.Integer
6Curve_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
7Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
8Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
9Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
10Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
11Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
12Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
13Excluded_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
14Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
15Activity at 0.0009732759 uM (0.000973276μM**)% Activity at given concentration.Float%
16Activity at 0.00292 uM (0.00291983μM**)% Activity at given concentration.Float%
17Activity at 0.00876 uM (0.00875954μM**)% Activity at given concentration.Float%
18Activity at 0.026 uM (0.0262786μM**)% Activity at given concentration.Float%
19Activity at 0.079 uM (0.0788357μM**)% Activity at given concentration.Float%
20Activity at 0.115 uM (0.115μM**)% Activity at given concentration.Float%
21Activity at 0.236 uM (0.236183μM**)% Activity at given concentration.Float%
22Activity at 0.368 uM (0.367706μM**)% Activity at given concentration.Float%
23Activity at 0.461 uM (0.460544μM**)% Activity at given concentration.Float%
24Activity at 0.786 uM (0.786366μM**)% Activity at given concentration.Float%
25Activity at 1.251 uM (1.25092μM**)% Activity at given concentration.Float%
26Activity at 2.296 uM (2.2963μM**)% Activity at given concentration.Float%
27Activity at 3.166 uM (3.16601μM**)% Activity at given concentration.Float%
28Activity at 5.612 uM (5.61242μM**)% Activity at given concentration.Float%
29Activity at 10.17 uM (10.1672μM**)% Activity at given concentration.Float%
30Activity at 11.51 uM (11.5149μM**)% Activity at given concentration.Float%
31Activity at 20.59 uM (20.5948μM**)% Activity at given concentration.Float%
32Activity at 31.22 uM (31.2225μM**)% Activity at given concentration.Float%
33Activity at 57.49 uM (57.4898μM**)% Activity at given concentration.Float%
34Activity at 80.69 uM (80.6934μM**)% Activity at given concentration.Float%
35Activity at 115.0 uM (115μM**)% Activity at given concentration.Float%
36Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH095569

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