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BioAssay: AID 435010

Fluorescence Cell-Free Homogeneous Dose Retest to Identify 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. For primary screening, RecA intein fusion protein was incubated with compounds more ..
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 Tested Compounds
 Tested Compounds
All(2128)
 
 
Active(1875)
 
 
Inactive(163)
 
 
Inconclusive(91)
 
 
 Tested Substances
 Tested Substances
All(2133)
 
 
Active(1879)
 
 
Inactive(163)
 
 
Inconclusive(91)
 
 
AID: 435010
Data Source: Broad Institute (2047-02_INHIBITORS_DOSE-TITRATION_MLPCN-CHERRYPICK)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-06-22
Modify Date: 2011-10-18

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 1875
Related Experiments
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AIDNameTypeComment
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2223Broad Institute MLPCN RecA Intein Splicing Activity Inhibition ProjectSummarydepositor-specified cross reference: Project Summary
489010Intein inhibitors as potential Tuberculosis drugsConfirmatorydepositor-specified cross reference
492950Intein inhibitors as potential Tuberculosis drugs, a cytotoxicity screenConfirmatorydepositor-specified cross reference
2119Summary of Probe Development Efforts to Identify Inhibitors of Glycogen Synthase Kinase-3 beta (GSK-3b)Summarysame project related to Summary assay
434947Luminescence Cell-Free Homogeneous Counter Screen to Identify Inhibitors of ADP-glo ReagentsConfirmatorysame project related to Summary assay
434954Luminescence Cell-Free Homogeneous Dose Retest to Identify Inhibitors of Glycogen Synthase Kinase-3 beta ActivityConfirmatorysame project related to Summary assay
623978Inhibition of Human GSK3 alpha Activity Measured in Biochemical System Using Microfluidics - 2046-11_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
623979Inhibition of Human CDK5 Activity Measured in Biochemical System Using Microfluidics - 2046-12_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
623989Inhibition of Human GSK3 beta Activity Measured in Biochemical System Using Microfluidics - 2046-10_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
623998GSK3beta Measured in Biochemical System Using Plate Reader - 2046-01_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
623999CDK5 Measured in Biochemical System Using Plate Reader - 2046-05_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624027GSK3beta Assay at Varied ATP concentrations Measured in Biochemical System Using Plate Reader - 2046-04_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624057Human p-Tau and Total Tau ELISA Measured in Cell-Based System Using Plate Reader - 2133-07_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624076Kinome profiling single dose point Measured in Biochemical System Using Plate Reader - 2133-09_Inhibitor_SinglePoint_DryPowder_ActivityOthersame project related to Summary assay
624086TCF/LEF Reporter Assay Measured in Cell-Based System Using Plate Reader - 2133-01_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624088PathHunter bCatenin (U2-OS) Measured in Cell-Based System Using Plate Reader - 2133-06_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624091Dose responses of selected compounds against GSK3b Measured in Biochemical System Using Plate Reader - 2133-10_Inhibitor_Dose_DryPowder_ActivityOthersame project related to Summary assay
624108GSK3beta Surface Plasmon Resonance (SPR) Assay Measured in Biochemical System Using Microfluidics - 2133-08_Inhibitor_Dose_DryPowder_ActivityOthersame project related to Summary assay
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
449749Fluorescence Cell-Free Homogeneous Secondary Screen to Identify Inhibitors of DnaB-Intein Splicing ActivityConfirmatorysame project related to Summary assay
449750Fluorescence Cell-Free Homogeneous Secondary Screen to Identify Non-Covalent Inhibitors of RecA-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

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. For primary screening, RecA intein fusion protein was incubated with compounds first in 1536 well plates. The RecA intein splicing activity was initiated by addition of reducing reagent TCEP. 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 inhibition of RecA-Intein splicing activity.
Protocol
GFP-RecA Splicing Assay in Dose

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 3-fold 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 TCEP to initiate the reaction
Add 3uL/well of TCEP(2mM) 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/Em.510nm)
Solutions:
Intein buffer
20 mM NaPi 7.0
500 mM NaCl
500 mM L-Arginine.HCL
0.01 % Tritin-100
1 % DMSO
ZnCl2 (in H2O, Sigma 229997-50G)
60 mM ZnCl2
TCEP (in intein buffer, Sigma 646547)
2 mM TCEP
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.
IC50 values were calculated using the curve fitting strategies in Genedata
Screener Condoseo (7.0.3). IC50 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(IC50)
PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when
IC50 > 1 log over the highest tested concentration
Activity_Outcome = 2 (active) when
IC50 <= 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
1EC50_Qualifier'>', '=', or '<'String
2EC50_uM*the concentration at which activity reaches 50% of the maximumFloatμM
3Log_EC50_Mthe log of the EC50 (molar)Float
4Log_EC50_M_Standard_Errorthe standard error for the log of the EC50 valueFloat
5Hill_Slopethe slope at EC50Float
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
Classification
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