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BioAssay: AID 449750

Fluorescence Cell-Free Homogeneous Secondary Screen to Identify Non-Covalent Inhibitors of RecA-Intein Splicing Activity

M. tuberculosis harbors three inteins, which interrupt the DnaB, RecA, and SufB proteins. GFP-RecA intein fusion protein, which is purified as the 4-thiopyridine derivative in 8 M urea, can be refolded by dilution and retains full competence to undergo protein splicing upon activation by a thiol reducing reagent. In primary screening, the RecA intein splicing activity was followed by GFP more ..
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 Tested Compounds
 Tested Compounds
All(2128)
 
 
Active(1380)
 
 
Inactive(737)
 
 
Inconclusive(11)
 
 
 Tested Substances
 Tested Substances
All(2133)
 
 
Active(1385)
 
 
Inactive(737)
 
 
Inconclusive(11)
 
 
AID: 449750
Data Source: Broad Institute (2047-05_INHIBITORS_DOSE-TITRATION_MLPCN-CHERRYPICK)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-07-13
Hold-until Date: 2010-09-14
Modify Date: 2011-12-21

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 1380
Related Experiments
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AIDNameTypeComment
2223Broad Institute MLPCN RecA Intein Splicing Activity Inhibition ProjectSummarydepositor-specified cross reference: Summary
489010Intein inhibitors as potential Tuberculosis drugsConfirmatorydepositor-specified cross reference
492950Intein inhibitors as potential Tuberculosis drugs, a cytotoxicity screenConfirmatorydepositor-specified cross reference
2221Fluorescence Cell-Free Homogenous Primary HTS to Identify Inhibitors of RecA Intein Splicing ActivityScreeningsame project related to Summary assay
434968Fluorescence Cell-Free Homogeneous Counter Screen to Identify Inhibitors of GFP Chromophore FormationConfirmatorysame project related to Summary assay
435010Fluorescence Cell-Free Homogeneous Dose Retest to Identify Inhibitors of RecA-Intein Splicing ActivityConfirmatorysame project related to Summary assay
449749Fluorescence Cell-Free Homogeneous Secondary Screen to Identify Inhibitors of DnaB-Intein Splicing ActivityConfirmatorysame project related to Summary assay
493229RecA Intein Measured in Biochemical System Using Plate Reader - 2047-02_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
493233GFP-RecA MOA-DTT Measured in Biochemical System Using Plate Reader - 2047-05_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
493242DnaB Intein secondary assay Measured in Biochemical System Using Plate Reader - 2047-04_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
493243GFP Counter Screen Measured in Biochemical System Using Plate Reader - 2047-03_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
Description:
Keywords:GFP-RecA intein, protein splicing, refolding, reducing reagent, GFP fluorescence, covalent

Assay Overview:
M. tuberculosis harbors three inteins, which interrupt the DnaB, RecA, and SufB proteins. GFP-RecA intein fusion protein, which is purified as the 4-thiopyridine derivative in 8 M urea, can be refolded by dilution and retains full competence to undergo protein splicing upon activation by a thiol reducing reagent. In primary screening, the RecA intein splicing activity was followed by GFP fluorescence. This current MOA assay is to distinguish covalent inhibitors and candidates for non-covalent inhibition. Specifically, The GFP-RecA intein was incubated first with compounds in 1536-well plates, the RecA splicing activity was initiated by addition of 5mM DTT, which is known to abrogate covalent inhibition by electrophiles. GFP fluorescence, produced as a result of protein splicing, was read with fluorescence plate reader.



Expected Outcome: Active wells will show a reduced GFP fluorescence intensity due to non-covalent inhibition of RecA-Intein splicing activity.
Protocol
GFP-RecA Splicing Assay with DTT in Dose(for mode of action)

1) Dispense positive control
Dispense 100 nL of positive control ZnCl2(60mM) using Combi nl in respective wells according to plate design to 1536-well assay ready plates (Aurora 00027830) that contain 22.5 nL/well of 10 mM compound in 8 doses with 3fold dilution(generated by Echo acoustic dispenser).

2) Dispense GFP-RecA Intein
Add 3 uL/well of GFP-recA intein(2uM) using Combi nl(Thermo), incubate at room temperature for 30 minutes.
3) Dispense DTT to initiate the reaction
Add 3ulL/well of DTT(10mM) with Combi nL (Thermo), incubate at room temperature for ~18 hours
4)Centrifuge
Centrifuge the plates at 1000 rpm x1min
5) Read on Envision for Fluorescence(Ex.405nm/Em510nm)
Solutions:
Intein buffer
20 mM NaPi 7.0
500 mM NaCl
500 mM L-Arginine.HCL
.0.1% Triton-100
1% DMSO
ZnCl2 (in H2O, Sigma 229997-50G)
60mM ZnCl2
DTT (in intein buffer, Sigma 43816)
10 mM DTT
GFP-RecA intein (in intein buffer, provided by Henry Paulus's lab)
2 uM GFP-RecA Intein
Comment
Dose Data Analysis
Neutral control wells (NC) and positive controls wells (PC) were included on every plate.
Active compounds result in decreased readout signal.
Normalization and Pattern Correction
The raw signal was normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate PC wells is set to a normalized activity value of -100.
The median raw signal of the intraplate NC wells is set to a normalized activity of 0.
The raw signal of each compound well is then scaled to these two reference points to obtain a normalized activity value.
No plate pattern correction algorithm method was applied.
AC50 values were calculated using the curve fitting strategies in Genedata
Screener Condoseo (7.0.3). AC50 values were extrapolated up to 1 log
over the highest tested concentration.
PubChem Activity Score and Outcome
PUBCHEM_ACTIVITY_SCORE:
Inactive compounds = 0
Active compounds = -10*Log(AC50)
Superactive compounds (full inhibition at all concentrations; constant curve fit) = 100
PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when
AC50 > 1 log over the highest tested concentration
Activity_Outcome = 2 (active) when
AC50 <= 1 log over the highest tested concentration
Activity_Outcome = 3 (inconclusive) when
Curve fitting strategy resulted in a constant fit with activity >30% but <70%
or
The fit was not valid due to poor fit quality.
Activity_Outcome = 4 (unspecified) when
no usable data was obtained from the tested compound
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AC50_Qualifier'>', '=', or '<'String
2AC50_uM*the concentration at which activity reaches 50% of the maximumFloatμM
3Log_AC50_Mthe log of the AC50 (molar)Float
4Log_AC50_M_Standard_Errorthe standard error for the log of the AC50 valueFloat
5Hill_Slopethe slope at AC50Float
6S0the fitted activity level at zero concentrationFloat%
7SInfthe fitted activity level at infinite concentrationFloat%
8Num_Pointsthe number of data points included in the plotInteger
9Max_Activitythe maximum activity value observedFloat%
10Activity_at_18nM (0.018μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
11Activity_at_0.05uM (0.05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.15uM (0.15μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.46uM (0.46μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_1.35uM (1.35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_4.2uM (4.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_12uM (12μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_0.038mM (38μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH087438-01

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