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BioAssay: AID 434968

Fluorescence Cell-Free Homogeneous Counter Screen to Identify Inhibitors of GFP Chromophore Formation

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 screenning, the RecA intein splicing activity was followed by GFP more ..
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 Tested Compounds
 Tested Compounds
All(2128)
 
 
Active(1764)
 
 
Inactive(349)
 
 
Inconclusive(15)
 
 
 Tested Substances
 Tested Substances
All(2133)
 
 
Active(1769)
 
 
Inactive(349)
 
 
Inconclusive(15)
 
 
AID: 434968
Data Source: Broad Institute (2047-03_INHIBITORS_DOSE-TITRATION_MLPCN-CHERRYPICK)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-06-03
Modify Date: 2010-07-26

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 1764
Depositor Specified Assays
AIDNameTypeComment
492950Intein inhibitors as potential Tuberculosis drugs, a cytotoxicity screenconfirmatory
2223Broad Institute MLPCN RecA Intein Splicing Activity Inhibition Projectsummary
489010Intein inhibitors as potential Tuberculosis drugsconfirmatory
Description:
Keywords: GFP, 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. In primary screenning, the RecA intein splicing activity was followed by GFP fluorescence. The inhibition of GFP fluorescence could arise either from inhibition from the inhibition of protein splicing or from the inhibition of GFP chromophore formation. In this counter screen, cpds were tested with denatured GFP protein purified as the 4-thiopyridine derivative in 8 M urea to rule out false positives which interupt GFP chromophore formation. Specifically, 4-thiopyridine modified GFP was incubated with compounds first in 1536 well plates. the GFP chromophore formation was initiated by addition of reducing reagent TCEP. GFP fluorescence was read with fluorescence plate reader

Expected Outcome: Active wells will show a reduced GFP fluorescence intensity due to inhibition of GFP chromophore formation.
Protocol
GFP refolding assay in Dose (counter screen)

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 denatured GFP
Add 3 uL/well of denatured GFP(0.6uM) using Combi nl(Thermo), incubate at room temperature for 30 minutes.

3) Dispense TCEP to initiate the reaction
Add 3ulL/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
20mM NaPi 7.0
500mM NaCl
500mM L-Arginine.HCl
0.01% Triton-100
1% DMSO

ZnCl2 (in H2O, Sigma 229997-50G)
60mM ZnCl2

TCEP (in intein buffer, Sigma 646547)
2mM TCEP

denatured GFP- (in intein buffer, provided by Henry Paulus's lab)
0.6uM GFP
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.

EC50 values were calculated using the curve fitting strategies in Genedata
Screener Condoseo (7.0.3). EC50 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(EC50)

PUBCHEM_ACTIVITY_OUTCOME:

Activity_Outcome = 1 (inactive) when
EC50 > 1 log over the highest tested concentration

Activity_Outcome = 2 (active) when
EC50 <= 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
Result Definitions
Show more
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)
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